Acute Myeloid Leukemia Stem Cells Cells Are Rare and Heterogeneous in Human Acute Myeloid Leukemia.

Abstract 390

Human leukemic stem cells are hypothesized to be rare, restricted to phenotypically immature hematopoietic cells and capable of incomplete differentiation. However, recent work in other tumors has challenged this hypothesis. We used a robust xenotransplantation model based on NOD-SCID-IL-2Rγcnull (NSG) mice to better characterize the frequency and heterogeneity of human SCID leukemia initiating cells (SL-IC). We performed an extensive analysis on primary specimens from 11 AML patients. First, we determined the frequency of SL-IC in un-fractionated AML specimens using transplantation (i.v.) in adult NSG mice for 12 weeks and limiting dilution analysis. Our results indicate that SL-IC are rare cells in primary AML and that the frequency of SL-IC varies greatly from patient to patient: one SL-IC per 0.14 to 4.5 × 106 mononuclear cells. Normal hematopoietic stem cells (HSC) are phenotypically characterized as lineage-, CD34+, CD38- and SL-IC were initially described as being restricted to the CD34+38- compartment. To determine in this model if SL-IC are restricted to this immature cell compartment, we sorted AML cells based on surface staining for a lineage cocktail, CD34 and CD38 expression. CD38+ cells were further sorted by expression of CD45RA and CD123. In contrast to previous results, mice injected with cells from multiple different fractions engrafted including fractions with a mature cell phenotype. Although some fractions did not engraft from individual patients, engrafting cells were found in multiple compartments from all individuals studied. For each engrafting fraction, the AML cells found in mice 12 to 16 weeks post-transplant had the same phenotypic heterogeneity (defined by expression of lineage, CD34 and CD38) as observed in primary specimens consistent with either de-differentiation or lineage infidelity for these cell surface markers. Secondary transplant experiments demonstrated that each engrafting fraction contains self-renewing leukemic stem cells. In order to compare the frequencies of SL-IC in each fraction, we sorted 4 different subsets (based on lineage and CD38 expression) from 1 AML patient and performed limiting dilution analysis (LDA) in NSG mice. SL-IC were detected in each subset, but their frequency was 10-fold higher (1 in 38,000 cells) in Lin-CD38- fractions compared to other fractions and un-fractionated samples. However, as Lin-CD38- cells represent only 3% of all leukemic cells, only 34% of SL-IC were present in this fraction. By comparison, the Lin+CD38+ cell compartment has a SL-IC frequency of 1 in 106 cells but 25% of SL-IC are found in this compartment. Overall, this data demonstrate that human AML stem cells are rare but they are not restricted to immature cell fractions. Rather, leukemic stem cells can be found at different frequencies in all cell fractions. These results suggest that efforts to therapeutically target leukemic stem cells specifically may require re-evaluation.