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Figure 2. Correlation between post-plerixafor CD34+ cell counts and baseline cell counts. The CD34+ level at 12 h correlated positively with the baseline level of CD34+ (P=0.0006) but not baseline levels of absolute neutrophil count (ANC, P=0.66) or white blood cells (WBC, P=0.49). The graphs show the association between the value of peripheral blood CD34 concentration at 12 h after plerixafor and the baseline values of (A) CD34, (B) ANC and (C) WBC in 15 patients with SCD treated with 80 (circles), 160 (squares) and 240 (triangles) μg/Kg of plerixafor. Patients on hydroxyurea are represented by filled circles, squares and triangles, patients off hydroxyurea are represented by open circles, squares and triangles.
Safety and efficacy of plerixafor in SCD patients
ABC
patient, with calculation of the absolute number of positive cells by multiplying the percentage of positive cells by the relevant number of cells obtained from a concurrent complete blood count. For plasma studies, whole blood is centrifuged at 2500 rpm for 15 min at 4°C and plasma frozen at ≤ -80°C until batch testing. The following coagulation system activation markers relevant to sickle vaso-occlusion are tested:26,29 prothrombin frag- ment 1.2 (Enzygnost F1+2, Dade-Behring) and factor VIII (STA®- ImmunoDef VIII, Stago).
Statistical analysis
Absolute concentrations as well as the fold increases (ratio of peak at 12 h to baseline absolute concentrations) of standard clinical as well as research parameters were analyzed. For patient 8(2), to avoid computing correlations on an infinite value, we replaced the baseline value of 0 by 0.2 in the analyses using CD34+ ratio (10/0.2 = 50). When applying non-parametric statistics, the chosen value does not affect the results, as long as it is close to 0. The presence of a trend in increases of CD34+, ANC, and WBC counts (both at 12 h and fold increases) with increasing dose was tested using the non-parametric Cuzick test for trend. Correlations between baseline values, and values at 12 h or fold increases were estimated using the Kendall tau. A dif- ference in the distribution of 12 h and fold increases according to the administration of hydroxyurea was investigated using a Wilcoxon test.
For analyses of cell activation and coagulation, Wilcoxon signed rank paired testing was performed on the combined dose cohorts for differences between 12 h and baseline values, where- as the presence of significant fold differences between dose lev- els was examined with the Kruskal-Wallis test. Correlations between values were estimated using the Kendall tau. Two- tailed P values <0.05 were considered statistically significant.
Results
Patients’ characteristics
Fifteen subjects have been recruited to date for the study at the first three dose levels of 80, 160 and 240 μg/kg. Fourteen patients were enrolled from Montefiore
Medical Center (New York, USA) and one patient from The Mount Sinai Hospital (New York, USA) (Table 1). Two patients enrolled at dose levels 1 and 2 were subse- quently re-enrolled in the study at a higher plerixafor dose (dose level 3). All subjects had a past history of mod- erate to severe acute chest syndrome, defined by requir- ing treatment with simple or exchange transfusion. Importantly, for safety and feasibility, patients were con- tinued on their standard outpatient treatment being used to control their disease. Ten of 15 patients were on hydroxyurea, with a median HbF level of 12.4% (Online Supplementary Table S1) and median baseline ANC of 4100/μL (Online Supplementary Table S2). Only one of the 15 subjects was receiving chronic transfusion therapy, with a HbF of 1.2% and HbA of 54%; this patient was also on deferasirox for the treatment of transfusion-relat- ed iron overload. HbA was absent in all other patients. In the non-transfused patients, HbF levels correlated strong- ly with hemoglobin concentration, hematocrit, and retic- ulocyte counts. Of nine patients for whom splenic imag- ing was available, seven had splenic atrophy (Online Supplementary Table S1).
Efficacy of CD34+ mobilization
Absolute WBC counts, neutrophil counts and CD34+ cell concentrations increased from baseline in all patients (Figure 1). Absolute monocyte and lymphocyte counts also increased from baseline (Online Supplementary Table S2). Our target goal of mobilizing at least 30 CD34+ cells/μL was, however, reached in only 50% of patients given the plerixafor dose of 80 μg/kg, 33% of patients given 160 μg/kg, and 33% of patients given 240 μg/kg. Peak ANC (P=0.03) and WBC count (P=0.05), but not CD34+ cell count (P=0.65), increased with increasing dose level. As previously reported in healthy donors,13,30,31 there was a strong correlation of peak CD34+ count with base- line CD34+ concentration (Kendall tau=0.68, P=0.0006) but no correlation was observed with baseline ANC (Kendall tau=0.09, P=0.66) or baseline WBC count (Kendall tau=0.13, P=0.49) (Figure 2). There was also no correlation, as previously reported,13 with baseline platelet
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