Expression of surface targets and effects of targeted drugs. (A) Expression of surface antigens on CD34⁺/CD38⁻ cells and CD34⁺/CD38⁺ cells in patients with AML, CML (both at diagnosis and relapse), and normal donors (nBM). Cells were stained with antibodies against CD33, CD52, CD117 (KIT), and CD135 (FLT3). Expression of these targets on CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cells was determined by multicolor flow cytometry, as described in supplemental Patients and methods. The dotted line represents clear expression (staining index > 3). The range of each box represents the 25th to 75th percentiles. The middle horizontal line represents the median expression level. Red asterisk (*): P < .05 vs normal CD34⁺/CD38⁻ BM cells; gray asterisk: P < .05 vs normal CD34⁺/CD38⁺ BM cells. Pairwise Wilcoxon test and P values were adjusted for multiple comparison using the Benjamini-Hochberg procedure. (B) Induction of apoptosis measured by annexin V staining after incubation of AML cells (n = 3) with GO (0.01-5 µg/mL) or medium control at 37°C for 48 hours (left panel). CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cells were then examined for signs of apoptosis by annexin V/DAPI staining. Analysis was performed by flow cytometry. Surviving CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cell fractions after incubation of AML MNCs with alemtuzumab (100-500 µg/mL) in the presence of 30% complement–containing human serum for 1 hour at 37°C (n = 4 patients) are shown (right panel). The absolute cell count of viable cells was measured with counting beads and DAPI staining by flow cytometry. Results are expressed as percentage of annexin V⁺/DAPI⁻ cells (left panel) or viable cells (percentage of control; right panel) and represent the mean ± SD from 3 or 4 patients. *P ≤.05 vs control. (C) MNCs from 4 patients with AML were incubated in medium (control), GO, alemtuzumab, or a combination of both drugs in the presence of 30% human serum. Cells were incubated with GO (1 µg/mL) for 47 hours before adding alemtuzumab (300 µg/mL) for 1 hour (total treatment 48 hours). Thereafter, the absolute numbers of viable cells in the CD34⁺CD38⁻ and CD34⁺/CD38⁺ fractions were measured with counting beads and DAPI staining by flow cytometry. Results are expressed as viable cells (percentage of control) and represent the mean ± SD from 4 patients. *P < .05 vs control. (D) CML MNCs were incubated with various concentrations of GO (0.01-5 µg/mL) (n = 5; left panel) or with denileukin-diftitox (10-100 µg/mL) (n = 5; right panel) for 48 hours at 37°C. Thereafter, apoptosis was analyzed in CD34⁺/CD38⁻ CML LSCs (left and right panels) and CD34⁺/CD38⁺ progenitor cells (left panel, green bars) using annexin V/DAPI staining and flow cytometry. Results are expressed as the percentage of annexin V⁺/DAPI⁻ cells and represent the mean ± SD from 5 experiments. *P < .05, ^#P = .06 (borderline significant). (E) MNCs from 3 patients with CML were incubated in medium (control), GO (1 µg/mL), alemtuzumab (100 µg/mL), or a combination of both drugs in the presence of 30% human serum. Cells were incubated with GO for 47 hours before adding alemtuzumab for 1 hour (total treatment 48 hours). Thereafter, the absolute numbers of viable cells in the CD34⁺/CD38⁻ and CD34⁺/CD38⁺ fractions were measured with counting beads and DAPI staining by flow cytometry. Results are expressed as viable cells (percentage of control) and represent the mean ± SD from 3 experiments. *P < .05 vs control. The table shows the expression of target antigens on CD34⁺/CD38⁻ AML and CML LSCs in all samples used for the in vitro drug-incubation experiments in panels B-E. Results were obtained using multicolor flow cytometry and are expressed as staining index (SI), as described in supplemental Patients and methods. SI values were graded using the following score: −, SI = 0 to 1.3; +/−, SI = 1.31 to 3; +, SI = 3.01 to 10; ++, SI > 10. Patient numbers (#) refer to patients defined in supplemental Tables 2 and 3. CP, chronic phase; D, diagnosis; n.t., not tested; R, relapse.