Clinical and functional impact of CD surface molecules on AML and CML LSCs. (A-B) Effects of GO and alemtuzumab on engraftment of CML and AML stem cells in NSG mice. A total of 0.5 to 1 × 10⁶ CD34 magnetic-activated cell sorting (MACS) cells from 3 patients with CML (A) or 1.5 to 15 × 10⁶ cells (T-cell–depleted MNCs by MACS) from 5 patients with AML (B) was preincubated with or without GO (5 µg/mL), alemtuzumab (500 µg/mL), or both drugs in the presence of 30% complement–containing human serum for 1 hour at 37°C (same cell number for all conditions). Then, cells were washed and injected into the lateral tail vein of irradiated NSG mice. After 25 to 27 weeks (for mice with CML; A) or 7 to 15 weeks (for mice with AML; B) mice were euthanized, BM cells were flushed from tibiae, femora, and humeri, and engraftment of human CD45⁺ cells was analyzed by flow cytometry. Results are expressed as percentage of human CD45⁺ cells (of all viable cells), and represent the mean ± SD from 3 to 5 independent experiments with 5 mice per group. *P < .05 vs control. (C) Overall survival of AML patients based on surface marker expression on LSCs. AML patients were split into 2 groups per marker, based on higher (yellow graphs) or lower (blue graphs) expression of surface antigens on CD34⁺/CD38⁻ stem cells: CD11a, CD25, CD26, CD33, CD44, and CD135 (FLT3). The thresholds of expression were selected based on patient distribution and formation of subsets in flow cytometry experiments (supplemental Patients and methods). OS of patients with AML was calculated using the Kaplan-Meier method. The P values were calculated using the log-rank test.