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direct assay, we measured CD36 expression in BM cells from three CML patients treated for three months with imatinib, bosutinib or dasatinib, respectively. All three sam- ples showed a substantial reduction of CD36 expression in the CD34+CD38low compartment as compared to matched diagnostic samples (Figure 4A). To assess the BCR/ABL1 sta-
tus of the cells during treatment, only patient #11 treated with imatinib had a sufficient number of cells to allow for FACS sorting and subsequent FISH analyses. The CD34+CD38lowCD36+ cells contained 44% BCR/ABL1 posi- tive cells, whereas CD34+CD38lowCD36- cells only con- tained 6% BCR/ABL1 positive cells (Figure 4B,C). This
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Figure 3. A subgroup of primitive CML cells less sensitive to imatinib express CD36 (A) Linear regression and Spearman’s rank correlation show significant correla- tion between IL1RAP and CD36 expression in primitive CML cells, Y=0.76X + 2.4; r=0.68, P=0.0048. (B) Contour plot of co-expression of IL1RAP and CD36 in a rep- resentative CML sample (CML #5). (C) FISH on sorted cells from three CML patients showed a mean of 98% BCR/ABL1 positive cells within CD34+CD38lowIL1RAP+CD36+ cells and 98% BCR/ABL1 positive cells within CD34+CD38lowIL1RAP+CD36– cells. In the CD34+CD38lowIL1RAP–CD36– cell fraction a mean of 3% were BCR/ABL1 positive; mean based on cells from two CML patients, the third patient had no cells with a CD34+CD38lowIL1RAP–CD36– phenotype. (D) FISH showing a BCR/ABL1 positive (upper panel) and negative (lower panel) cell. (E) CD34+CD38lowIL1RAP+ CML cells FACS sorted according to CD36 expression does not appear to differ in cell growth and survival in vitro. The mean of three CML samples is shown; error bars depict standard deviation. (F) CD34+CD38lowIL1RAP+ CML cells FACS sorted according to CD36 expression and treated with imatinib at a concentration of 5μM show that CD36 expressing cells are more resistant to imatinib treatment in vitro. The mean of three CML samples is shown; error bars depict standard deviation. (G) Representative histograms from cell cycle analysis using DRAQ5 to determine DNA content show a majority of both CD36+ and CD36– cells in G0/G1 phase within the CD34+CD38lowIL1RAP+ population. (H) Data on cell cycle status from three CML patient samples are summarized showing mean and standard deviation. *P<0.05. ns; not significant.
haematologica | 2018; 103(3)