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Myeloid leukemia in children with Down syndrome (ML-DS) is associated with young age and somatic GATA1 mutations. Because of high event-free survival (EFS) and hypersensitivity of the leukemic blasts to chemotherapy, the prior Children’s Oncology Group protocol ML-DS protocol (AAML0431) reduced overall treatment intensity but lacking risk stratification, retained the high-dose cytarabine course (HD-AraC), which was highly associated with infectious morbidity. Despite high EFS of ML-DS, survival for those who relapse is rare. AAML1531 introduced therapeutic risk stratification based on the previously identified prognostic factor, measurable residual disease (MRD) at the end of the first induction course. Standard risk (SR) patients were identified by negative MRD using flow cytometry (

Biallelic CEBPA mutations are associated with favorable outcomes in acute myeloid leukemia (AML). We evaluated the clinical and biologic implications of CEBPA-basic leucine zipper (CEBPA-bZip) mutations in children and young adults with newly diagnosed AML. CEBPA-bZip mutation status was determined in 2958 patients with AML enrolled on Children’s Oncology Group trials (NCT00003790, NCT0007174, NCT00372593, NCT01379181). Next-generation sequencing (NGS) was performed in 1863 patients (107 with CEBPA mutations) to characterize the co-occurring mutations. CEBPA mutational status was correlated with disease characteristics and clinical outcomes. CEBPA-bZip mutations were identified in 160 (5.4%) of 2958 patients, with 132 (82.5%) harboring a second CEBPA mutation (CEBPA-double-mutated [CEBPA-dm]) and 28 (17.5%) had a single CEBPA-bZip only mutation. The clinical and laboratory features of the 2 CEBPA cohorts were very similar. Patients with CEBPA-dm and CEBPA-bZip experienced identical event-free survival (EFS) of 64% and similar overall survival (OS) of 81% and 89%, respectively (P = .259); this compared favorably to EFS of 46% and OS of 61% in patients with CEBPA-wild-type (CEBPA-WT) (both P < .001). Transcriptome analysis demonstrated similar expression profiles for patients with CEBPA-bZip and CEBPA-dm. Comprehensive NGS of patients with CEBPA mutations identified co-occurring CSF3R mutations in 13.1% of patients and GATA2 mutations in 21.5% of patients. Patients with dual CEBPA and CSF3R mutations had an EFS of 17% vs 63% for patients with CEBPA-mutant or CSF3R-WT (P < .001) with a corresponding relapse rate (RR) of 83% vs 22%, respectively (P < .001); GATA2 co-occurrence did not have an impact on outcome. CEBPA-bZip domain mutations are associated with favorable clinical outcomes, regardless of monoallelic or biallelic status. Co-occurring CSF3R and CEBPA mutations are associated with a high RR that nullifies the favorable prognostic impact of CEBPA mutations.

We enrolled 150 patients in a prospective multi-center study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplant (HSCT) comparing detection of measurable residual (MRD) disease by a “Difference from Normal” flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT-1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients being screened for HSCT had detectable residual disease by ΔN (0.04–53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (0.04–14%) by ΔN. The disease free survival of this group was 10% (0–35%), compared to 55% (46–64

The CBFA2T3-GLIS2 (CBF/GLIS) fusion is a product of a cryptic translocation exclusively seen in refractory infant AML. Lack of relevant model systems that accurately recapitulate this infant AML has limited progress. To overcome this barrier, we developed an endothelial cell (EC) co-culture system to support malignant transformation, self-renewal, and propagation of leukemia-initiating cells (LIC) in CBF/GLIS-transduced human cord blood hematopoietic stem/progenitor cells (CB HSPCs) ex vivo. Lack of recurrent cooperating mutations suggests that CBF/GLIS fusion might be sufficient for malignant transformation. To test this, we expressed the CBF/GLIS fusion or GFP control in CB HSPCs (CBF/GLIS-CB or GFP-CB) by lentiviral transduction and placed transduced cells in either EC co-culture or myeloid-promoting culture (MC). CBF/GLIS-CB cells expanded faster with prolonged lifespan in EC co-culture compared to MC (Figure 1A). Proliferation of CBF/GLIS-CB cells declined after transfer to either an EC trans-well culture or in suspension culture (Figure 1B), suggesting that direct contact as well as secreted factors are required for optimal growth of transduced cells. The CBF/GLIS fusion has been shown to confer enhanced megakaryocytic differentiation. At 6 weeks, CBF/GLIS-CB cells in EC co-culture formed significantly more megakaryocytic colonies than CBF/GLIS-CB cells grown in MC or CBF/GLIS-GFP cells grown in either condition (Figure 1C). At 12 weeks, CBF/GLIS-CB cells cultured in EC co-culture continued to produce numerous megakaryocytic colonies, demonstrating long lived self-renewal and enhance megakaryocytic differentiation of CBF/GLIS-CB cells co-cultured with ECs. To determine whether the EC niche promotes generation and propagation of LICs, we evaluated the murine engraftment of CBF/GLIS-CB cells expanded on ECs or in MC following 3, 6, 9 and 12 weeks of culture. CBF/GLIS-CB cells cultured in EC co-culture at each time point exhibited robust engraftment that progressed to frank leukemia in vivo (Figure 1D), demonstrating that EC co-culture promotes long-term maintenance of functional LICs. CBF/GLIS-CB cells grown in MC also induced leukemia from 3- and 6-week cultures but then became senescent at 9 and 12 weeks, suggesting limited preservation of the LICs. Flow cytometric analysis of CBF/GLIS-CB cells identified a malignant population that is of the RAM immunophenotype (CD56 hi, CD45 dim, and CD38 dim/-) previously reported in infants with CBF/GLIS AML in both culturing conditions. However, CBF/GLIS-CB cells in EC co-culture constituted an almost homogeneous population that expressed the RAM immunophenotype, whereas only a subset was detected in MC at week 6 (Figure 1E). To determine the fidelity of transformation to primary leukemia, we performed RNA-sequencing of CBF/GLIS-CB cells cultured with ECs or in MC. Unsurpervised clustering analysis demonstrated that the CBF/GLIS-CB cells from weeks 6 and 12 in EC co-culture clustered with primary CBF/GLIS-positive patient samples, but not CBF/GLIS-CB cells cultured in MC nor GFP controls (Figure 1F). Further transcriptome analysis revealed CBF/GLIS and HSC signature genes, previously identified to be associated with CBF/GLIS AML, were both significantly enriched in CBF/GLIS-CB cells grown in EC culture relative to MC (Figure 1G). These results suggested that the signaling pathways that are aberrantly dysregulated in primary CBF/GLIS leukemia are faithfully recapitulated in CBF/GLIS-CB cells co-cultured with ECs. Despite concerted efforts, previous attempts to model CBF/GLIS AML in murine hematopoietic cells have failed to generate overt leukemia. In this study, we demonstrate that in an EC co-culture system, the CBF/GLIS oncogenic fusion is sufficient to transform human CB HSPCs that faithfully recapitulates the morphology, transcriptome and immunophenotype of CBF/GLIS AML as well as highly aggressive leukemia in xenograft models. Furthermore, the EC co-culture system provides a tractable model system to further interrogate the mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy in CBF/GLIS AML. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Introduction: It has long been known that Down syndrome is associated with an increased risk for hematologic malignancies. One such disease is myeloid leukemia associated with Down syndrome (ML-DS), a disease that almost always occurs during the first 5 years of life. As research on ML-DS has progressed, understanding has grown that after the initiation of therapy, the non-neoplastic myeloid progenitor cells in ML-DS patients have a characteristic immunophenotype with the expression of CD56 on a subset of the CD34+ myeloid progenitor cells being one of the most notable features. The discovery that this immunophenotype is normal in ML-DS patients post-therapy has been of the utmost importance as it has led to such patients being properly classified as being negative for measurable residual disease. Plasmacytoid dendritic cells (pDCs) are another cell type in which CD56 expression is often part of the neoplastic immunophenotype, and we hypothesized that CD56 may also be differentially expressed in ML-DS pDCs post-therapy. Herein, we investigated the immunophenotype of pDCs in ML-DS patients found to be negative for measurable residual disease. Methods: A total of 10 bone marrow specimens from ML-DS patients post-treatment initiation and 7 bone marrow specimens from patients that did not have DS, were aged 0-4 years (matching the age range of ML-DS), had a myeloid neoplasm and were post-treatment initiation (non-DS) were included in this study. All specimens were found to be negative for measurable residual disease by difference from normal (ΔN) flow cytometry (the gold standard for the determination of residual disease in the Children's Oncology Group 1531 study on ML-DS) and were evaluated for CD56 and CD303 expression on pDCs. pDCs were defined as HLA-DR+/CD123++ (high intensity). Results: As expected, the ML-DS patients had a significantly greater percentage of CD34+CD56+ myeloid progenitor cells than the non-DS group, both in terms of percent total non-erythroid cells (0.9% vs 0.006%, P Conclusions: The data from this preliminary study indicate that much like the myeloid progenitor cells, the pDCs in ML-DS patients after the initiation of therapy have an immunophenotype that could be mistaken as abnormal. Importantly, they show that the setting of cutoff values for the determination of abnormal pDC CD56 expression, even relatively high ones, could lead to false positive results in ML-DS specimens post-treatment initiation. The dissemination of this knowledge is of increased importance as more flow cytometry laboratories begin to increase their investigation of pDCs. Further studies are needed to delineate common mechanisms between the expression of CD56 in myeloid progenitor cells and pDCs in ML-DS patients post-treatment initiation. Disclosures Pardo: Hematologics, Inc.: Current Employment. Lott: Hematologics, Inc.: Current Employment. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership.

Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p3% was strongly associated with MDS/CMML (286/293, 98%, p Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

Introduction: Despite advances in cytotoxic and targeted therapies, recurrent disease remains the most significant obstacle to long-term survival in patients with childhood and adult leukemias. As part of our efforts to identify therapies that can be repurposed for immediate use in patients with leukemia, we interrogated a library of all available antibody-drug conjugates (ADCs) whose targets are expressed in leukemias (AML or ALL). A list of targets with available ADCs was merged with transcriptome data from over 3000 pediatric and adult leukemias (AML and ALL) to identify targets that are expressed in a large cohort of leukemias with immediate therapies for repurposing. The transcriptome data set included nearly 2000 pediatric AML cases sequenced as part of Target Pediatric AML (TpAML), 419 adult AML cases from TCGA LAML and the Beat AML program, as well as 853 ALL cases from COG and St. Jude trials. In this repurposing endeavor, CD74 emerged as the most expressed transcript in AML and ALL. CD74 encodes for a cell surface protein that associates with the class II major histocompatibility complex and is involved in the regulation of antigen presentation for immune response and B-cell differentiation. STRO-001 (Sutro Biopharma) is a CD74-directed, site-specific ADC developed for the treatment of multiple myeloma and lymphomas. Given broad expression of CD74 in leukemias, we evaluated the efficacy of STRO-001 in AML and ALL preclinical models. Methods: To characterize CD74 expression, RNA-seq data obtained from pediatric and adult AML and ALL patients was examined. Cell surface expression of CD74 was determined by flow cytometry using PE labeled anti-human CD74 antibody. The CD74-targeting ADC (STRO-001) was obtained from Sutro Biopharma.The preclinical efficacy of STRO-001 was evaluated against AML and ALL cell lines and patient samples expressing various levels of CD74 in vitro and in vivo. For in vivo studies, AML and ALL cell lines were transduced with GFP/Luciferase construct, and GFP+ cells were injected intravenously into NSG mice. Leukemia burden was measured by bioluminescence (IVIS) imaging weekly. Results: Transcriptomics analysis showed CD74 expression in a majority of adult and pediatric AML (>99% of cases) and at a much higher level compared CD33 and CD123 (targets currently developed for AML, Fig. 1A). CD74 is also broadly expressed in pediatric ALL, with a significant increase in expression observed compared to CD19 and CD22 (known targets in ALL, Fig. 1B). We confirmed that CD74 is expressed on the cell surface of AML blasts in primary patient samples (Fig. 1C) as well as AML and ALL cell lines (Fig. 1D). Given confirmation of cell surface expression of CD74, we investigated whether targeting CD74 can effectively eliminate leukemia cells. We evaluated the in vitro cytotoxicity of STRO-001 against K562 (a CML cell line that does not express CD74), AML cell lines (MV4;11 and NOMO-1), and ALL cell lines (REH1 and RS4;11) with varied CD74 expression. STRO-001 demonstrates target-specific cytotoxicity against CD74-expressing AML and ALL cell lines, but not K562 cells (Fig. 1E). STRO-001 exhibited high potency in CD74 expressing cells, with IC-50s of 41nM (MV4;11), 1.3nM (NOMO-1), 0.7nM (REH-1) and 3nM (RS4;11). In vivo studies in NSG mice transplanted with AML and ALL cell lines showed high potency. Treatment with STRO-001 at 3mg/kg once a week for 3 weeks effectively eradicated the leukemia in NOMO-1, REH-1, and RS4;11-bearing xenograft mice, while disease progression was observed in untreated control mice (Fig. 1F). We further evaluated the efficacy of STRO-001 in primary patient samples. Primary leukemia specimens from 3 patients with varied CD74 expression (Fig. 1G) were incubated with increasing concentrations of STRO-001 for 3 days. STRO-001 exhibited potent anti-leukemia activity against primary AML cells with IC-50s of 17.4nM, 12.8nM, and 4.07nM, respectively (Fig. 1H). Conclusion: Through transcriptomics profiling and validation of the cell surface expression by flow cytometry, we have identified CD74 as a viable therapeutic target for AML and ALL in children and adults. We further demonstrate that targeting CD74 with STRO-001 effectively eliminates leukemia cells both in vitro and in vivo, providing the preclinical data to compel evaluation of STRO-001 in clinical trials for childhood and adult leukemia. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Background: A rare but highly aggressive type of AML that is only seen in infants with a unique immunophenotype (RAM phenotype) is caused by cryptic CBFA2T3-GLIS2 (CBF/GLIS) fusion. This infant AML is highly refractory to conventional chemotherapy with near uniform fatality despite highly intensive and myeloablative therapy (PMID 23153540). Transcriptome profiling of CBF/GLIS AML has revealed new insights into the pathogenesis of the fusion and uncovered fusion-specific molecular biomarkers that could be used for risk stratification and to inform treatment (PMID 30592296). Studying the largest cohort of these high-risk infants, we demonstrated several alterations in gene expression and transcriptional networks in these CBF/GLIS-positive patient samples that have potential for therapeutic targeting (PMID 31719049). FOLR1, which encodes for folate receptor alpha, was highly and uniquely expressed in CBF/GLIS AML but was entirely absent in AML with other cytogenetics abnormalities and in normal hematopoietic cells. Furthermore, we recently demonstrated that forced expression of CBF/GLIS enhances the proliferation and alters differentiation in cord blood (CB) CD34+ early precursors towards megakaryocytic lineage that recapitulates acute megakaryocytic leukemia seen in infants (PMID 31719049). Of significance, we showed that FOLR1 surface expression is causally linked to CBF/GLIS-induced malignant transformation, thus making it an attractive antigen for targeted therapies against CBF/GLIS AML cells. Given that chimeric antigen receptor (CAR) T cells are extremely effective at eradicating relapsed/refractory B-ALL malignancies, we developed FOLR1-directed CAR T cells for pre-clinical evaluation in CBF/GLIS AML. Methods: We generated a FOLR1-directed CAR using anti-FOLR1 binder (Farletuzumab), IgG4 intermediate spacer and 41-BB/CD3zeta signaling domains. The pre-clinical efficacy of FOLR1 CAR T cells was evaluated against CBF/GLIS AML cell lines in vitro and in vivo. CBF/GLIS AML models include CB CD34+ cells transduced with CBF/GLIS expression construct (CBF/GLIS-CB) and WSU-AML cell line. We also engineered Kasumi-1 cell line to express FOLR1 (Kasumi-1 FOLR1+) to evaluate target specificity (Figure 1A). Results: We tested the target specificity of FOLR1-directed CAR T cells against FOLR1-positive (CBF/GLIS-CB, WSU-AML, Kasumi-1 FOLR1+) and FOLR1-negative (Kasumi-1) cells. CD8 FOLR1 CAR T cells demonstrated cytolytic activity against FOLR1 positive but not FOLR1 negative cells (Figure 1B). Furthermore, both CD8 and CD4 FOLR1 CAR T cells produced higher levels of IL-2, IFN-γ, and TNF-α and proliferated more robustly than did unmodified T cells when co-incubated with FOLR1 positive but not FOLR1 negative cells (Figure 1C). These results indicate highly specific reactivity of FOLR1 CAR T cells against AML cells expressing FOLR1. We next investigated the in vivo efficacy of FOLR1-directed CAR T cells. In CBF/GLIS-CB, WSU-AML, and Kasumi-1 FOLR1+ xenograft models, treatment with FOLR1 CAR T cells induced leukemia clearance, while disease progression occurred in all mice that received unmodified T cells (Figure 1D). Activity of FOLR1 CAR T cells in vivo was target specific, as they did not limit the leukemia progression nor extend the survival of Kasumi-1 xenografts (Figure 1D). To determine whether FOLR1 is expressed on normal HSPCs, we characterized FOLR1 expression in normal CB CD34+ samples. FOLR1 expression was entirely silent in HSPC subsets (Figure 1E). Consistent with lack of expression, no cytolytic activity was detected against HPSCs Moreover, FOLR1 CAR T cells did not affect the self-renewal and multilineage differentiation capacity of normal HSPCs as compared to unmodified control T cells (Figure 1F), whereas significant eradication of colonies were detected in the CBF/GLIS-CB cells (Figure 1G). Conclusion: In this study, we demonstrate that FOLR1 CAR T effectively eradicates CBF/GLIS AML cells without compromising normal HSPCs, providing a promising approach for the treatment of high-risk CBF/GLIS AML. Transition of this CAR T to clinical development for infant AML is underway. