Introduction. The E-selectin ligand is a carbohydrate structure (sialyl Lewis x) expressed on myeloid cells and plays a role in cell adhesion and activation by binding specifically to E-selectin on endothelial cells. Acute myeloid leukemia (AML) blasts express the E-selectin ligand on glycoforms of CD44, CD62L and CD65. Interaction of AML blasts in the marrow microenvironment is believed to involve a number of receptors, including CXCR4, VLA4, and CD44. E-selectin in the vascular niche regulates dormancy for hematopoietic stem cells (Winkler IG et al. 2012), and chemoresistance and survival in AML. E-selectin inhibitors are now being studied in clinical trials in combination with chemotherapy for hematologic malignancies. Here, we evaluate the clinical significance of the expression of the E-selectin ligand using a panel of AML patient blood and bone marrow samples.Methods. We studied 89 serially acquired AML patient samples obtained with informed consent on an IRB approved protocol, 40 from new diagnosis patients, and 49 from relapsed or refractory patients. Five patients had favorable, 30 intermediate, and 43 unfavorable risk cytogenetics. We used multicolor flow cytometry to examine binding of an E-selectin-Fc chimeric protein or staining by HECA452 antibody that recognizes sialyl Lewis a/x carbohydrate epitopes. The blast population was gated by forward scatter vs. side scatter, then CD45 vs. side scatter. Leukemia stem cells (LSCs) were defined as CD34+CD38-CD123+.Results. Percent E-selectin-Fc functional binding for the AML blast population ranged from 0.4 to 99.4%, mean 32.1% and median 20.9%. Percent staining by HECA452 antibody ranged from 0.9 to 99.6%, mean 54.3% and median 58%. These data suggest that the epitope recognized by the HECA452 antibody is present, but not always functionally active. The E-sel-Fc binding % was correlated with HECA452 % expression by Pearson's product-moment correlation, p=1.543e-06, correlation coefficient 0.50. We found a number of statistical correlations between expression of E-selectin ligand and clinical characteristics. There were statistically significant differences for mean fluorescence intensity (MFI) based on disease status, newly diagnosed vs. relapsed/refractory patients for E-sel-Fc binding, with values of 2884 vs. 11764 (p=0.0026 by Welch two sample t-test, p=0.000074 by Wilcoxon rank sum test with continuity correction) and HECA452 staining 3387 vs. 8994 (p=0.00038 by t-test, p=0.0012 by Wilcoxon rank sum test) as depicted in left side figure, respectively. By Pearson's product-moment correlation, the MFI for E-sel-Fc binding was correlated with the MFI for HECA452 staining, p=5.815e-08, correlation coefficient 0.55. There were also significant differences for E-sel-Fc binding % favorable/intermediate vs. unfavorable risk cytogenetics, 24% vs. 39%, p=0.019 (t-test), p=0.033 (Wilcoxon rank sum test) (middle figure), but no difference for MFI. There was a tight correlation between expression of E-selectin ligand by the blast population and the leukemia stem cell population defined as CD34+CD38-CD123+, with p< 2.2e-16 (Pearson's product-moment correlation) and correlation coefficient 0.90 for % expression by E-sel-Fc binding as depicted in right side figure, and by MFI for E-sel-Fc binding, p= 3.24e-09 and correlation coefficient 0.65. E-sel-Fc binding MFI correlated with Flt3 mutation status (ITD and TKD), with mutation 9919 vs. 3858 without, p=0.042, and with a trend toward significance by HECA staining MFI, with mutation 8733 vs. 5101 without, p=0.055. There were no statistically significant differences for other features such as age, gender, secondary vs de novo AML, or presenting WBC.Conclusions. E-selectin ligands are variably expressed by AML blasts and LSCs. Mean fluorescence intensity of E-selectin-Fc binding is 4-fold higher for relapsed/refractory patients than for newly diagnosed patients (p=0.0026), suggesting that sequestration in the vascular niche of the marrow is associated with chemotherapy resistance. Percent E-selectin-Fc binding is higher in patients with unfavorable than favorable/intermediate risk (p=0.019) and MFI correlated with Flt3 mutation status (p=0.042), again correlating with worse prognosis. Moreover, expression of E-selectin ligands by LSCs is correlated with blasts from the same patients, suggesting a role for E-selectin in leukemia survival and propagation. [Display omitted] DisclosuresMagnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Fogler:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership. Becker:Novartis: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Abbvie: Research Funding; Rocket Pharmaceuticals: Research Funding; Amgen: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding; Trovagene: Research Funding; GlycoMimetics: Research Funding.