P463: database guided strategy for the characterization of leukemic stem cell in acute myeloblastic leukemia. implications in the diagnosis and prognosis of the disease.

Background: In recent years, advances in different diagnostic techniques have made it possible to characterize the most immature compartments in acute myeloblastic leukemia (AML). The leukemic stem cell (LSC) is identified as the fraction of aberrant CD34+ CD38- progenitors, the actual chemoresistant reservoir of the disease. There are already data that confer a prognostic value to the proportion of SCL present in a sample at diagnosis. However, information about this population and its clinical significance is still limited. Aims: • To detect, quantify and characterize LSCs in samples of AML by flow cytometry using standardized procedures and automatized analysis tools. • To investigate the association between LSCs and the different molecular subtypes of AML. • To correlate the proportion of LSCs at diagnosis with the clinical evolution. Methods: Study of 145 patients with newly diagnosed AML (88 cases with recurrent alterations and 57 cases classified as NOS or associated with dysplastic changes). Standardized flow cytometry protocol (AML EuroFlow panel) and automated analysis, based on the comparison of the blastic compartments with specifically designed normality databases. Hierarchical analysis and classification of the cases according to the different patterns of expression. Comparative survival study using Kaplan-Meier curves and Log Rank test. Results: The 16.6% of patients presented a LSCs phenotype in more than the 1% of leukemic blast cells. The presence of a high percentage of LSCs was associated with leukemias of immature phenotype, classified as NOS or with myelodysplasia-related changes, and complex karyotypes (24/57; 42.8%). This finding was exceptional in AMLs with recurrent genetic alterations (2 cases out of 88, 2.3%). LSCs phenotype showed highly variable alterations, highlighting the low expression of CD71 or HLA-DR and the increased expression of CD133, CD123 and CD7. To a lesser extent, alterations in the expression of CD13 and CD64 were also detected. (Figure 1) The presence of a high proportion of LSCs at diagnosis was associated with a particularly unfavorable evolution, including frequent chemoresistance and early relapses. (Figure 2). Summary/Conclusion: • The use of automated tools and standardized procedures allows the objective characterization of the LSCs compartment and its comparison with the normal precursors. • The results obtained in our study provide useful information for the early identification of patients with an extremely unfavorable evolution. • LSCs express differentiable phenotypic profiles with respect to normal medullar precursors, which represent potential targets for the development of targeted therapies.Figure 1. Dendrogram of the serie. The yellow, green, and red colors refer to the normal, decreased, or increased expression of markers, respectively.Figure 2. Patients who presented a population of LSCs at diagnosis (red) vs those in whom the presence of SCL 34+/38- was not identified (blue). Medssian PFS of 23 months in the SCLs+ group vs 3 months in the SCLs- group (Test Log Rank, p<0.001). Keywords: Leukemic stem cell, AML, Flow cytometry