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Hematopoietic stem cells (HSC) are defined by the expression of the CD34 antigen on the cell surface whereby CD34+/CD38- repopulating cells have been the hallmark. This combination of antigens defining normal HSC has also been used to define leukemic stem cells (LSC). Although there is recognition of variability of CD34 expression in AML and lack of CD34 expression in a subset of AMLs (e.g., NPM1c), accurate and functional consequence of CD34 expression in AML has not yet been fully defined. We examined CD34 transcript (RNA seq) and protein expression (flow cytometry) as well as methylation status in a cohort of 2185 adult and pediatric AML cases. We demonstrate that CD34 is silenced in a significant proportion of AML through epigenetic mechanism and correlate CD34 expression with disease characteristics, fusions, mutations, and clinical outcome. Ribodepleted RNA seq data from 1048 pediatric AML (Children's Oncology Group, AAML1031), 390 adult AML (TCGA and BeatAML), 68 bone marrow, and 16 CD34+ PBMC from healthy donors were interrogated for CD34 expression. CD34 negative cohort ( A correlation of CD34 expression with fusion and SNV subtypes revealed striking lack of CD34 expression in patients with KMT2A (54%), NUP98-KDM5A (81%), KAT6A-CREBBP (83%) fusions and NPM1 mutation (62%; Fig. 1B). The paucity of CD34 expression in KMT2A-r cohort was also seen in adults with a frequency of 61% CD34-Neg in KMT2A-r. Outcome evaluation of all patients based on CD34 expression failed to demonstrate differences in EFS or OS. However, significant variability in CD34 expression within KMT2A-r cases prompted further analysis. KMT2A-r patients with and without CD34 expression had an OS of 40% and 60%, respectively (p=0.0013, Fig. 1C). Despite the recent allocation of some KMT2A-r to high risk status based on fusion partners, the majority of KMT2A-r patients remain standard risk (SR). We examined the clinical outcome for SR KMT2A-r patients (which represents 71% of KMT2A-r). Using the same cutoffs, CD34-Neg group showed dramatically better outcomes, with 5 yr estimates of 42% vs. 24%, EFS and 67% vs. 38%, OS (p=0.0019, p The observation by transcriptome was validated by multi-dimensional flow cytometry (MDF) conducted at Hematologics, Inc. CD34 expression data by MDF was available from 1613 patients enrolled on COG AAML0531 and AAML1031. CD34 expression Given CD34 silencing in such a large cohort of patients, we inquired the underlying mechanism of such significant silencing. We evaluated the corresponding methylation data (Illumina EPIC array) to determine whether CD34 expression may be modulated through epigenetic mechanisms. Correlation of CD34 transcript expression with CD34 promotor methylation status demonstrated significant hypermethylation of the CD34 promotor sites (Fig. 1F). The CD34 antigen plays an important role as a marker of HSCs and LSCs, however, at least 25% of patients lack expression. CD34-Neg is markedly correlated with KMT2A-r AML and specifically enriched in fusion partners which lack risk stratifying features. CD34-Neg also exhibit significantly improved OS and EFS over CD34-expressors. These outcome observations may have dramatic and immediate implications in the clinical management of the SR KMT2A cohort. Flow based CD34 quantification may be an immediate tool used to identify KMT2A-r patients with CD34 expression; these patients may benefit from intensive or targeted therapies. Figure 1 Figure 1. Disclosures Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Pardo: Hematologics, Inc.: Current Employment. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Cryptic inv(16)(p13.3q24.3) leading to the CBFA2T3-GLIS2 (CBF/GLIS) oncogenic fusion is exclusively seen in infants with AML and is associated with adverse outcome. Infants with this fusion are uniformly refractory to conventional therapies and despite intensive and myeloablative therapies, virtually all patients relapse with survival less than 15% (Smith et. al. 2020). In the effort to discover actionable targets for this highly refractory leukemia, we interrogated the genome and transcriptome of nearly 3,000 AML cases, including 45 cases of CBF/GLIS AML, and contrasted this to the transcriptome in normal hematopoietic tissues. Initial computational effort to identify CBF/GLIS-specific targets focused on genes that are overexpressed in CBF/GLIS AML and silent in normal hematopoietic tissues providing us with 194 candidate genes. Filtering this library of genes based on cell surface expression and prevalence in target population narrowed the target gene list to six cell surface encoding genes (FOLR1, FRAS1, HPSE2, KLRF2, MEGF10 and PCDH19). FOLR1 encodes folate receptor alpha and given available therapeutic options with FOLR1-targeting agents, this gene was selected for further studies and therapeutic assessment of available targeted therapies. Based on RNA-seq data, FOLR1 is uniquely expressed in CBF/GLIS AML as it is absent in other AML and in normal hematopoietic cells (Figure 1A) providing an opportunity to specifically target leukemia cells while sparing normal hematopoiesis. Cell surface expression of FOLR1 on AML blasts was confirmed by flow cytometry, which further shows AML-restricted expression of FOLR1 on AML cells but not in normal hematopoietic subsets in individual CBF/GLIS patient samples (Figure 1B, C). We previously showed that forced expression of CBF/GLIS fusion transcript in cord blood hematopoietic stem/ progenitor cells (CB HSPCs) induces malignant transformation that fully recapitulates primary CBF/GLIS AML (Hylkema et. al. 2019 ASH). We also demonstrated that CBF/GLIS-transduced CB HSPCs upregulate FOLR1 expression indicating that FOLR1 expression is causally associated with CBF/GLIS expression (Figure 1D). Given cell surface expression of FOLR1 on CBF/GLIS AML cells, we next investigated the preclinical efficacy of targeting FOLR1 using FOLR1-directed site-specific antibody-drug conjugate (STRO-002, Sutro Biopharma). In vitro efficacy of STRO-002 was tested against MV4;11 AML cell line engineered to express FOLR1 (MV4;11 FOLR1+, Figure 1D) and CBF/GLIS-transduced CB HPSCs. STRO-002 exhibited high cytotoxicity against MV4;11 FOLR1+ and CBF/GLIS-transduced CB HPSCs with IC50s of 0.1nM and 4.2nM, respectively (Figure 1E). In vivo efficacy of STRO-002 was evaluated in MV4;11 FOLR1+ NSG xenograft models, which showed potent activity that led to complete leukemia clearance after 3 weekly doses of STRO-002 at 2.5 and 5 mg/kg (Figure 1F). The anti-leukemia effects of STRO-002 resulted in significant increase in survival (p=0.002, Figure 1G). STRO-002 showed similar in vivo efficacy in the xenograft mice bearing CBF/GLIS-transduced CB HSPCs (Figure 1H) suggesting highly potent activity of STRO-002 against FOLR1+ AML cells. In this study, we utilize a computational approach to discover AML-restricted targets (high expression in AML, silent in normal) that are previously elusive to identify actionable targets for high-risk CBF/GLIS AML. From this discovery pipeline, we demonstrate FOLR1 to be highly and uniquely expressed in CBF/GLIS AML but not in normal counterparts, providing a strategy to target leukemia cells without impacting normal hematopoiesis. Finally, we demonstrate that targeting FOLR1 with STRO-002 antibody drug conjugate effectively eliminates CBF/GLIS-positive AML in vitro and in vivo, providing a promising approach to eradicate leukemia and improve outcomes in this high-risk AML subtype. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

The quantitative expression of cell surface antigens and light scattering properties of five cellular reference populations in stressed bone marrow specimens were compared between pediatric and adult patients treated for acute myeloid leukemia (AML). The mean intensity of each antigen as well as the within patient and between patient variability showed striking consistency between the two different age groups. The only difference between the groups of specimens was the proportion of progenitor cells in the adult cohort averaged less than three times the proportion in the pediatric cohort. These data show that the amounts of gene products expressed on bone marrow cells are invariant with age. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Introduction: CD74 is a type II transmembrane protein expressed on antigen-presenting cells and an MHC class II chaperone. It has been associated with tumor progression and metastasis in solid tumors, and its expression has been suggested to serve as a prognostic factor in many cancers, with higher relative expression associated with oncogenesis. As the expression of CD74 has been associated with response to bortezomib in multiple myeloma, we inquired whether such correlation might be seen in pediatric AML. We prospectively evaluated CD74 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome as part of Children's Oncology Group protocol AAML1031 that randomized patients younger than 30 years of age with de novo AML to standard treatment with (Arm B) or without (Arm A) bortezomib. The addition of bortezomib to standard chemotherapy increased toxicity but did not improve survival. Given the association of CD74 with B-lymphoid neoplasms and bortezomib's known efficacy in B-cell neoplasms and multiple myeloma, we hypothesized that within Arm B, the patients with CD74 expression would have a more favorable outcome. Methods: In total, 1,139 newly diagnosed pediatric patients with de novo AML were randomized to standard chemotherapy (n=561, Arm A) or standard chemotherapy with bortezomib (n=578, Arm B). All patients received the identical chemotherapy backbone with either four intensive chemotherapy courses or three courses followed by allogeneic hematopoietic stem cell transplantation for high-risk patients. To qualify for this correlative study, 991 patients satisfied 2 criteria: (1) submitting a bone marrow aspirate for ΔN at diagnosis and (2) providing consent for correlative biology studies. