LSCs in CML express CD26. (A) Heat map analysis of gene array data of RNA isolated from sorted CD34⁺/CD38^─ SCs (left) or CD34⁺/CD38⁺ progenitor cells (right) obtained from patients with chronic phase (CP) CML (n = 4) and cord blood (CB) SC. Gene array analyses were performed as described in the supplemental material available on the Blood Web site. Expression levels were normalized to CB control. Top-regulated genes of interest are shown. (B) Heat map analysis of pPCR data (upper panel). mRNA analyses were performed on sorted CD34⁺/CD38^─ SC (upper left) and CD34⁺/CD38⁺ cells (upper right) obtained from 3 CB samples and 4 patients with CML. Top-regulated genes of interest are shown. Lower panel: qPCR analysis of expression of CD26 mRNA in sorted CD34⁺/CD38^─ SC (lower left) and CD34⁺/CD38⁺ cells (lower right) obtained from 7 patients with CML and 3 CB samples. CD26 mRNA levels are expressed as percent of ABL. Results represent the mean ± SD (CML, n = 7; CB, n = 3). (C) Multicolor flow cytometry analysis of CD26 expression on CD34⁺/CD38^─ SC in patients with CML (CP, n = 33; AP, n = 3; BP, n = 2), normal/reactive BM (n = 33), and BM of patients with idiopathic cytopenia of unknown significance (ICUS) (n = 15), AML (n = 39), myelodysplastic syndrome (MDS) (n = 12), JAK2 V617F⁺ myeloproliferative neoplasms (MPN) (n = 6), MDS/MPN overlap disorders (n = 5), and indolent systemic mastocytosis (ISM) (n = 7). The upper panel shows the percentage of CD26⁺ SC. Boxes represent the 25th to 75th percentile. The lower subpanel shows representative staining examples: red indicates a positive staining-reaction, blue indicates a negative stain. The isotype-matched control antibody is also shown (black open histograms). (D) The upper panel shows a FISH analysis of purified CD34⁺/CD38⁻/CD26⁺ CML LSC (left) and CD34⁺/CD38⁻/CD26⁻ SC (right) in one CML patient. FISH was performed on cytospin slides using a triple color–staining approach, with 1 probe specific for ABL1 (red) and 2 (green and blue) specific for BCR. Red-green and red-blue fusion-spots indicate BCR/ABL1. Quantitative FISH data are shown in supplemental Table 13. Lower panel: Percentage of BCR/ABL1⁺ cells by FISH (left panel) and percent BCR/ABL1 mRNA relative to ABL mRNA (right panel) in sorted CD34⁺/CD38⁻/CD26⁺ LSC and CD34⁺/CD38⁻/CD26⁻ SC from the same patients. FISH data are expressed as mean ± SD from 3 patients, and BCR/ABL1 mRNA levels from sorted cells as mean ± SD from 4 different CML patients.