The CXCR4 and adhesion molecule expression of CD34+ hematopoietic cells mobilized by "on-demand" addition of plerixafor to granulocyte-colony-stimulating factor

Abstract

Background: Granulocyte-colony-stimulating factor (G-CSF) is routinely used for mobilization of hematopoietic stem and progenitor cells preceding autologous transplantation after high-dose chemotherapy in hematologic malignancies. However, due to high mobilization failure rates, alternative mobilization strategies are required.

Study design and methods: Patients who poorly mobilized CD34+ hematopoietic cells (HCs) with G-CSF additionally received the CXCR4 antagonist plerixafor. The phenotype of CD34+ HCs collected after this plerixafor-induced "rescue" mobilization, in regard to adhesion molecule and CD133, CD34, and CD38 expression in comparison to CD34+ HCs collected after traditional G-CSF administration in good mobilizers, was analyzed flow cytometrically. To confirm previous studies in our patient cohort, the efficiency of mobilization and subsequent engraftment after this "on-demand" plerixafor mobilization were analyzed.

Results: Pronounced mobilization occurred after plerixafor administration in poor mobilizers, resulting in similar CD34+ cell yields as obtained by G-CSF in good mobilizers, whereby plerixafor increased the content of primitive CD133+/CD34+/CD38- cells. The surface expression profiles of the marrow homing and retention receptors CXCR4, VLA-4, LFA-1, and CD44 on mobilized CD34+ cells and hematopoietic recovery after transplantation were similar in patients receiving G-CSF plus plerixafor or G-CSF. Unexpectedly, the expression levels of respective adhesion receptors were not related to mobilization efficiency or engraftment.

Conclusion: The results show that CD34+ HCs collected by plerixafor-induced rescue mobilization are qualitatively equivalent to CD34+ HCs collected after traditional G-CSF mobilization in good mobilizers, in regard to their adhesive phenotype and engraftment potential. Thereby, plerixafor facilitates the treatment of poor mobilizers with autologous HC transplantation after high-dose chemotherapy.

Figure 1

CXCR4 and adhesion molecule expression profiles in G-CSF and G-CSF plus plerixafor–mobilized HSPCs. (A) Representative density and dot plots illustrate the gating strategy for HSPCs based on forward and side scatter (FS and SS) and CD34 expression. (B) Representative fluorescence histograms (left), MFIRs of surface CXCR4 levels (middle), and percentages of CXCR4+ cells (right) of CD34+ cells collected after G-CSF– (n = 23) or G-CSF plus plerixafor–induced mobilization (n = 7). Representative fluorescence histograms of CD49d (C, left), CD11a (D, left), and CD44 (E, left) expression on CD34+ cells mobilized with G-CSF or G-CSF plus plerixafor. The corresponding MFIRs of CD49d (C, right, n_(G) = 24, n_(G+P) = 7), CD11a (D, right, n_(G) = 24, n_(G+P) = 7), and CD44 (E, right n_(G) = 15, n_(G+P) = 6) are shown in box plot format. G = G-CSF; G+P = G-CSF plus plerixafor. ns = not significant.

Figure 2

CD34 and CD38 expression on CD133+ HCs in leukapheresis products. (A) The dot plots show the surface expression of CD34 and CD38 on pregated CD133+ HCs and are representative for 11 leukapheresis products collected after G-CSF (top) and seven leukapheresis products collected after G-CSF plus plerixafor administration (bottom). (B) Percentages of CD38– cells within the CD133+/CD34+ cell population in G-CSF (n = 11) compared to G-CSF plus plerixafor (n = 7). *p < 0.05.

Figure 3

Plerixafor rescues HSPC mobilization in poor mobilizers. (A) Numbers of preapheresis CD34+ cells (×10⁶/L PB) 1 day before and the next morning after plerixafor injection in poor mobilizers as determined by flow cytometry (n = 8). (B) Comparison of preapheresis CD34+ cell counts (×10⁶/L PB) in good mobilizers receiving G-CSF (n = 28) and poor mobilizers receiving G-CSF plus plerixafor (n = 8). (C) Numbers of CD34+ cells/kg collected after G-CSF– (n = 26) or G-CSF plus plerixafor–induced mobilization (n = 8). Results are illustrated in box plot format, indicating the median. The 25th and 75th percentiles are marked by the edges of the box and the minimum and maximum observations (excluding outliers) are marked by the whiskers. Outliers are defined as cases that are more than 1.5-fold the interquartile range away from the edges of the box and are indicated as dots. (D) Preapheresis CD34+ cell numbers (×10⁶/L PB) of good mobilizers who did (n = 22) or did not (n = 4) receive chemotherapy immediately before G-CSF. (E) Total CD34+ cell numbers/kg collected from good mobilizers, who received chemotherapy before G-CSF (n = 22) or not (n = 4). (F) Correlation of preapheresis CD34+ cell numbers (×10⁶/L PB) with total CD34+ cells/kg collected via leukapheresis. (■) Patients treated with G-CSF (n = 27); () patients treated with G-CSF plus plerixafor (n = 8). *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant.

Figure 4

CXCR4, CD49d, CD11a, and CD44 expression on mobilized CD34+ cells are independent of mobilization efficiency. Preapheresis CD34+ cell numbers (×10⁶/L PB) were correlated with MFIRs of CXCR4 (A, left, n = 31), percentages of CXCR4+ cells within the CD34+ population (A, right, n = 31) and MFIRs of CD49d (B, n = 32), CD11a (C, n = 32), and CD44 (D, n = 21) on CD34+ cells. (●) Patients receiving G-CSF; () patients receiving G-CSF plus plerixafor.

Figure 5

Transplantation with HCs mobilized via G-CSF or G-CSF plus plerixafor results in similar time to engraftment. (A) The days to neutrophil (left, n_(G) = 20, n_(G+P) = 6) and PLT engraftment (right, n_(G) = 19, n_(G+P) = 5) after transplantation with G-CSF– or G-CSF plus plerixafor–mobilized HCs are shown in box plot format. (B) Correlation of transplanted CD34+ cell numbers/kg (mobilized by G-CSF or G-CSF plus plerixafor) with time to neutrophil (left, n = 26) and PLT engraftment (right, n = 24). Correlations of neutrophil (left) and PLT engraftment (right) with surface levels of CXCR4 (C, n_{(Neutrophils)} = 23 and n_(PLTs) = 21), CD49d (D, n_{(Neutrophils)} = 24 and n_(PLTs) = 22), CD11a (E, n_{(Neutrophils)} = 24 and n_(PLTs) = 22), and CD44 (F, n_{(Neutrophils)} = 15 and n_(PLTs) = 14). G = G-CSF; G+P = G-CSF plus plerixafor. *p < 0.05; **p < 0.01; ns = not significant.