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD74 by ΔN. AML was considered to be CD74-positive if the MFI was more than two times above background autofluorescence and more than 40% of the leukemia was above background autofluorescence. Results: Among 991 patients, 263 were CD74-positive (26.5%) by ΔN, with similar prevalence in Arm A (27.9%) and Arm B (25.2%). Correlation of CD74 expression with clinical characteristics showed that those with CD74 expression had higher median age (p For patients in Arm A (no bortezomib), the differences in OS (66.1% vs 61.0%, p=0.138) and EFS (48.9% vs 41.7% p=0.088) were not significant between those that were CD74-positive and those that were CD74-negative (Figure 1). However, patients in Arm B receiving bortezomib that were CD74-positive showed a significant improvement in OS (75.3% vs 62.5%, p=0.006) and EFS (53.6% vs 44.3%, p=0.028) compared to those who were CD74-negative (Figure 1). Comparison of the outcomes for CD74-positive patients with and without bortezomib exposure showed a difference in OS of 66.1% vs. 75.3% for those in Arm A vs. Arm B but did not reach significance (p=0.155). Multivariable analysis for OS yielded a hazard ratio of 0.67 (95% CI: 0.44 - 1.02) and p=0.061, approaching, but not reaching, statistical significance. Conclusions: These data demonstrate that CD74 expression is associated with more favorable disease characteristics and survival. Patients receiving bortezomib that were CD74-positive showed a superior response to therapy compared to patients who did not express CD74, by both OS and EFS, suggesting that CD74-positive childhood AML patients stand to benefit from bortezomib therapy. Bortezomib may induce a mechanistic response in CD74-positive AMLs similar to that in bortezomib-treated B-cell neoplasms and/or multiple myeloma, where bortezomib has proven to be beneficial. Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019). Pollard:Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

E-selectin (E-sel) is a cell adhesion glycoprotein that is expressed on endothelial cells and has been implicated in therapeutic resistance. In most myeloid leukemias, leukemic blasts express E-sel ligands (EsL), which contain the glycan epitope of the carbohydrate sialyl Lex (sLex). This expression increases the likelihood of adhesion to vascular endothelial cells and facilitates sequestration in the bone marrow vascular niche, leading to cell adhesion-mediated drug resistance and poor clinical outcome. E-sel antagonists like uproleselan, interrupts leukemic cell homing to the vascular niche, increases susceptibility to cytotoxic and targeted therapies and can be potent adjuncts to therapeutics. Recent data demonstrated a correlation between leukemic cell surface levels of EsL and response to uproleselan, linking EsL expression to response. We questioned whether transcriptome profiling of EsL-forming glycosylation genes can be used to identify elevated EsL expression in patients with acute myeloid leukemia (AML), and subsequently which patients might best respond to uproleselan. RNA-seq data from patients treated in COG AAML1031 (N = 1,074) was available for evaluation. We examined transcriptome expression of 24 genes that code for enzymes involved in glycosylation of EsL. All analyses were performed in R. Cox proportional hazards models were generated using the survival package. Multidimensional flow cytometry (MDF) was used to detect cell surface EsL expression by two techniques: direct binding of an E-sel/hIg, PE labeled chimera, and the anti-sLex antibody HECA-452. Seven of the 24 genes examined had minimal expression (mean To verify surface protein expression of the two genes, leukemic specimens from SFhigh patients (N = 10) and SFlow patients (N = 10) underwent cell surface expression evaluation of glycosylated EsL using two MDF assays. SFlow patients had low or undetectable levels of cell surface EsL by both assays, whereas SFhigh patients had significantly higher expression of EsL (p < 0.001, Fig. 1F). This suggests a strong correlation between transcriptome measurements of EsL glycosylation genes and cell surface glycosylation levels of EsL. In summary, we have shown that multiple genes involved in the glycan synthesis of EsLs are highly expressed in pediatric AML. Two of these genes, ST3GAL4 and FUT7, are associated with poor outcome. Additionally, high expression of these genes is detectable at the transcript level and associated with cell surface EsL expression. These genes provide novel targets for overcoming drug resistance induced by the tumor microenvironment, and lend support for the use of EsL glycosylation genes as predictive biomarkers. These data also confirm the importance of E-sel in disease progression in AML and its potential as a therapeutic target. Figure 1 Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Fogler:GlycoMimetics Inc: Employment, Equity Ownership. Loken:Hematologics, Inc: Employment, Equity Ownership.

CBFA2T3-GLIS2 fusions are uniquely expressed in infants with AML and its presence is associated with refractory disease with high induction failure and nearly uniform fatality. Recent studies into varying features of this refractory leukemia have defined a unique immunophenotype that is referred to as the RAM phenotype with exceptionally high CD56 expression. Further, deep sequencing of patients with this fusion has failed to identify additional recurrent sequence or structural variants, suggesting that the presence of this fusion might be sufficient for malignant transformation. Given its presence uniquely in infants and lack of cooperating events, the premise of malignant transformation by a single, highly penetrant event might be justified. Despite discovery efforts to define genes, pathways, and networks that are dysregulated in this AML subset, lack of appropriate model systems has limited pre-clinical evaluation of potential therapies. Prior efforts in generating cell lines and xenograft models have met with limited success. We inquired whether primitive CD34+ cord blood cells might provide the appropriate developmental milieu to study fusion-induced transformation. To investigate this possibility, we transduced human CD34+ cord blood cells with a lentivirus encoding CBFA2T3-GLIS2 fusion transcript and GFP. Transduced and mock-transduced control cells were cultured in bulk in a myeloid promoting condition (IMDM with 15% FBS and 10 ng/mL of each growth factor SCF, TPO, FLT3L, IL3, and IL6). The transduction efficiency was approximately 9% at MOI of 50 and expression of fusion transcript was confirmed by RT-PCR. Growth kinetics and surface expressions were assessed by flow cytometry. Evaluation of the growth properties showed that CBFA2T3-GLIS2 transduced cells exhibited enhanced proliferation, with a doubling time (DT) of 93 hours after 5 weeks, whereas DT for mock-transduced control cells was 124 hours (Figure 1A). Further, CBFA2T3-GLIS2 transduced cells had significant expansion of the GFP+ cells, an increase from 9% at the initiation of cultures to 95% by week 8 (Figure 1B). After 12 weeks in culture, fusion-positive cells continue their rapid expansion. Surface CD34 expression was lost after the first week of culture for both CBFA2T3-GLIS2 and mock-transduced cells (Figure 1C), indicative of rapid differentiation in myeloid promoting condition. Morphological evaluation of cells expressing CBFA2T3-GLIS2 reveal a homogenous population with open chromatin, occasional multinucleated cells, prominent nucleoli, and abundant focally basophilic and vacuolated cytoplasm with cytoplasmic blebs (Figure 1D) which are morphologic features suggestive of megakaryocytic differentiation. Megakaryocytic lineage of the fusion transduced cells was confirmed by demonstration of high expression of CD41 and CD61 on the surface of transduced cells (Figure 1E). We further evaluated immunophenotypic alterations as a result of CBFA2T3-GLIS2 fusions, by comparing the surface expression of 16 different markers including CD11b, CD33, CD34, CD36, CD38, CD56, CD64, CD117, CD123, and HLA-DR between CBFA2T3-GLIS2 and mock transduced cells. Mock-transduced cells showed expression of antigens associated with monocytic differentiation (CD36, CD64, and CD11b) (Figure 1F) whereas the CBFA2T3-GLIS2 transduced cells were consistent with more immature cells expressing CD117 and lacking CD11b, CD36 and CD64 (Figure 1G). Of significance, fusion-transduced cells had remarkably high CD56 expression (mean linear MFI of 43 and 3556 for the mock-transduced and transduced cells respectively), similar to that observed in patients with fusion-positive RAM phenotype (average MFI=2040) (Figure 1H), suggesting that CD56 expression is causally linked to the expression of the fusion transcript. This study demonstrates that the CBFA2T3-GLIS2 fusion is sufficient for malignant transformation and recapitulates megakaryocytic AML that is seen in infants. This further demonstrates that engineered primitive cord blood cells can be utilized for generation of appropriate cell lines that can be used for therapeutic development. Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Cell surface antigen expression in AML is becoming focus of investigation as it relates to prognostic impact as well as potential therapeutic implications. The CD117 (c-KIT) receptor tyrosine kinase is expressed on the cell surface of hematopoietic stem cells and its signaling is important in cell survival, proliferation and differentiation. CD117 is expressed in a majority of AML cases. In addition, mutations in c-KIT have been identified in a subset of patients, specifically those with CBF AML (inv(16)/t(16;16) and t(8;21)). As there is increasing interest in using tyrosine kinase inhibitors with anti-KIT activity based on surface CD117 expression as treatment in AML, we sought to evaluate the association of CD117 expression with biologic and clinical features in pediatric and young adult (YA) AML. A total of 1803 pediatric and YA patients (ages 0.01-29.2 years) treated on the Children's Oncology Group (COG) phase III trials AAML0531 (n=762) and AAML1031 (n=1041) with complete cyto/molecular and outcome data available were included in the analysis. The primary aim of AAML0531 evaluated the addition of gemtuzumab ozogamicin to a 5-course MRC-based backbone, while AAML1031 evaluated the addition of bortezomib to a similar 4-course backbone. Outcomes between the 2 primary randomized arms on each trial were similar and thus were included together for each trial. Multidimensional flow cytometry was used to determine the CD117 mean fluorescence intensity (MFI) of myeloid progenitor cells as defined by CD45 low and side scatter using a CD117-PE antibody with stable intensity detection since 2002. Patients were divided into quartiles based on CD117 expression and clinical characteristics and outcome were evaluated across the quartiles. CD117 MFI distribution was similar across both trials (Fig 1A). Analysis of CD117 expression with cytogenetics demonstrated that t(8;21) was significantly associated with higher CD117 (p Outcome analysis demonstrated that CD117 surface expression was not associated with CR, with similar CR rates across the quartiles (p=0.726). In a univariate analysis, overall survival (OS) was associated with higher CD117 expression (p We show here that CD117 expression was higher in patients with favorable risk CBF AML and inversely associated with KMT2A rearrangements, which are generally considered to be neutral or unfavorable depending on the specific translocation partner. Higher CD117 expression was associated with improved outcomes, which may in part be explained by the association with CBF and inverse association with KMT2A. Although patients with higher CD117 MFI generally have favorable risk genomic alterations, the strategy of c-KIT inhibition warrants further investigation in AML given the potential for facilitating de-escalation of conventional therapeutics with significant toxicity in future clinical trials if found to be safe and effective. Disclosures Eidenschink Brodersen: Hematologics, Inc: Employment. Pardo:Hematologics, Inc: Employment. Dai:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Introduction: Despite maximally intensified chemotherapy, cure rates remain suboptimal for children and young adults with acute myeloid leukemia (AML). As CD123 (IL3RA) is expressed on the majority of AML cells, it is a promising immunotherapeutic target. Increased CD123 expression has been linked to high-risk disease characteristics in adult AML, but clinical implications for childhood AML are less well-defined. Methods: The Children's Oncology Group AAML1031 phase 3 trial (NCT01371981) tested the efficacy of the addition of bortezomib to standard chemotherapy in a randomized fashion. A total of 1400 children and young adults were enrolled on study. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD123 by multi-dimensional flow cytometry (MDF) at Hematologics, Inc. Patients were stratified to either a low-risk (LR) or high-risk (HR) arm of the therapy based on cytogenetic and molecular alterations and end-of-induction (EOI) minimal residual disease (MRD) measured by MDF. LR patients received four cycles of chemotherapy, while HR patients received 3 courses of chemotherapy followed by best allogeneic hematopoietic stem cell transplantation (HSCT). Patients with high allelic ratio FLT3-ITD enrolled on arm C received sorafenib in combination with standard chemotherapy followed by HSCT. Here we provide data on expression of CD123 across all patients and correlate this expression with disease characteristics and clinical outcomes. Results: Surface CD123 expression on AML cells was available for 1040 patients, and expression level varied significantly across the study population with a median CD123 molecules per cell of 1300 (range 120-13,100 molecules per cell). For analysis, the study population was divided into quartiles (n=260 each) based on CD123 expression levels. Significant variation in cytogenetic/molecular characteristics was observed across the four quartiles, where those with highest CD123 expression had a lower prevalence of t(8;21), inv(16), and CEBPA mutations (p Conclusions: CD123 expression is strongly associated with disease-relevant cytogenetic and molecular alterations in childhood AML. Patients with highest CD123 surface expression are more likely to have HR genetic alterations and a paucity of LR features. Further, despite similar induction remission rates, those with high CD123 expression had inferior clinical outcomes compared to patients with lower CD123 expression. However, despite an association with HR features, expression of CD123 appears to be independently associated with therapeutic response given that outcome differences were maintained in multivariate regression analysis. This suggests that CD123 expression may provide additional prognostic information, as highlighted by the inferior outcomes in LR patients that had high CD123 expression. In pediatric and young adult patients with the highest risk disease, the higher CD123 expression represents a valuable therapeutic target in the development of immunotherapies for childhood AML. Disclosures Eidenschink Brodersen: Hematologics, Inc: Employment. Pardo:Hematologics, Inc: Employment. Tasian:Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corportation: Research Funding. Loken:Hematologics, Inc: Employment, Equity Ownership.

The cryptic fusion CBFA2T3-GLIS2 was initially described in infants with acute megakaryoblastic leukemia (AMKL) and is associated with adverse outcome. This fusion was subsequently shown to be present in other AML subsets with similar adverse associations. In order to define genes and pathways that are dysregulated and define therapeutic targets in these high-risk patients, diagnostic specimens from 1,049 children and young adults with AML underwent whole transcriptome sequencing (mRNA and miRNA Seq). Initial interrogation of the mRNA transcriptome identified 25 patients (2.4%) with the cryptic CBFA2T3-GLIS2 fusion. Patients with CBFA2T3-GLIS2 were significantly younger (median age 1.51 vs 10.2 years, p < 0.001), with 92% of fusion-positive patients < 3 years old, and an overall incidence of 9% in all patients < 3 years (23/255, p < 0.001). Half of all CBFA2T3-GLIS2 cases were classified as AMKL (13/25, 52%). Patients with CBFA2T3-GLIS2 lacked other risk features and the overwhelming majority (88%, p < 0.001) were classified as standard risk by conventional cytogenetic and molecular classifications. However, fusion-positive patients had an overall survival of 10% compared to 67.3% for the fusion-negative cohort (hazard ratio (HR) = 3.31, p < 0.001, Fig. 1A), with similar adverse event-free survival (HR = 2.48, p < 0.001). Genomic interrogation of fusion-positive patients by whole genome or targeted exome capture sequencing showed a lack of recurrent somatic mutations or other structural events in this patient population (Bolouri et al. 2017). Transcriptional profiling contrasting CBFA2T3-GLIS2 positive (N = 25) and negative cohorts (N = 1,024) identified several differentially enriched pathways, including TGFB/BMP (FDR < 0.001), WNT (FDR < 0.001), and Hedgehog signaling (HH, FDR = 0.002). Fusion-positive cases highly expressed GLIS2, the HH pathway genes, HHIP, PTCH1, and GLI1, as well as BMP2 (Fig. 1B). In addition, CASP1 and TRAIL-R2, which are involved in apoptosis, as well as the tumor suppressor GLIPR1 were significantly down-regulated. Integration of the miRNA with mRNA transcriptome demonstrated that expression of both CASP1 and TRAIL-R2 were significantly anti-correlated with and both are putative targets of the overexpressed miR-181b-5p; while downregulation of GLIPR1 was associated with an increase in miR-130a-3p, as well as being a predicted target (Fig. 1C). Gene-set enrichment analysis revealed highly up-regulated cell-adhesion and cell-surface markers, including extracellular matrix binding (FDR < 0.001), cell-adhesion molecule binding (FDR < 0.001), and integrin binding genes (FDR < 0.001). Investigating the most highly differentially expressed genes (90th percentile log2 fold-change (LFC), FDR < 0.001) with demonstrated localization to the plasma membrane and extracellular matrix, showed that CBFA2T3-GLIS2 exhibits a unique expression profile with a distinct set of dysregulated genes with plasma membrane localization (Fig. 1D). One of the most highly expressed cell-surface genes included NCAM1 (aka CD56; LFC = 6.97, FDR < 0.001). Given the extreme level of CD56 transcript expression in fusion-positive cases, we studied CD56 protein level using multidimensional flow cytometry (Hematologics, Seattle, WA). Surface CD56 in fusion-positive cases was massively over-expressed, with a median mean fluorescent intensity (MFI) of 1,960.0 versus 9.0 MFI for those without the fusion (p < 0.001, Fig. 1E). We evaluated CD56 as a potential therapeutic target using an anti-CD56 antibody-drug conjugate (m906-PBD-ADC) developed by NCI. Leukemic blasts or control lymphocytes from a patient with relapsed fusion-positive AML were incubated with varying doses of m906 and cytotoxicity was assessed after 72 hours (Notable Labs, Foster City, CA). The