Hematopoietic stem cell (HSC) transplantation is a crucial treatment option for hematological malignancies. The use of peripheral blood stem cells not only for autologous but also for allogeneic transplantation is constantly increasing over the recent years. Current mobilization regimens that include cytokine (granulocyte colony stimulating factor, G-CSF) alone or in combination with chemotherapy frequently result in inadequate numbers of hematopoietic progenitor cell (HPC). The problem is of importance especially in heavily pretreated patients. Unfortunately around up to 35% patients fail to mobilize the successful number of stem cells.

Plerixafor is a CXCR4 antagonist approved for mobilization of peripheral blood stem cells in non-Hodgkin’s lymphoma and multiple myeloma patients (1, 2). The chemokine receptor CXCR4 and ligand SDF-1 are integrally involved in homing and mobilization of HPCs (1, 2). Disruption of the SDF-1/CXCR4 axis by the CXCR4 antagonist, plerixafor, was demonstrated in clinical trials to improve mobilization when it was included in the mobilization regimen (3,4). Chemotaxis of CXCR4 toward SDF-1α plays an important role in the trafficking and homing of HPCs to the bone marrow compartment (1, 2). Plerixafor reversibly blocks the ability of HPCs to bind to the bone marrow matrix. When used with granulocyte colony-stimulating factor (G-CSF), plerixafor helps increase the number of these progenitor cells in the peripheral blood. Plerixafor (AMD3100, Mozobil, Genzyme) has been recently approved by the US Food and Drug Administration (FDA; December 2008) and the European Medicines Evaluation Agency (EMEA; July 2009) to enhance mobilization of hematopoietic stem cells in combination with G-CSF in patients with lymphoma and multiple myeloma (MM).

Plerixafor was originally developed as a potential anti-HIV agent because it antagonizes the chemokine receptor 4 (CXCR4), which serves as a coreceptor for the entry of T-lymphotropic HIV strains into host T-lymphocyte cells (5). During pharmacokinetic studies of the drug, leukocytosis was observed. Hendrix et al. (5) reported that the plasma concentration of plerixafor declined gradually after a single

intravenous dose, while the white blood cell count gradually increased, reaching a maximum count of ?3 times its baseline at 6 h. This leukocytosis appeared to result from CD34+ cell mobilization. Further work demonstrated that the administration of plerixafor resulted in a consistent increase in the number of CD34+ cells in the peripheral blood, suggesting that it could be used as a potential CD34+-cell-mobilizing agent in the setting of autologous transplantation (5).

It was proved that plerixafor synergistically augments the mobilization effect of G-CSF on CD34+cells (6). Hematopoietic differentiation of transplanted CD34+cells was similar after plerixafor or G-CSF mobilization methods. Studies on patients with NHL or MM indicated that mobilization of CD34+/CD38– cells, a more primitive subset of CD34+ cells, was eightfold higher with the addition of plerixafor to a G-CSF regimen when compared to G-CSF alone (6).

This is a single center prospective study evaluating the outcome of mobilization regimen and autologous stem cell transplantation in myeloma and lymphoma patients mobilized using plerixafor based regimen. Total of 21 patients aged 52 (22-69 years) were mobilized with plerixafor in our institution over two-year period. 6/21(28,5%) patients received plerixafor as a rescue mobilization in the Compassionate Use Program (CUP). All patients signed the informed consent form before administration of plerixafor. The remaining 15 patients were given plerixafor in a commercial way. All of the patients failed to mobilize the satisfactory number of stem cells after standard mobilizing regimen or who failed to proceed to apheresis due to low peripheral blood CD34+ cell counts (“poor mobilizers”). We assessed the outcome of autologous transplantation (ASCT) in this group of patients.

The protocol included a daily injection of G-CSF (10 μg/kg) administered subcutaneously each morning for 4 consecutive days, followed by plerixafor administration (240 μg/kg s.c.) starting on the fourth day at around 10 p.m. The following morning the patients underwent apheresis procedure (blood volumes as per site preference). Administration of G-CSF and plerixafor injection were repeated the following day. Three patients were given chemo-mobilization regimen (plerixafor and G-CSF plus chemotherapy – high-dose of cyclophosphamide in all three cases). These patients were predicted to be poor mobilizers on the basis of low CD34+ cell numbers in the peripheral blood at optimal time points following chemotherapy and G-CSF. Two of them collected the satisfactory number of CD34+ cells.

