Quantitative assessment of the CD26+ leukemic stem cell compartment in chronic myeloid leukemia: patient-subgroups, prognostic impact, and technical aspects

Abstract

Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26− HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.

CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy

“…These findings verify CML LSCs are not absolutely dependent on BCR-ABL activity for their survival, and may determine disease persistence, highlighting those patients who are at high risk of molecular recurrence on TKI withdrawal [6,7]. Although many labs have performed extensive analyses to identify potential surface markers of primitive cell populations in the preclinical setting, including CD26 [8][9][10], and IL1-RAP [11,12], these markers show variability and have, therefore, not yet been translated into routine clinical practice. However, CD26 is promising, with recent data suggesting a correlation between CD26 expression and treatment response, as well as a Lin − CD34 + CD38 −/low CD45RA − cKIT − CD26 + population being identified as a potential therapeutic target at a single-cell level [13]; the diagnostic potential of CD26 is currently being evaluated within clinical trials [14,15].…”

FOXM1 Transcription Factor: A New Component of Chronic Myeloid Leukemia Stem Cell Proliferation Advantage

“…Multiple events enhancing b-catenin stability and triggering FOXM1 phosphorylation contribute to the interaction between b-catenin and FOXM1 (Fig. Conversely, PLK1 inhibitors (BI 2536 belonging to the same family of BI6727 and rigosertib [ON-01910]) have been already advanced for clinical use and found to synergize with TK inhibitors in IM-sensitive and IMresistant CML cells [Jamieson et al,2004;Gleixner et al, 2010;Okabe et al, 2015;Culen et al, 2016]. FOXM1 inhibitors (still in early phase of development) and PLK1 inhibitors have comparable effects on K562 cells, at least according to the parameters we have investigated (Fig.…”

“…2). Conversely, PLK1 inhibitors (BI 2536 belonging to the same family of BI6727 and rigosertib [ON-01910]) have been already advanced for clinical use and found to synergize with TK inhibitors in IM-sensitive and IMresistant CML cells [Jamieson et al,2004;Gleixner et al, 2010;Okabe et al, 2015;Culen et al, 2016]. They might be, therefore, adopted for a pilot study to define whether they complement IM or other TK inhibitors in eroding LSC compartment through FOXM1 inhibition.…”

Flow Cytometry Assessment of CD26+ Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia

“…For compensation setup, BD OneFlow™ Setup Beads (Ref 658620; BD Biosciences, San Jose, CA) and BD™ FC Beads 8‐color Kit (Ref 658621; BD Biosciences, San Jose, CA) were used. The CD26 + population was identified by sequential gates with the aim to exclude debris and doublets, using the analysis procedure described and published in our previous work . As shown in Figure , firstly, the CD34 gate was performed on viable cells identified by FSC and SSC light properties (a–b); then, exclusively CD34 + /CD38 − population was gated (d).…”

“…Analysis of BM and PB samples was performed on two 3-lasers, 8-colors BD FACSCanto™ II flow cytometers using the FACSDiva 8 software version 8.0.1 BD™ (BD Biosciences, San Jose, CA) in order to reach a sensitivity of 10 −5 , and the acquisition and analysis of at least 1.0 × 10 6 cells. Instruments setup were monitored daily and, to ensure reproducible results over time, we followed a standardized protocol that implied adjustments of FACS internal parameters, using the BD The CD26 + population was identified by sequential gates with the aim to exclude debris and doublets, using the analysis procedure described and published in our previous work (16). As shown in Figure 1, firstly, the CD34 gate was performed on viable cells identified by FSC and SSC light properties (a-b); then, exclusively CD34 + /CD38 − population was gated (d).…”