CD56-ADC exhibited a dose-dependent and CD56-specific toxicity on leukemic blasts (LIN-CD33+CD14-CD38-, p < 0.001, Fig. 1F), suggesting that CD56 might prove to be a therapeutic target in this high-risk cohort of patients. This study provides comprehensive transcriptome profiling of CBFA2T3-GLIS2 that defines a distinct and highly lethal subset of AML predominantly seen in infants. We also identify pathways and networks that further provide potential therapeutic options. Most importantly, CD56 may present the most immediately actionable target in this cohort of high risk patients. Figure 1. Figure 1. Disclosures Gekas: Notable Labs: Employment. Kolb:Roche- Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Eidenschink Brodersen:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Background: CD33 is variably expressed on leukemia blasts in most patients with acute myeloid leukemia (AML). Efforts to target CD33 therapeutically have focused on gemtuzumab ozogamicin (GO), an antibody-drug conjugate delivering a DNA-damaging calicheamicin derivative. Qualification for GO therapy has been determined by expression of CD33 by multidimensional flow cytometry (MDF); patients with positive CD33 expression receive GO. Previous studies from the AAML0531 protocol demonstrated that the cell surface intensity expression of CD33, determined by MDF, predicts the response to treatment with GO, and that in part this expression is regulated by a pair of CD33 single nucleotide polymorphisms (SNPs) in linkage disequilibrium that also, independently, predict response to therapy. Patients with CC genotype for rs12459419 have lower relapse risk and improved disease-free survival, compared to CT and TT genotypes. Because GO therapy is associated with treatment-related toxicity, it is important to identify biologic variables associated with benefits and risks. To date, there has been no report associating SNP status among CD33 positive versus CD33 negative patients that could assess how the combination of these biomarkers for determining administration of GO therapy could improve patient outcomes. Objective: In an effort to determine which children would benefit most from GO treatment in future prospective pediatric AML protocols, we aimed to elucidate if CD33 SNP genotype status should be combined with quantitative CD33 cell surface antigen expression on the diagnostic leukemia cells (CD33+ versus CD33-) to determine GO eligibility, with a retrospective analysis of children enrolled in Children's Oncology Group AAML0531. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 666 satisfied two criteria for this study: (1) submission of a blood or bone marrow sample for MDF at diagnosis with corresponding CD33 SNP genotype data available, and (2) proper consent for specimen testing. CD33 Expression Levels on Leukemic Blasts The diagnostic AML leukemia population was identified by gating on CD45 versus log-side scatter and CD33 expression levels were determined by measuring the mean fluorescence intensity (MFI) by flow cytometry. For a patient to be considered CD33+ two criteria were required: intensity of CD33 on blasts was at minimum four times the MFI of its correspondent autofluorescent control, and at least 80% of the total blasts were greater than this minimum. Genotyping CD33 SNPs CD33-coding SNP rs12459419-Ala14Val and linked promoter SNP rs3865444 were genotyped using the Sequenome (San Diego, CA) platform at the Biomedical Genomics Center, University of Minnesota. Both SNPs had a call rate of 0.98 and were in Hardy-Weinberg equilibrium (P=0.05). Results: Association of Genotype and cell surface expression of CD33 for AAML0531 patients Of 666 patients, 84% (560/666) were CD33+. CC genotype was observed in 54.5% (305/560) of CD33+ cases, 37.5% (210/560) had CT genotype and 8% (45/560) TT genotype. Of the 16% (106/666) of patients who were CD33 negative, 33% (35/106) had CC genotype, 47% (50/106) CT genotype, and 20% (21/106) had TT genotype. Comprehensively, 340/666 (51%) had CC genotype (51%) and 10% were CD33 negative (35/340). Conversely, out of a total of 66 patients with TT genotype, 45 (68%) were CD33+ and 32% (21/66) were CD33 negative. Conclusions: These results clarify the relationship between the amount of CD33 expressed on AML at diagnosis as measured by MDF and CD33 SNP genotype status. While correlation with clinical outcome analysis is ongoing, these data support inclusion of CD33 SNP genotyping for eligibility of GO therapy. Therefore, the current recommendation for future COG AML clinical protocols is that GO will be administered to patients with CD33 expression as determined by MDF and CC genotype patients regardless of CD33 expression. Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Paine:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of 0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Background: Down syndrome (DS) acute myeloid leukemia (DS-AML) is predominantly found in young children (under the age of 4) and responds well to chemotherapy. The difference in clinical features suggests that despite the predominance of megakaryoblastic disease, DS-AML blasts are different from non-DS AML blasts with megakaryoblastic morphology (AMKL). The immunophenotypes of DS-AML blasts at diagnosis were compared to those of non-DS AML patients using unsupervised hierarchical clustering analysis (HCA) and assessed for their immunophenotypic overlap with known morphologic subgroups. In addition, residual disease monitoring of non-DS AML patients using "Difference from Normal" flow cytometry has been applied to DS-AML with unexpected results. Methods: A standardized antibody panel was used to define quantitative gene product (antigen) expression in bone marrow (BM) specimens from patients with AML at diagnosis and following chemotherapy. The quantitative phenotypic expression of multiple cell surface markers and physical characteristics defined a position in 15 dimensional data space for each patient. HCA was performed to mathematically cluster DS-AML patients enrolled on Children's Oncology Group study AAML1531 with non-DS patients from study AAML0531 based on 15 dimensional profiles. Five patients with DS who were older than 4 years of age and therefore treated on AAML0531 were also included. Data regarding morphology were appended to the data set. The same antibody panel was used to assess measurable residual disease (MRD) in 147 follow up BM aspirates from 50 DS patients ages 1 to 21 undergoing treatment for either AML or B-ALL (n=46, n=4 respectively) who were not included in the clinical studies. When possible, cell sorting was performed for FISH or SNP/CGH microarray studies. Results: Diagnostic Clustering: HCA of 17 DS-AML diagnostic phenotypes with 769 non-DS AML patients revealed a tight immunophenotypic correlation between 12 (71%) DS-AML patients (Figure 1). Further analysis of this diagnostic subset showed a consistent mean antigen expression profile that was distinct from non-DS AML diagnostic phenotypes. Similarly, the DS-AML patients clustered separately from the 5 older DS patients (>4 years of age) with AML, who were treated on AAML0531 and exhibited consistently different identifying diagnostic markers despite the shared constitutional trisomy 21. Additionally, the phenotype associated with the DS-AML cluster does not match that of patients with AMKL morphology. The largest immunophenotypic cluster of non-DS patients with AMKL morphology were classified with the RAM phenotype[1], which segregate into a mathematically unique branch of the dendogram distinct from the DS-cluster. Observation of a consistent DS-specific non-leukemic myeloid regenerative phenotype: Of the 147 post chemotherapy specimens submitted for MRD analysis off clinical study, 141 (96%) had a clearly identifiable CD34+/CD56+ population present between 0.06-6.0% total non-erythroid composition. A second abnormal population expressing CD34+ but not HLA-DR was also observed in these patients but at a lower level (0.02-2.66%) compared to the CD34+/CD56+ cell population. Two patients had a reproducible absence of 56+, DR- progenitors across all follow up specimens (6/147) and may represent unique clinical cases with disease features beyond our knowledge. In four DS patients undergoing treatment for B-ALL, the same immunophenotypic features (CD34+/CD56+ and negative HLA-DR) were observed. Genetic data: In 4 cases, cell sorting of CD34+/CD56+ post treatment progenitors revealed a lack of cytogenetic markers present in the clone of leukemic blasts at diagnosis. Conclusions: The blasts of patient with typical DS-AML (i.e under the age of 4 years at diagnosis) are immunophenotypically distinct from those of non-DS-AML patients and exhibit consistent immunophenotypic features that differ from DS patients with AML who are older than 4 years of age. The normal myeloid progenitors in DS patients who have undergone chemotherapy for AML or ALL express an immunophenotype that is different from normal BM of non-DS patients recovering from chemotherapy. [1] Brodersen, Lisa Eidenschink, et al. "A Recurrent Immunophenotype at Diagnosis Independently Identifies High Risk Pediatric AML: A Report from the Children's Oncology Group Trial AAML0531." Disclosures Wilson: Hematologics: Employment. Brodersen:Hematologics: Employment. Zehentner:Hematologics: Employment, Equity Ownership. Pardo:Hematologics, Inc: Employment. Wells:Hematologics: Employment, Equity Ownership. Loken:Hematologics: Employment, Equity Ownership.

