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C. Lagresle-Peyrou et al.
production, leukocyte migration, phagocytosis and adap- tive immune response) (Figure 2B). Major differences in gene expression (>900 differentially expressed genes) were observed when comparing mobilized and BM sam- ples (Table 3). The genes downregulated in mobilized ver- sus BM HSPC are involved in cell cycle-related processes (e.g. DNA replication, chromosome segregation, and nuclear division) – confirming that mobilized samples contain more quiescent cells, presumably HSC, than pro- genitors (Figure 2B, Online Supplementary Figure S4A and Online Supplementary Table S2). Interestingly, mobilized populations poorly expressed genes that are typical of committed hematopoietic progenitors, relative to BM samples (Figure 2C, Online Supplementary Figure S4A and Online Supplementary Table S2). Conversely, a large propor- tion of the genes involved in HSC biology were strongly expressed in mobilized HSPC (Figure 2C, Online Supplementary Figure S4A and Online Supplementary Table S2). Importantly, some HSC markers (e.g. THY1, HLF and FLT3) were more strongly expressed in Plerixafor-mobi- lized SCD samples – confirming the latter’s high HSC con- tent – than in BM samples and Filgrastim- or Plerixafor- mobilized HSPC from healthy donors (Figure 2C).
Fewer than 300 genes were differentially expressed between Filgrastim- or Plerixafor-mobilized samples (Table 3). Of note, genes encoding transcriptional regula- tors and surface markers of plasmacytoid dendritic cell progenitors are upregulated in Plerixafor-mobilized sam- ples compared to Filgrastim-mobilized samples (Online Supplementary Figure S4B). FACS analyses showed the appearance of a CD133-CD34dim cell population preferen- tially in Plerixafor-mobilized samples (Online Supplementary Figure S2 and data not shown). This popula- tion might contain plasmacytoid dendritic cell progenitors that are mobilized by Plerixafor, as recently described.16
Proof of stemness was further confirmed by transplan- tation into conditioned, immunodeficient mice. Human chimerism in the BM and spleen was similar in recipients of Filgrastim-mobilized CD34+ cells from healthy donorss and Plerixafor-mobilized CD34+ cells from SCD patients (Figure 3A,B). Similar counts of lymphoid and myeloid subsets were detected in both groups (Figure 3A,B). The numbers of human CD34+ cells, HSC and multipotent progenitors in bone marrow were comparable between the two groups (Online Supplementary Figure S5). After sec- ondary transplantation, all recipients of Plerixafor-mobi- lized SCD samples and Filgrastim-mobilized CD34+ cells from healthy donors displayed engraftment - further demonstrating the presence of true HSC in HSPC mobi- lized with Plerixafor in SCD patients (data not shown).
Discussion
Successful transplantation of autologous gene-corrected cells primarily depends on the collection and effective genetic modification of a sufficient number of true stem cells. Thus, poor harvesting of BM or mobilized peripheral stem cells limits the success of this procedure. To over- come the need for two or more BM aspirates, we initiated a phase I/II clinical trial with the objective of establishing whether Plerixafor-induced stem cell mobilization in SCD patients can avoid the increased risk of vaso-occlusive crises observed with Filgrastim mobilization and of vali- dating the efficiency of HSPC harvesting with this proce-
Table 3. Number of differentially expressed (up- or downregulated) genes in HSPC from different sources (false discovery rate < 0.05).
Comparison
Differentially Upregulated Downregulated expressed
SCDBMvs.HDBM 134 52 82
SCD Pler vs. HD Pler
SCD Pler vs. HD Filg
SCD Pler vs. HD BM
SCD Pler vs. SCD BM
HD Pler vs. HD BM
HD Filg vs. HD BM
HD Pler vs. HD Filg
HD Filg vs. SCD BM
165 133 32 265 188 77 1685 761 924 1544 753 791 923 315 608 1893 789 1104 47 20 27 1683 800 883 1024 398 626
dure.26 Because of the risks incurred by the patients and the absence of a direct benefit, the trial was restricted to patients with <10x109 granulocytes/L, knowing their role in vaso-occlusive crises.
We decided to discontinue hydroxyurea treatment 3 months before the mobilization in P3 and to submit all the patients to monthly transfusions. The rationale for this decision was based on the following observations: (i) hydroxyurea has no beneficial role in CD34+ cell mobiliza- tion in thalassemic patients;27 (ii) hydroxyurea withdrawal is associated with an increase in the number of circulating CD34+ cells in SCD patients;28 and (iii) in various clinical settings hydroxyurea has been associated with myelosup- pression29,30 suggesting BM toxicity and potential impair- ment of HSC.
In order to optimize the safety of the mobilization pro- cedure in SCD patients, we reduced HbS levels to below 30% via erythrocyte exchanges, and we closely moni- tored white blood cell counts and serum levels of inflam- matory cytokines. Furthermore, the use of Plerixafor avoided the adverse events associated with Filgrastim: vascular events and splenic rupture after the administra- tion of this latter have been extensively reported in clinical populations and even in healthy stem cell donors.31 Although these events are usually rare, their frequency may be higher in the presence of other vascular risk fac- tors (such as SCD).31 Thus the benefit/risk ratio of using a hematopoietic growth factor such as Filgrastim (especially at the high doses required in patients without a malignant blood disease) appears to be unacceptably low. Hence, we gave our three patients Plerixafor at the standard dose. No adverse events occurred, serum levels of inflammatory cytokines were in the normal ranges (data not shown) and the white blood cell counts did not exceed 40x109/L (i.e. a value often reported in the literature, and far from the 50 to 75x109/L often observed in healthy donors after 5 days of Filgrastim treatment).32
The rapid mobilization with Plerixafor alone (compared with Filgrastim) is also an important advantage. The cur- rent guidelines on mobilization in patients with a malig- nant disease recommend initiating apheresis 11 h after Plerixafor administration;33 this contrasts with the 5 to 7 days required for collection after Filgrastim administra- tion. Moreover, rapid, transient stem cell mobilization by
HD Pler vs. SCD BM
SCD: sickle cell disease. HD: healthy donor. BM: bone marrow. Pler: Plerixafor. Filg:
784
Filgrastim.
haematologica | 2018; 103(5)