Method of Mobilization: Implications on Graft Composition and Immune Reconstitution Post Autologous Hematopoietic Cell Transplantation

Research Article

Ann Hematol Oncol. 2016; 3(4): 1089.

Method of Mobilization: Implications on Graft Composition and Immune Reconstitution Post Autologous Hematopoietic Cell Transplantation

Solh M1,2*, Rathmann K², Chang-Fong S², Lamontagne D², Oyer J³, Copik A³ and Khaled Y²

¹Blood and Marrow Transplant Group of Georgia, Northside Hospital, USA

²Blood and Marrow Transplant Center, Florida Hospital Cancer Institute, USA

³University of Central Florida, School of Medicine and Biomedical Research, USA

*Corresponding author: Melhem Solh, Blood and Marrow Transplant Group of Georgia, Northside Hospital, University of Central Florida, 5670 Peachtree Dunwoody Rd NE, Atlanta, Ga 30342, USA

Received: June 01, 2016; Accepted: July 06, 2016; Published: July 08, 2016


Background: Plerixafor, a reversible CXCR4 antagonist, is used in conjunction with G-CSF for stem cell mobilization prior to autologous hematopoietic cell transplantation (AHCT). The effect of adding plerixafor to growth factors during mobilization on graft composition and early myeloid and immune recovery has not been widely described.

Methods: 49 adult AHCT recipients were enrolled on a single arm prospective trial where blood samples were freshly collected from the pheresis product and from patients’ peripheral blood on days 30 and 60 post AHCT. Flow cytometric analysis was done to quantify CD3+ T cells, CD3+ CD56+ NK-like T cells, CD56+ CD16+ and CD56+ CD16- NK cells as well as CD19+ B cells.

Results: Compared to patients mobilized with G-CSF, patients mobilized with Plerixafor plus G-CSF (G+P) required less number of collection days (1.9 vs 1.4 days; p=0.05) to reach the target CD34+ cell dose. Both groups had similar median times to neutrophil and platelet engraftment. G+P group had a higher percentage of CD4 (12.9% vs 9.2%), similar CD3+, NK and B cells in the graft compared to G-CSF mobilization. Both G+P and G-CSF groups had similar peripheral hematologic and immune recovery at days 30 and 60 post AHCT.

Conclusion: Our study shows that patients mobilized with G+P have similar immune and hematologic recovery to G-CSF mobilization post AHCT.

Keywords: Plerixafor; Mobilization; G-CSF; Autologous; Immune


Autologous Hematopoietic Cell Transplantation (AHCT) is an established treatment for patients with multiple myeloma and chemo sensitive, relapsed or refractory lymphomas [1]. As more than 98% of AHCTs in adults are performed using peripheral blood stem cell grafts, the success of this procedure depends largely on the ability to collect enough hematopoietic stem cells for adequate engraftment [2]. The quantity of CD34+ cells has traditionally been used as a surrogate for the number of hematopoietic stem cells, and the infused CD34+ dose is correlated with successful neutrophil and platelet engraftment, progression free survival and overall survival post high dose chemotherapy and AHCT [3-5]. The International Myeloma working Group Suggested collection of at least 4x106 CD34+ cells/ kg for a single AHCT and 8x106 CD34+ cells/kg to allow for two transplants if feasible [6]. In many centers, a minimum dose of 2x106 CD34+ cells/kg is considered acceptable to proceed with AHCT for myeloma or lymphoma patients.

The optimal mobilization method for either myeloma or lymphoma patients is still debatable and strategies for graft collection vary between different centers. The mobilization strategy may affect the stem cell graft that can be associated with overall patient outcomes [1,7]. Chemotherapy followed by granulocyte-colony-stimulating factor (G-CSF) or G-CSF alone has been the standard for CD34+ cells mobilization into the peripheral blood. Myeloma patients were traditionally mobilized with high dose cyclophosphamide (4-7g/m²) followed by G-CSF [8]. Inadequate mobilization using traditional strategies among myeloma and lymphoma patients can be seen in 5-30% of the cases [9]. Lately, alternative strategies for CD34+ cells mobilization include lower dosages of cyclophosphamide followed by G-CSF, G-CSF alone or G-CSF combined with Plerixafor with or without chemotherapy [10,11].

Plerixafor (AMD3100), a reversible and selective antagonist of the CXCR4 chemokine receptor that blockes CXCR4 and stromalcell derived factor 1-a interactions, was originally synthesized for activity against human immune deficiency virus. In initial studies, plerixafor was found to cause an increase in peripheral blood counts and mobilization of CD34+ from the bone marrow to the peripheral blood [12]. Addition of Plerixafor to G-CSF has been shown to be superior to G-CSF alone in myeloma and lymphoma patients in terms of mobilization as measured by CD34+ counts, collection yield and number of collection days to achieve the target yield [10,13]. The effect of Plerixafor on graft composition was assessed in cryopreserved grafts of NHL patients and it was found to mobilize more CD3+ cells, Helper CD4+ cells and CD8+ cells [14]. This study was conducted to evaluate the effect of plerixafor on graft composition of freshly collected stem cell aphaeresis product and to further delineate the implication of adding plerixafor on count recovery and immune reconstitution markers in the first 60 days post AHCT.