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse cell surface antigen expression. Multidimensional flow cytometry (MDF) quantifies the expression of such surface antigens on neoplastic cells, permitting an objective means for sub-classification of this disorder. Objective: To investigate the relationship between the presenting immunophenotype and response to therapy in a large, controlled study of pediatric AML patients. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for MDF at diagnosis, (2) proper consent for specimen testing and (3) leukemia comprising >10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs. Unsupervised hierarchical clustering analysis was performed to mathematically define groups of patients with similar diagnostic IEPs. Selection of the appropriate number of clusters was accomplished by minimizing within-cluster variation while maximizing between-cluster variation. The rate of measurable residual disease (MRD) by MDF at the end of initial induction therapy (EOI1) and event free survival (EFS) was determined for each group and compared to the mean MRD rate and EFS for the entire cohort. Of the 769 total patients, 643 (84%) had bone marrow specimens submitted for MRD by MDF at EOI1. Results: Of the 643 patients with bone marrows submitted for MRD by MDF at EOI1, 223 (35%) were positive. The 5 year EFS rate was 49% for the study (N=769). Hierarchical clustering analysis of the IEPs defined 11 groups of patients (A-K) with similar quantitative diagnostic immunophenotypes. Five immunophenotypic groups of patients were associated with comparableEOI1 MRD rates (15%-36%) and EFS (50%-69%) to the study average. These 5 groups (A-E) consisted of immunophenotypes close to the normal counterpart of myeloid progenitor cells (Groups A, B, C; N=266, 77 and 106) or normal monocytes (Groups D and E; N=41 and 68). Two immunophenotypic groups (Group F and G) of patients were associated with significantly higher rates of MRD (Group F: 54%, p Three immunophenotypic groups (H-J) had low rates of MRD (20%-24%) but poor EFS (27%-39%). The defining different-from-normal immunophenotypic features of Group H (N=52) were expression of CD56 and CD13 with a lack of CD34. The defining different-from-normal immunophenotypic features of Group I (N=11) were CD34 positive expression with a lack of CD117. The defining different-from-normal features of Group J (N=28) were bright CD11b expression with a lack of CD34 and CD117. The majority of patients in these groups were stratified as standard risk (94%, 100%, and 75% respectively). One immunophenotypic group (K) had a high rate of MRD (50%) and comparable EFS (52%) to the study average. The defining different-from-normal features of Group K (N=23) were expression of CD56 with a lack of CD34 and CD13. Conclusions: IEPs can provide a powerful tool to discriminate patient cohorts that have a high probability of relapse and subsequently elucidate when MRD by MDF at EOI1 is predictive of patient outcome. Notably 2 IEP groups (97 patients, 13% of total) had significantly higher MRD rates and were associated with a high probability of disease relapse. IEPs can identify these 2 groups of patients otherwise stratified as good or standard risk, which can provide clinicians with an additional analytical method to more appropriately stratify patients. Disclosures Loken: Hematologics Inc.: Equity Ownership. Voigt:Hematologics Inc.: Employment. Brodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Pardo:Hematologics Inc.: Employment.

Background: Risk stratification of pediatric patients with acute myeloid leukemia (AML) combines cytogenetics and molecular markers at diagnosis with detection of measurable residual disease (MRD) by multidimensional flow cytometry (MDF) after initial induction chemotherapy (EOI1). However, using MDF to distinguish between favorable and poor risk groups by diagnostic immunophenotype has not been sufficiently reproducible for clinical implementation. Objective: We sought to characterize a recurrent, unique diagnostic immunophenotype identified among patients treated on Children's Oncology Group trial AAML0531 and determine the clinical characteristics, frequency, and outcome of patients with this immunophenotype in a large cohort. Patients and Methods: Of the 1022 newly diagnosed pediatric patients with de novo AML who enrolled in protocol AAML0531 (exclusive of patients with promyelocytic leukemia and Down syndrome), 812 who submitted a specimen for MDF at diagnosis and gave consent to MRD testing were eligible for the study. Results: A recurrent diagnostic phenotype was identified by a combination of high intensity CD56 expression, a lack of HLA-DR, dim to negative CD38 expression and dim to negative CD45 expression (designated as RAM phenotype). At diagnosis 19 patients (2.3%) expressed this phenotype and MRD was subsequently detected by MDF in 16/19 patients at EOI1 at a level of 0.02%-41% (median 0.3). Eleven patients with this phenotype submitted bone marrow specimens for MDF analysis at all 3 additional monitoring time points during therapy and in 8 patients MDF analysis revealed disease persistence throughout treatment. The experimental cohort (RAM cohort, N=19) and 2 control cohorts, one consisting of all other patients with CD56 positive AML (CD56+ control, N=166) and the other of patients with CD56 negative AML (CD56- control, N=627), were analyzed for clinical outcomes. Patients in the RAM cohort were significantly younger with a median age at diagnosis of 1.26 years [range 0.75-16.89] than those in the CD56+ control (median 7.57 years [range 0.01-29.8], p=0.002) and the CD56- control (median 10.64 [range 0.02-23.94], p MDF analysis detected MRD in 84% of RAM cohort compared to 29% (p Conclusions: A definitive diagnostic phenotype is identified that is highly predictive of poor outcome in pediatric AML. The survival of patients with the RAM phenotype is similar to or worse than those with high-risk molecular features. It occurs in 10% of infants ( Disclosures Brodersen: Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Pardo:Hematologics Inc.: Employment. Voigt:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with high genomic and phenotypic complexity. Although a relationship between phenotype and t(15;17) is recognized, the effect of other genotypic abnormalities on phenotype remains to be elucidated. Objective: To explore the relationships between genotypic and phenotypic abnormalities in diagnostic specimens in pediatric AML. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for multidimensional flow cytometry (MDF) at diagnosis, (2) consent to blood bank specimen testing and (3) leukemia comprising >10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs. Unsupervised hierarchical clustering was performed to mathematically cluster patients with similar IEPs. The underlying karyotypic and mutational profile of each patient was compared to the IEPs to identify clusters of patients with relationships between phenotype and identifiable genetic abnormalities. Supervised bagged tree-based models were used to quantify the importance of each cell-surface antigen in identifying these clusters. Levene's test was used to assess the homogeneity of mean cell-surface antigen expression for important markers. Results: Analysis of hierarchical clustering results revealed 7 immunophenotypic clusters (A-G) of patients with relationships between IEP and 4 common genetic abnormalities (Figure 1). Cluster A and Cluster B were both strongly associated with the t(8;21) karyotype (Figure 1). 54/62 patients in Cluster A (87%) and 24/26 patients in Cluster B (92%) were positive for t(8;21). The most important cell-surface antigens in identifying patients with the Cluster A associated phenotype were dim CD33, expression of CD56, expression of CD34 and expression of HLA-DR. The mean antigen expression of these markers was significantly more homogenous for patients in Cluster A compared to all other patients for CD33 (p = 0.02), CD34 (p < 0.01) and HLA-DR (p Cluster C was strongly associated with chromosome 16 abnormalities (Figure 1). Of patients in this cluster, 50/55 (91%) were positive for inv(16)/t(16;16). The mean high-expression of CD13 was the most important antigen in identifying patients with this phenotype, and patients in Cluster C had more consistent mean CD13 expression compared to all other patients (p Cluster D was strongly associated with 11q23 abnormalities (Figure 1). Of patients in this cluster, 105/135 (78%) were positive for 11q23 translocations. The most discriminating markers in identifying these patients were dim-to-negative CD34 and dim-to-negative CD117. 69% of all patients with 11q23 abnormalities comprised this cluster. Clusters E, F and G were associated with FLT3 ITD mutations (Figure 1). Of patients in these clusters, 44%, 49% and 53% were positive for FLT3 ITD mutations. Cumulatively, 63% of all patients with FLT3 ITD mutations were grouped into one of these three clusters. Conclusion: Hierarchical clustering algorithms identify IEPs associated with common genetic abnormalities in pediatric AML, providing insight into the consistent dysregulation of cell-surface gene product expression in patients with common genetic abnormalities. Further analysis may reveal that IEPs can identify patients otherwise classified as good or standard risk that will have a poor outcome. Disclosures Voigt: Hematologics Inc.: Employment. Brodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