The following parameters were recorded: age, sex, body weight, diagnosis, stage of the disease, the number of prior chemotherapy lines, prior radiotherapy, previous mobilization regimens, number of apheresis days, CD34+ yield, WBC yield, the volume of frozen apheresis product. During the transplant and post-transplant period the following data were collected: the count of CD34+ in the graft, time to granulocyte and platelet engraftment and progression free survival (PFS). Engraftment was assessed using conventional EBMT criteria considering the transplant day as day 0. Neutrophil engraftment was defined as the first of three consecutive days on which the neutrophil count reached ≥ 0.5 x 10⁹/L. Platelet engraftment was defined as the first of three consecutive days on which the platelet count reached ≥ 20 x 10⁹/L without platelet transfusion for at least 48 h.

The PFS was estimated by the Kaplan-Meier method. All statistical analysis was performed using Statistica10 software.

The prospective analysis included 21 patients (“poor mobilizers”), 6 multiple myeloma patients, including one patients with POEMS, and 14 lymphoma patients including diffuse large B-cell (3 pts), follicular (2 pts), nodal marginal zone lymphoma (1 pt), Waldenstroem’s disease (1 pt), malignant lymphocytic lymphoma (4 pts) and Hodgkin’s lymphoma (3 pts), also 1 pt with Kostmann syndrome. The disease status at the time of mobilization was defined as complete remission (CR), partial remission (PR), stable disease (SD), or progressive disease (PD) according to commonly accepted criteria (7, 8). The table 1 summarizes the patient characteristics.

All the patients had been treated previously for their primary disease. Four lymphoma patients (19%) underwent radiotherapy before. Two patients (9.5%) in the lymphoma group and one patient (4, 7) in the multiple myeloma group had a history of autologous stem cell transplantation. All the patients received chemotherapy before the attempt at mobilization. Patients had received a median of three lines of chemotherapy (range: 1-8) before plerixafor based mobilization regimen. A chemotherapy regimen was defined as one or more courses of chemotherapy of a certain type, including conditioning regimen and chemotherapy used for previous stem cell mobilization. The number of chemotherapy courses was recorded accordingly. Within the MM group, these lines included lenalidomide in two patients (9.5%) and a previous ASCT in two (9.5%). Among the lymphoma patients, six had been treated with combination chemotherapy containing fludarabine or cladribine (28%), one patient had a history of previous ASCT.

Overall, there were 19 failed previous mobilization attempts in this series. All of these attempts combined chemotherapy and G-CSF. A total of 15 patients (71%) had one, and four (19%) had 2 failed mobilization attempts before plerixafor administration. No patient had a history of bone marrow harvest. As a result of these previous attempts, 11 patients (5 – MM and 6 – lymphoma) had collected variable numbers of PBSCs, for a median of 0.6 x 10⁶ per kg CD34+ cells (0.16-0.94). Two patients entered the plerixafor group without a previous mobilization attempt.

The satisfactory number of PB CD34+ cells to allow stem cell collection was defined as 10 CD34+ cells/μL. Treatment with plerixafor increased the levels of circulating CD34+ cells before PBSC collection, with a median of 20 cells per μLafter first plerixafor administration (range: 1-121) and 17 (range: 1-148) after the second injection of plerixafor. Overall, 71% (6 MM patients and 9 lymphoma patients) met the target of ≥10 CD34+ cells per μL. Mobilization with plerixafor plus G-CSF in our patients had a good safety profile which is consistent with previous reports. We did not observe any adverse events. One patient was mobilized with plerixafor twice – each time responded to plerixafor injection well, collecting the satisfactory amount of cells.

Successful mobilization was defined as the collection of a total of ≥ 2.0 x 10⁶ CD34+ cells per kg.Stem cell aphaeresis was performed in all patients receiving plerixafor and a median of 1.45 x 10⁶CD34+ per kg (range: 0.03-6.74) cells were collected with a median of two aphaeresis (2-4).The apheresis were done twice in most cases (in 8 cases apheresis was performed three times, and in once case – four times). Ten patients (47.5%) collected ≥ 2 x 10⁶ CD34+ per kg. The median number of collected WBC was 11.35 x 10⁸ per kg (range: 6.92-21.94). The median volume of frozen product of aphaeresis was 1600 ml (range: 800-3100).Apheresis blood volumes were processed following site standard protocols. The numbers of CD34+ cells collected during each apheresis session were recorded. The apheresis and cell processing was done according to local regulations.

The timing and protocol of ASCT was determined according to local regulations. Conditioning regimen for auto-SCT, support care and posttransplant follow-up were carried out as per site preference. A total of 13 (62%) of the patients had proceeded to transplant at the time of analysis, 5 patients with MM (38%) and 16 patients with lymphoma (54%), 1 patient with Kostmann syndrome. The conditioning regimens included carmustine, etoposide, cytarabine, and melphalan (BEAM) in 3 patients, melphalan 200 mg/m2 (MEL200) in 3 patients; melphalan 140 mg/m2 (MEL140) in 2 patients); and cyclophosphamide, etoposide, and carmustine (CBV) – in 4 patients, cyclophosphamide and alemtuzumab in one patient. The median dose of PBSCs infused was 2.67 x 106 CD34+ cells per kg and median WBC was 10.73 x 108 per kg. Eight patients received pooled cells, plerixafor-mobilized PBSCs and products from previous collections to reach ≥ 2 x 106 CD34+ cells per kg. All other patients received plerixafor-mobilized PBSCs only. All patients engrafted neutrophils and platelets after plerixafor-mobilized auto-SCT, with no differences between plerixafor-only cells and pooled cells, or between MM and lymphoma patients. Neutrophil engraftment was carried out at a median of 12 days (range: 9-13). Platelet engraftment was performed at a median of 15 days (range: 9-18). Two patients with multiple myeloma underwent tandem transplantation (second ASCT was three months after the first procedure). All transplanted patients were alive at a median follow up period of 12.4 months (range: 1-23) after transplant. Two patients suffered from relapse: one of them was a Hodgkin lymphoma patient, heavily pretreated, the other one experienced the relapse of CLL (enlargement of lymph nodes in the neck region confirmed on PET scan), was successfully treated with radiotherapy and a year later achieved a complete remission. Two multiple myeloma patients were given maintenance therapy with thalidomide after the transplant and are in good condition at the time of analysis with no need of escalation of chemotherapeutic treatment. Three patients are planned for ASCT in the nearest future. Only five patients altogether could not proceed to ASCT due to mobilization failure with plerixafor plus G-CSF. Progression free survival (PFS) was 78% at the time of analysis with a median follow up time of 12.4 months (range: 1-23). The PFS for a group of patients matched by sex, age, diagnosis, status of the disease treated in our Department of Hematology and BMT was 55% with a median follow up time of 22.5 months (range 1-33) after autologous transplantation.

Autologous stem cell transplantation is a method of choice in many hematological malignancies enabling the patients to achieve remission (in lymphoma patients) or to prolong life (in myeloma patients). Commonly used mobilizing regimens include high dose chemotherapy followed by G-CSF administration.

Plerixafor registration clinical trials proved this drug to be efficient in mobilizing peripheral blood stem cells enabling the patients to achieve the target stem cell levels and allowing more patients to qualify for transplant procedure (3, 4). Moreover, the drug turned out to be well tolerated (9). It is worth to stress that the registration studies included patients with no history of previous mobilization failure. Before the drug was made available commercially, it was provided to American and European transplant centers free of charge, within the Compassionate Use Program (CUP) (1014).Itwas the first study to include the patients who failed previous mobilization regimen procedures. The first study conducted in the United States within the CUP revealed that the minimum target of 2.0 x 106 CD34+ +cells/kg b.w. was collected in 66% of patients (12). Worldwide data identified plerixafor as a competent drug in the setting of stem cell collection (15, 16). The apheresis yield in German and Austrian study ranged between 69.4% to 76.6% of a target minimum of ≥ 2.0 x 106 CD34+ cells/μL. In the Polish CUP experience theminimum target of ≥ 2.0 x 106 CD34+ cells/kg was collected in 40 patients (65.6%). The following factors were identified on the basis of multivariate analysis as the factors that contributed to an inferior outcome of stem cell collection: the diagnosis of NHL and previous radiotherapy (11). The data collected by Central European Leukemia Group (CELG) indicated the following factors that may contribute to plerixafor-based mobilization failure: an age ≥ 65 years, a diagnosis of NHL, and treatment with more than four chemotherapy regimens (17). In this group of 197 patients the target of ≥ 2.0 x 106 CD34+ cells/kg was collected in 133 cases (67.5%). Several studies results confirmed the effectiveness of plerixafor as a mobilization agent working alone (18), with G-CSF (3, 4, 10, 16, 19) or being added to a standard mobilization regimen (so called salvage mobilization therapy) (20-23). Our data indicate that only 47.5% collected the satisfactory number of CD34+ cells to proceed to autologous transplantation procedure. Discrepancies in apheresis schememight contribute to this outcome. In other studies plerixafor was given for up to 7 days and the apheresis was done up to seven times.

In the French study evaluating the effectiveness of stem cell collection in patients treated previously with lenalidomide or purine analogue containing regiments the minimum target number of CD34+ cells was collected from 69% of patients in a median of 2 days (24). Salvage mobilization was successful in the majority of patients previously treated with lenalidomide. In fludarabine-exposed patients, only 58% of patients will achieve successful salvage mobilization with plerixafor plus G-CSF, suggesting the need for novel mobilization regimens algorithms in this subgroup of patients (24).

We found that the proportion of CD34+ cells among total nucleated cells (NC) was significantly lower in the plerixafor group compared to the matched historic control group of patients mobilized in a standard way (0.22 vs 1.72, p <.004). In our setting, high nucleated cell count translated into high volume of stem cell product.

Potential side effects of plerixafor administration include: hematological effects (leukocytosis, thrombocytopenia), potential for tumor cell mobilization (plerixafor is contraindicated in leukemias), splenic enlargement and potential for rupture (only in prolonged administration or when overdosed). The patients may experience also some mild side effects (≥ 5%): nausea, diarrhea, fatigue, injection side reactions, headache, arthralgia vomiting, dizziness (1, 2).

Theoretically, plerixafor mobilizes mostly the stem cell fractions that are responsible for short-term reconstitution of hematopoiesis, which may result in late graft failure (9). We did not observe such an event within the study period. In 2011 The EMA-directed Plerixafor Safety Survey was introduced. The aim of the study is to monitor the outcome of patients receiving plerixafor-mobilized autologous stem cells and compare these to equivalent patients transplanted without the use of plerixafor. The CALM project was designed as a non-interventional European study aiming to include more than 5000 patients. Moreover, additional data will be collected when patients are mobilized with plerixafor outside the recognized indications (off-label). The main issue of this study is to evaluate the relapse rate after autologous transplantation with plerixafor-mobilized hematopoietic cells. The results will be probably available in the nearest future as the data collection is planned up to 2013.

The Polish CUP programme revealed that the median number of plerixafor ampoules used during one mobilization regimen was 1.8 (range 0.5-4.3) (11). This fact is worth noting due to high cost of this drug. Shaughnessy et al (25) proved that plerixafor/G-CSF and cyclophosphamide/G-CSF for upfront mobilization of CD34+ cells resulted in similar numbers of cells collected, costs of mobilization, and clinical outcomes. Additionally, plerixafor/G-CSF mobilization resulted in more predictable days of collection, no weekend apheresis procedures, and no unscheduled hospital admissions. On the contrary Li et al stated that the targeted use of plerixaforincreased the overall charges associated with stem cell collection in all patients by an average of 17% (21).

Stem cell mobilization with plerixafor and G-CSF provides solution for majority of patients requiring autologous hematopoietic stem cell transplantation and failing mobilization with G-CSF in combination with chemotherapy. However, due to high leukocytosis, this protocol may require modification of stem cell collection and freezing procedures in order to avoid large volumes. Further studies regarding higher number of patients and longer follow up period are needed to establish the relapse and survival rate.