A total of fifty one patients eligible for AHCT and stem cell mobilization at our center were enrolled on a prospective trial to evaluate graft composition and immune reconstitution markers at day 30 and day 60 post AHCT. Only patients with multiple myeloma or non Hodgkin’s lymphoma were included in this analysis and 2 patients with Hodgkin’s disease were excluded. This study was conducted according to the declaration of Helsinki and was approved by our institutional IRB. All subjects included in this study signed an IRB approved informed consent prior to participation. Thirty three patients received plerixafor + G-CSF (G+P) and 16 patients received G-CSF alone. A small portion (2.5mL) of the autologous peripheral blood stem cell product was collected prior to transplant and peripheral blood (~10 mL) was collected on days +30, and +60 post transplantation.

Mobilization and Collection for Stem Cells

The mobilization regimen consisted of filgrastim 10 μg/kg/day for 4 consecutive days. Daily measurements of blood CD34+ and total white counts were started on day 4. CD34+ levels were determined using flow cytometry (BD FACS Canto II) using a single platform assay (Beckman-Coulter stem Kit) based on recommendations by the international society of hematopathology and graft engineering (ISHAGE). Patients who had a peripheral blood CD34+ level = 20/ μl on day 4, received plerixafor 0.24 mg/Kg at 10 pm of that day in addition to the scheduled filgrastim dose. Peripheral blood aphaeresis was started in the morning of day 5 and continued till the target cell dose or patient failed to collect. The minimum acceptable cell dose was = 2 x106 CD34+cell/Kg and patients who did not reach this cell dose after 3 days of collection, were considered mobilization failures. Patients who had received plerixafor on the night of day 4, continued to receive plerixafor through the mobilization period. Collection was performed with a COBE Spectra auto PBSC machine. The daily blood volume processed during the aphaeresis was 20 liters.

High Dose Chemotherapy and Transplant course

All patients who had a successful collection were admitted to the inpatient unit for high dose chemotherapy and autologous stem cell infusion. Non-Hodgkin’s lymphoma patients received a conditioning regimen consisting of R-BEAM (Rituximab 375 mg/m² on day -7, Carmustine 300 mg/m² on Day -6, etoposide 200 mg/m² days -5 to -2, cytarabine 300 mg/m² days -5 to -2 and melphalan 140 mg/m² on day -2). Multiple myeloma patients received conditioning regimen with high dose melphalan (melphalan 200 mg/m² on day -2). CD34+ cells were infused on day 0. All patients received G-CSF at 5 μg/kg/day starting day +5 after AHCT till neutrophil engraftment or until the first day with absolute neutrophil count (ANC) >2500x109/l. All patients received antibacterial, antiviral and antifungal prophylaxis and blood product and nutritional support per institutional guidelines.

Neutrophil engraftment was defined as ANC =0.5x109/l for 3 consecutive days. Platelet engraftment was defined as platelet level of =20x109/l without transfusion. All patients remained in the bone marrow transplant unit till neutrophil engraftment and were followed in the clinic until at least 100 days post transplantation.

Graft and Post Transplant Peripheral blood Cell subset Analysis

Samples were drawn from the aphaeresis product (2.5 ml) and from transplant recipients’ peripheral blood (10 ml) on days +30 and +60 post AHCT. The CD34+ content of the graft was analyzed by flow cytometry (BD FACS Canto II). A single platform assay was used (Beckman-Coulter Stem kit) via ISHAGE protocol. This kit contains CD34 and CD45 monoclonal antibodies, 7- aminoactinomycin D (7- ADD), NH4CL, and stem-kit fluorospheres. The data was analyzed using FACS Diva software (BD biosciences).

Samples drawn on collection day, and days +30 and +60 post AHCT were immediately processed, stained with antibodies and analyzed for lymphocyte content. Peripheral blood cells were depleted of red blood cells using a red blood cell lysis solution, washed twice in PBS and re-suspended in staining buffer ( PBS + 2mM EDTA + 0.5% BSA). Next, cells (0.5 x 106) were stained with antibody cocktail (30 min at 4oC), washed and analyzed by flow cytometry. The antibody cocktail contained the following pre-conjugated monoclonal antibodies: CD56-PE (Miltenyi Biotech, Auburn, CA), CD3-APC, CD16-FITC, (Beckman Coulter, Brea, CA), CD19-PE-CY7 (BD Biosciences, San Jose, CA). Data were acquired using BD FACS Canto II (BD Biosciences) and analyzed with the FACSDiva software (BD Biosciences) to quantify CD3+ T cells, CD3+ CD56+ NK-like T cells, CD56+ CD16+ and CD56+ CD16- NK cells as well as CD19+ B cells.

Statistical Analysis and Data collection

Cell subset data were prospectively collected as per the study protocol. The clinical and demographic data was collected from the clinical program database and was subsequently merged into the cell subsets data for analysis. All calculations and statistical analysis were conducted using SPSS statistics 21.0 for windows. Continuous numerical variables were described with their medians and ranges. The Mann-Whitneu U test was used to analyze differences between quantitative variables and where the variables were not normally distributed. This test was also used due to low number of observations. The Chi-square test was used to compare categorical variables. A p-value of less than 0.05 was considered significant.


A total of 51 patients were enrolled on this study. Two patients with Hodgkin’s disease were excluded from the analysis. Of 49 eligible patients, 16 were mobilized with G-CSF alone (G-CSF group) and 33 with G-CSF plus plerixafor (G+P). The median age for the study group was 58 years (range 21-75 years).Thirty five patients (71%) had multiple myeloma (MM) and all received high dose melphalan conditioning before stem cell infusion. 14 patients (29%) had nonhodgkins lymphoma (NHL) and all received R-BEAM conditioning. There was no difference between the two groups (G-CSF versus G+P) in any of the basic demographic and disease parameters (Table 1).