Differential expression of CD25 and CD26 on CD34⁺/CD38⁻AML LSCs. (A) Expression of CD26 on CD34⁺/CD38⁻ cells (blue boxes) and CD34⁺/CD38⁺ cells (gray boxes) in 22 patients with normal BM, 115 patients with FLT3 wt, and 40 AML patients exhibiting FLT3 ITD (mutated). Samples were obtained at diagnosis or relapse. Cells were stained with antibodies against CD26, CD34, CD38, and CD45 by multicolor flow cytometry, as described in supplemental Patients and methods. Results represent the percentage of CD26⁺ cells in each donor. The median percentage of positive cells in all donors in each group are shown as horizontal lines. The dotted line represents clear expression (>20% positive cells). The range of each box represents the 25th to 75th percentiles. The difference in the percentage of CD26⁺ cells in the 2 groups of AML donors tested was significant (P < .001, Wilcoxon rank-sum test). Corresponding results were obtained when comparing expression levels (defined as stimulation index) in the 2 groups of patients (data not shown). (B) Staining results obtained with an antibody against CD25 in normal BM (n = 25), in patients with FLT3 wt AML (n = 115), and in patients with FLT3 ITD AML (n = 39; diagnostic or relapse samples). In a majority of the patients, CD34⁺/CD38⁻ LSCs (blue boxes) and CD34⁺/CD38⁺ progenitor cells (gray boxes) stained positive for CD25 in AML patients exhibiting FLT3 ITD, whereas cells failed to stain positive in most patients with AML exhibiting wt FLT3. Red asterisk (*): P < .05 vs normal CD34⁺/CD38⁻ BM cells; black asterisk (*): P < .05 vs normal CD34⁺/CD38⁺ BM cells.