Background:Flow cytometric studies are useful in the diagnostic workup of patients with unexplained cytopenias and it has been demonstrated that bone marrow aspirates with immunophenotypic abnormalities by flow cytometry but not diagnostic morphologic or cytogenetic findings frequently evolve into myelodysplastic syndromes (MDS) (Kern 2013). Two flow cytometric scoring systems (FCSSs), the Wells FCSS and the Ogata FCSS, have diagnostic and prognostic utility. The Wells FCSS utilizes a difference from normal algorithm incorporating more than ten phenotypic parameters. The accumulation of these abnormalities is not only useful in diagnosis but is predictive of patient outcome (Wells 2003, Scott 2008, Alhan 2014). The recommended Ogata FCSS has evolved to include four cardinal parameters: (1) CD45 intensity on the myeloid progenitors, (2) frequency of lymphoblasts, (3) frequency of myeloid progenitors, and (4) granularity of the maturing myeloid cells. The Wells FCSS is more comprehensive as it uses more phenotypic characteristics, while the Ogata score is considered straightforward to implement in a routine setting (Della Porta 2012, Ogata 2009). This study compares the Wells FCSS and Ogata FCSS for sensitivity and specificity to detect clonal abnormalities documented by SNP/CGH microarray and conventional cytogenetics. Patients and Methods: The cohort included 99 patients with unexplained cytopenias whose bone marrow aspirates were submitted for SNP/CGH microarray and flow cytometry (HematoLogics). The immunophenotypic data were independently assigned a Wells FCSS (Cutler 2012) and an Ogata FCSS (Della Porta 2012). SNP/CGH microarray was assessed for MDS-associated genetic abnormalities. The findings were further correlated with conventional cytogenetic findings. Results: Of the 99 bone marrow aspirates, 20 exhibited clonal abnormalities associated with MDS. The Wells FCSS identified immunophenotypic abnormalities suggestive of MDS for 18 of 20 CGH positive specimens (sensitivity of 90%) and did not detect phenotypic abnormalities suggestive of MDS in 68 of 79 CGH negative specimens (specificity of 86%). In contrast the Ogata FCSS identified immunophenotypic abnormalities suggestive of MDS for 13 of 20 CGH positive specimens (sensitivity of 65%) and did not detect phenotypic abnormalities suggestive of MDS in 64 of 79 the CGH negative specimens (specificity of 81%). In an attempt to improve the sensitivity and specificity of the Ogata score, the granularity parameter was modified from side scatter channel mode of the granulocytes (compared to the side scatter mode of the lymphocytes) to the side scatter channel at the 15thpercentile of granulocytes (compared to the mean of lymphocytes). This modified parameter detected all specimens defined as hypogranular by the side scatter mode, and detected an additional 11 specimens as hypogranular. All of these specimens were detected as hypogranular by the Wells definition. This modified granularity method was then used along with the other three cardinal parameters to create a modified Ogata FCSS. The granularity modification resulted in improved sensitivity (70% versus 65%); specificity was unchanged. While the modified method outperformed the original, it did not match the performance of the Wells FCSS. Conclusions: In patients with unexplained cytopenias, the Wells FCSS demonstrates superior specificity and sensitivity than the Ogata FCSS for detecting myeloid immunophenotypic clones associated with SNP/CGH array and cytogenetic abnormalities. Modifying the Ogata granularity parameter marginally improves the sensitivity but does not improve the specificity. Implementation of the Wells FCSS requires a comprehensive understanding of phenotypic intensities and relationships in non-clonal hematopoiesis for patients with cytopenias. While the relative ease of implementing the Ogata FCSS is attractive, improvements are essential for diagnostic accuracy; improving the granularity parameter alone is not sufficient. Adding measurements for the maturing myeloid and erythroid compartments may increase the diagnostic utility of the Ogata FCSS but requires further study. Disclosures Brodersen: Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Zehentner:HematoLogics Inc.: Employment, Equity Ownership. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Singleton:Hematologics Inc.: Employment. Hartmann:Hematologics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership.

Background: Single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) microarray analysis is a powerful tool to assess myelodysplastic bone marrow specimens for the presence of genomic gains and losses as well as loss of heterozygosity (LOH) (reviewed by Nybakken & Bagg, JMD 2014). Its application can be a valuable addition to conventional cytogenetic analysis and may be superior to FISH testing for MDS assessment. Currently, microarray analysis does not have widespread use in an MDS work-up. Several groups have demonstrated that flow cytometric analysis can detect phenotypic aberrations in bone marrow aspirates with cytopenias with more abnormalities identified in patients with poor prognosis or with multiple genotypic abnormalities (Loken et al. 2008; Cutler et al. 2011; van de Loosdrecht et al. 2013). In this study SNP microarray results were compared with conventional cytogenetic and MDS panel FISH findings as well as phenotypic abnormalities detected by flow cytometry. Patients and Methods: 185 bone marrow aspirate specimens submitted to our laboratory for MDS work-up were analyzed by SNP/CGH studies. 36 of these (19.5%) were positive by SNP/CGH microarray analysis. 32 of the positive microarray cases (88.9%) were also analyzed by conventional cytogenetic studies, 35 (97.2%) by MDS FISH panel (5p, 7q, +8, -17p, -20q) and 31 (86.1%) were assessed by multidimensional flow cytometry (FCM) and were assigned an FCSS score (Wells et al. 2003). Results: Of the specimens in which the SNP/CGH array demonstrated genotypic abnormalities, 11/32 (34.4%) were negative by conventional cytogenetic analysis while 12/35 (34.3%) showed no abnormalities by MDS FISH panel analysis. SNP/CGH analysis revealed additional chromosomal gains and losses in 18/32 (56%) in comparison to cytogenetic analysis and in 22/35 (63%) in comparison to FISH analysis. Loss of Heterozygosity regions were detected in 28/36 cases (78%) with 96.4% (27/28) of these being larger than 2 Mb and 53% (19/28) spanning a significant chromosomal region (e.g. 1p, 5q, 7q and 17p) with known oncogenic and other MDS related genes. In 10/32 cases (31%), microarray analysis was able to characterize the origin of marker chromosome material, previously reported with unknown identity by conventional cytogenetic analysis. In an additional subset of 10 out of 32 cases (31%), cytogenetic analysis was able to either characterize balanced translocations or low level sub-clonal abnormalities not identified by microarray analysis alone. In 11/36 (31%) microarray analysis was able to detect clonal heterogeneity and evolution. In none of the specimens did FISH analysis detected abnormalities not revealed by microarray analysis. Flow cytometry performed on 31 of the array positive specimens revealed 6 to have >20% abnormal myeloid progenitor cells (classified as AML) while 23 the remaining 25 cases showed phenotypic abnormalities consistent with MDS (FCSS ranging from 1-6). In two specimens with a FCSS of 0, LOH regions on 16q or 1p and 21q were found, respectively, without the presence of numerical aberrations. A FCSS score of 1 with minimal phenotypic abnormalities (n=3), was comprised of one specimen with del(5q), one with LOH of 7q and one with trisomy 8, 1p loss and 1q gain. Specimens with an FCSS of 2 (n=7) showed only one specimen classified as complex (5 or more abnormalities). The two FCSS =3 specimens showed del(5q) with del(12p) and several LOH regions, not complex findings. One of the 4 specimen with FCSS = 4 was classified as complex while the other 3 specimens showed monosomy 7, LOH of 7q or LOH of 1p, respectively. Genotypic abnormalities were also related to phenotypic abnormalities in 4/7 (57%) specimens in the FCSS = 5/6 category which revealed complex microarray findings. Half (3/6) of the AML class had complex findings as well. Conclusions: These results emphasize the additional value that CGH/SNP microarray analysis adds to conventional cytogenetic analysis. Our dataset confirms that FISH studies do not provide additional information for MDS specimens positive by cytogenetic and/or microarray analysis. Most importantly, a high correlation between our phenotypic flow cytometric scoring system for myeloid abnormalities and microarray findings has been identified. Higher flow cytometric abnormality scores correlate with increasing complexity of genomic abnormalities. Disclosures Zehentner: HematoLogics Inc.: Employment, Equity Ownership. Brodersen:Hematologics Inc.: Employment. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Hammock:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Hartmann:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership.