Patients
Patients with high-risk hematologic diseases (acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, high-grade B cell lymphomas, Hodgkin’s lymphoma, myelodysplastic syndrome, and myelofibrosis) and candidates for myeloablative allogeneic HSCT were eligible for participation. Patients were 18 years of age or older and had an human leukocyte antigen (HLA)-matched sibling or HLA-matched unrelated donor. The trial was approved by the Duke University and Medical University of South Carolina Institutional Review Board and was carried out in accordance with the Helsinki Principle. The clinical trial was registered at www.clinicaltrials.gov as NCT01280955. Informed consent was obtained from all participants.
A cohort of 95 patients from the Duke HSCT program was identified for comparison during analysis. These patients received transplantation concurrently with the study group, as to control for supportive care and standard management practices of the time. Although eligible for the study, these patients were not enrolled for various reasons, including declining participation in the clinical trial, lack of insurance approval for participation, or managing physician preference. This control group had comparable diseases, underwent similar conditioning regimens, and received the identical graft-versus-host disease prophylaxis. Hematopoietic growth factor support was not routinely administered in either group.
Study design
The study was a two-stage, phase 1–2, open-label, two-center trial. Patients were conditioned with a myeloablative regimen such as total body irradiation (TBI) (1350 cGy)/cyclophosphamide, TBI/VP-16, busulfan/cyclophosphamide, or busulfan/fludarabine (Table 1). The donor stem cell grafts were from either granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells or BM from 8/8 or 7/8 HLA-identical family members or 8/8 (HLA A, B, C, DRβ1) allele-level matched unrelated donors. The target CD34+ cell dose for mobilized peripheral blood stem cell and BM stem cell recipients was 5 × 106/kg and 2 × 106/kg recipient ideal body weight. Prophylaxis for acute graft-versus-host disease was provided with tacrolimus and methotrexate in all patients. After donor cell infusions, patients received plerixafor given subcutaneously at 240 μg/kg every other day beginning at day +2 after transplant until day +21 or neutrophil engraftment, whichever came first. Phase I was a standard 3 + 3 design to evaluate safety. Three patients were enrolled in phase I portion of the study, and after all three patients demonstrated no dose-limiting toxicities, the study proceeded to phase II. The phase II portion involved the same study schema (Fig. 1). A subset of patients had a BM aspirate and biopsy at approximately day +30 after HSCT.
Table 1 Patient and transplant characteristics
Endpoints and assessments
The primary endpoint was safety determined according to frequencies and severities of adverse events and was assessed daily during the study. Duke Cancer Institute safety oversight committee evaluated all serious adverse events and events leading to patient withdrawal from the study. Other primary objectives of the trial were to determine plerixafor-associated adverse events, time to neutrophil recovery, and time to platelet recovery. Toxicities were graded using CTCAE version 4.0.3. Neutrophil engraftment was defined as absolute neutrophil count (ANC) of ≥0.5 × 109/L (500/mm3) for three consecutive laboratory values obtained on different days with ANC recovery being the first of the three consecutive days. Platelet engraftment was defined as platelet count ≥20 × 109/L for three consecutive days without a platelet transfusion for the preceding 7 days with platelet recovery being the first of the three consecutive days.
The secondary objectives were the incidence of grade II–IV acute graft-versus-host disease graded per NIH consensus criteria, overall survival at day +100, immunological reconstitution as determined by CD4 count, CD8 count, NK cells, and B cell pre-transplant, day +30, +60, and +90, and correlative laboratory studies assessing plasma cytokines/chemokines (IL-12, IFN-λ, TNF-α, and macrophage inhibitory protein-1) measured at day +7, +14, and +30.
Immune reconstitution
Quantification of the following subsets was performed by flow cytometry on fresh peripheral blood before transplantation and the 30, 60, and 90 days after transplantation: B cells (CD19+, CD3−,CD16−,CD56−), natural killer (NK) (CD3−,CD16+/CD56+) and natural killer T (NKT) (CD3+,CD16+/CD56+) cells, CD3+,CD4+,CD8+, regulatory (CD4+, CD25+, CD62L+), plasmacytoid dendritic cells (DCs) (CD123+, CD11c−), and myeloid DCs (CD123−,CD11c+).
Cytokine assay
Blood and BM samples obtained at the indicated time points were assayed for IL-12, IFN-λ, TNF-α, and macrophage inhibitory protein-1β (MIP-1β) using ELISA. The coating antibodies and the capturing antibodies for IL-12, IFN-λ, TNF-α, and macrophage inhibitory protein-1 (MIP-1β) as well as the standards were purchased from Novex™ by Life Technologies, and the ELISA was performed as per the manufacture’s recommendation.
Statistical analysis
All patient characteristics were compared using the Student’s t test for continuous variables and the chi-square test for categorical variables. The cumulative incidence for platelets and neutrophils was compared using Gray’s test. The Wilcoxon rank-sum test was used to compare immune recovery parameters at the different time intervals. Since the distributions of the cytokine levels are skewed to the right with extremely large values, we performed a logarithm transformation on these variables before analysis to make the distributions close to normal. Then, the cytokine levels were compared using the Wilcoxon rank-sum test between the two groups, and univariate linear regression models were fit to correlate times to ANC and platelet recovery with each of the (log) cytokine levels. Kaplan-Meier estimates of the survival curves for time to relapse, progression-free survival, and overall survival were obtained, and the log-rank test was used to compare survival curves. A logistic regression model with ordinal responses was fitted to compare the occurrence of graft-versus-host disease (GVHD) between the two groups.