Flow-cytometry evaluation of residual Leukemia Stem Cells in Chronic Myeloid Leukemia patients: feasibility and clinical application.

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the presence of the BCR-ABL1 oncogene, deriving from the t(9;22)-(q34;q11) balanced translocation. The deriving BCR-ABL1 fusion protein has tyrosine kinase activity, and it is capable of inducing the proliferation and expansion of myeloid precursors. Tyrosine kinase inhibitors, drugs capable of blocking the malignant proliferation of cells by directly targeting the BCR-ABL1 kinase activity, have dramatically changed patients’s outcome and improved survival rate. Dormant CML-Leukemia Stem Cells (LSCs) are intrinsically refractory to TKIs and for this reason, only few patients can discontinue TKI therapy. CML LSCs are transcriptionally silent and probably not detectable by qRT-PCR. Several studies tried to find a specific marker able to detect CML LSCs and distinguish them from normal hematopoietic stem cells (HSCs). CD26 (dipeptidylpeptidase IV) was very recently identified as the most specific surface marker for flow-cytometry quantification and isolation of CML LSCs mainly in the bone marrow while rare samples of peripheral blood have been tested so far. Thus, principal aim of this study was to evaluate the presence of circulating LSCs in peripheral blood samples of patients during TKI therapy, avoiding in this way the need of routine bone marrow aspirate. Secondary we investigated a correlation, if any, between LSCs value and degree of molecular response. The analysis was conducted on 202 CML subjects. We have tested by flow-cytometry analysis the expression on peripheral blood cells of stem cells markers such as CD45, CD34, CD38 and, in particular, we have exploited the co-expression of the PE-conjugated anti-CD26. In our experiments we confirmed that CD26 is a specific marker able to discriminate normal HSCs from CML LSCs also in peripheral blood and that it is not expressed on LSCs of other myeloid disorders. The results of our study also showed that circulating CD26+ LSCs are easily detected in CML patients while on TKIs treatment and when comparing bone marrow and peripheral blood actual amount of CD26+ LSCs in the same patient, values were superimposable. However, no correlation was found between CD26+ LSCs count and BCR-ABL/ABLIS ratio molecular analysis. This study assesses for the first time that flow-cytometry analysis of circulating CD45+/CD34+/CD38-/CD26+ LSCs is a powerful tool that could be of great help to identify the residual leukemic cells during follow-up of CML patients, just with routine peripheral blood testing. Further future investigations are required to establish possible roles of flow-cytometry LSCs detection during management of CML patients and to evaluate a potential relationship between undetectable CML LSCs and safe treatment discontinuation.