P655: limitations of using cd26+ leukemia stem cell quantitation in cml patients to assess residual leukemia clone

Background: Recent findings show that there is a population of stem cell (SC) highly specific for patients with chronic myeloid leukemia (CML). These cells are “immature” CD34+CD38low/neg SC and show expression of CD26, which is atypical for normal SC. These cells supposed to be CML leukemic stem cells (LSC). The presence of residual LSCs detected by flow cytometry (FC) in the peripheral blood of CML patients with a deep molecular response (DMR) has been demonstrated by several recent studies. The influence of residual LSCs on treatment-free remission and the reproducibility of methods for their determination require further study. Aims: To determine the representation of residual LSCs in patients with DMR and their significance for maintaining remission after stopping therapy. Methods: We analyzed 185 samples from 125 patients in chronic phase CML (133 blood and 52 bone marrow samples). The analysis included patients with DMR (at least MR4) and a control group of patients with de novo CML. The group of patients with DMR consisted of patients who were scheduled to discontinue therapy (n=43, blood samples n=52, bone marrow samples n=17) and patients who successfully discontinued TKIs (n=24, blood samples n=24). The control group consisted of 58 de novo CML patients (blood samples n=57, bone marrow samples n=35). The median level of BCR-ABL in the group of patients in the onset of CML was 81.1% (IQR 66.9-91.5). Detection of CML LSCs was performed by FC on BC CytoFlex flow cytometer. The analysis included 4 antigens (CD45, CD34, CD38 and CD26) and viability dye. At least 106 viable cells per sample were analysed (median 1.7×106). Descriptive statistics methods were used in the analysis. Comparative analysis of LSCs concentration was performed using the Man-Whitney test. Results: In the control group of patients with de novo CML, the median concentration of LSC cells in the bone marrow was 50.76 per µl (IQR 16.37-116.96) and 26.29 per µL (IQR 4.67-83.93) in blood samples (p=0.09). In one patient, at the onset of CML, CD26+ LSCs was not detected. None of patients with DMR, both on treatment and after discontinuation of TKI therapy, showed CD26 + LSCs in the blood or bone marrow samples (fig. 1). Summary/Conclusion: The CD26+LSCs compartment is detected in the blood and bone marrow in 98% (57/58) of patients at the onset of CML and is absent in patients with DMR both to TKI therapy and after discontinuation of therapy. This method is neither an alternative nor an adjunct to quantitative real-time PCR to select patients for stopping TKI therapy. The main cause in controversial results between different research groups is a lack of standardization in FC of CML LSC which includes gating strategy, reagents selection, protocols and flow cytometers on which analysis is performed. Keywords: Chronic myeloid leukemia, Leukemic stem cell