Low or Undetectable Numbers of Philadelphia Chromosome Positive (Ph+) Leukemia Cells in the Primitive (CD34^posCD38^neg) Stem Cell Fraction in Chronic Myeloid Leukemia (CML) Patients during Tyrosine Kinase Inhibitor Therapy.

Targeted tyrosine kinase inhibitors (TKIs) efficiently induce rapid hematologic and cytogenetic remission in most chronic myeloid leukemia (CML) patients. However, in vitro experiments have suggested that the most primitive CML stem cells residing in the CD34^posCD38^neg fraction are relatively resistant to TKIs. The prevalence of these stem cells in vivo in patients under TKI therapy is unclear. The aim of this project was to analyze the effect of TKI therapy on Ph+ leukemia stem cell pool in patients and to analyze the proportion of Ph+ cells in different stem cell fractions. A total of 26 chronic phase CML patients were included in the study. 18 patients were treated with imatinib, 5 with dasatinib, and 3 with bosutinib. The median time of TKI treatment was 20 months (range 3–72 months). Large volume (median 30 ml, range 5–55 ml) of bone marrow (BM) aspirate was collected and mononuclear cells (MNC) were isolated. CD34^pos cells were separated with paramagnetic beads and further sorted into CD34^posCD38^pos and CD34^posCD38^neg cell populations with multicolor flow cytometry in order to analyze progenitor cell fractions of different maturation stage. Proportion of Ph+ cells was determined with interphase FISH by counting 1000 cells in each fraction. The median yield of MNCs from 30 ml of BM aspirate was 280x10⁶ cells resulting in a median of 32 000 CD34^posCD38^neg cells (range 1000–91000). High-sensitivity counting of the proportion of Ph+ cells was feasible with a median number of counted interphase nuclei of 1005. During TKI therapy the CD34^pos cells expressing highest CD38 antigen level were already mostly differentiated into B-cell lineage (CD19 positive). The CD34^pos cells expressing low CD38 antigen levels expressed markers of more primitive cells such as C-kit (CD117) and CD133. Of 26 patients with CML, 19 were in complete cytogenetic remission (CCyR) when assessed by metaphase FISH of non-fractionated BM cells (1000 cells analyzed). Only 3 patients had single Ph+ cells in CD34^pos cell fractions (less than 1%). In remainder of patients, all progenitor cell fractions, including the most primitive CD34^posCD38^neg cells, were negative for Ph+ cells. 3 patients had 0–1% of Ph+ cells in non-fractionated BM sample. One of them had 0.2% of Ph+ cells in CD34^posCD38^neg fraction, but the other 2 patients had 0/1000 Ph+ stem cells. 4 patients had a partial cytogenetic response (5–20% of Ph+ cells in non-fractionated BM sample). Again, the proportion of Ph+ cells was not increased in the most primitive CD34^posCD38^neg cell fraction. Interestingly, patients who had discontinued imatinib treatment had lower level of Ph+ cells in different CD34^pos fractions (median 0.1%) when compared to non-fractionated BM (median 9.3%). Based on our data, in chronic phase CML patients, TKI therapy eradicates most Ph+ CD34^pos progenitor cells. Unexpectedly, leukemic stem cells were not enriched in the most primitive CD34^posCD38^neg cell fraction in vivo. These results differ from the in vitro studies, where CD34^posCD38^neg cells have been shown to be resistant to TKIs. This could be due to non-physiological conditions (growth factor sensitivity, other cytokines) in cell culture assays. In addition, leukemic stem cells in vivo may be located in the subcortical hypoxic stem cell niche in the BM and are less likely to be aspirated. Our data underline the tremendous proliferative potential of very rare stem cells in CML patients in CCyR, as is evident after discontinuation of TKI therapy. Future studies evaluating the kinetics of disappearance of Ph+ cells from stem cell fractions during TKI therapy and the location of residual Ph+ stem cells in the BM are warranted and may give important information on the depth of the therapy response. Furthermore, this knowledge may aid in targeting therapy to these cells and finding curative treatment strategies in CML.