Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission

Abstract

Chronic myeloid leukemia (CML) patients in sustained "deep molecular response" may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34⁺/CD38^- LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26⁺ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45⁺/CD34⁺/CD38^- stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26⁺ LSCs (median 19.20/μL, range 0.27-698.6). PB CD26⁺ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012-0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006-0.76). Notably, no correlation between BCR-ABL/ABL^IS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that "circulating" CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26⁺ LSCs during TKI treatment and the role of a "stem cell response" threshold to achieve and maintain TFR are ongoing.

Keywords: CD26; TKI; chronic myeloid leukemia; flow cytometry; leukemic stem cells; minimal residual disease.

Figure 1

Peripheral blood (PB) CD26⁺ leukemic stem cells (LSCs) flow cytometry evaluation in chronic myeloid leukemia (CML) patients at diagnosis, during TKI treatment and during treatment-free remission. (Panel A) PB CD26⁺ LSC evaluation in a representative CML patient at diagnosis. CD34⁺CD38⁻CD26⁺ cells were identified using sequential gate with the aim to exclude debris and doublets. First, CD34 gate was performed only on viable cells identified by FSC and SSC light properties (a); then, exclusively CD34⁺CD38⁻ population was gated (b). Both CD34⁺ and CD34⁺CD38⁻ populations show CD45^dim expression (c). Negative control (d); CD26 expression on CD34⁺CD38⁻ population (e), and CD90 co-expression on CD34⁺CD38⁻CD26⁺ cell (f). In the case here depicted, the percentage of CD26⁺ cells within the CD34⁺CD38⁻ fraction was 93%. (Panel B) PB CD26⁺ LSC evaluation in a representative CML patient during TKI treatment. Circulating CML LSCs were documented in about 70% CML patients on treatment regardless of type of TKI, length of treatment, and molecular response. This flow cytometry panel shows the presence of PB residual CD26⁺ LSCs in a CML patient on imatinib for 29 months. In this case, the percentage of CD26⁺ cells within the CD34⁺CD38⁻ fraction was 8% corresponding to 0.131/μL of circulating CD26⁺ LSCs. (a) control tube; (b) test tube CD26 expression; (c) co-expression of CD90 on CD26⁺LSCs. (Panel C) PB CD26⁺ LSC evaluation in a representative patient during treatment free remission (TFR). This flow cytometry panel shows the presence of PB residual CD26⁺ LSCs in a CML patient during TFR. In this case, the percentage of CD26⁺ cells within the CD34⁺CD38⁻ fraction was 1.2% corresponding to 0.0054/μL of circulating CD26⁺ LSCs. (a) control tube; (b) test tube CD26 expression; (c) co-expression of CD90 on CD26⁺LSCs.

Figure 2

Peripheral blood CD26⁺ leukemic stem cells and BCR-ABL1^IS transcript in the whole cohort of treatment-free remission patients. BCR-ABL1^IS neg = undetectable BCR-ABL1 transcript [no BCR-ABL1 copies/32,000 ABL copies (MR^4.5) or no BCR-ABL1 copies/100,000 ABL copies (MR⁵)].

Figure 3

Representative histograms of the expression of isotype control and CD26 on CD34⁺CD38⁻ fraction in peripheral blood (PB) of normal subjects and patients affected by other blood disorders. (A) Negative control, (B) CD26 in a representative case of idiopathic myelofibrosis, (C) negative control, (D) CD26 in a representative case of a Ph+ acute lymphoblastic leukemia, (E) negative control, (F) CD26 in a representative case of acute myeloid leukemia, (G) negative control, (H) CD26 in a representative normal donor after receiving G-CSF.