Thirty-one patients, who underwent autologous peripheral blood stem cell transplantation (APBSCT), were randomized to receive GM-CSF (Molgramostim, Leucomax; Sandoz-Schering-Plough Laboratories, Paris, France) or not to receive it. The study was approved by the local ethic committee. Written informed consent was obtained from all patients.
Patients
The diagnoses of the enrolled patients were breast cancer (n=13), osteosarcoma (n=5), relapsed Hodgkin’s disease (n=5), relapsed non-Hodgkin’s lymphoma (n=4), relapsed testicular cancer (n=2), small cell lung cancer (n=1) and multiple myeloma (n=1). GM-CSF group consisted of 16 patients (10 female, 6 male; mean age of 36.8±12.4 years) and -GM-CSF group consisted of 15 patients (6 female and 9 male; mean age of 37.0±12.2 years). Four patients in the GM-CSF group and 5 patients in the -GM-CSF group received previous radiotherapy. One patient in the GM-CSF group and 2 in the -GM-CSF group underwent second transplant. Interval between diagnosis and transplantation (time to transplantation) was 452.5±446.1 days in the GM-CSF group and 930.3±151.5 days in the no GM-CSF group. The patients’ characteristics are shown in table 1.
Stem Cell Mobilization and Apheresis
Recombinant human G-CSF (Filgrastim, Neupogen, Roche, Basel, Switzerland) was given at a total dose of 10- 15 µg/kg/day with twice-daily s.c. injections, beginning fourteen days after the completion of the last cycle of induction chemotherapy and continuing until the collection of PBSC was completed. Using a continuous flow cell separator (COBE Spectra, Lakewood, CO, USA), the leukapheresis procedure was performed wit a three-way central catheter on days 4, 5 or 6. Each apheresis continued 4 to 6 hours every day. Single apheresis was sufficient for all but one patient [10]. Harvested autologous plasma was mixed with dimethylsulfoxide (DMSO) to yield a final DMSO concentration of 10%. The final suspension was transferred into freezing bag and frozen to -100 ºC using a computerized freezing device (R 201 Planar) and then stored in liquid nitrogen at -196 ºC following standard methods.
Flow Cytometry
CD34+ cells in the leukapheresis product were enumerated by flow cytometry (FACScan; Becton Dickinson, Heidelberg, Germany) using direct CD34 immunofluorescence. A minimum of 1×106 mononuclear cells was incubated for 30 minutes at 4 ºC with fluorescence conjugated to fluorescein isothiocyanate (Becton Dickinson, Heidelberg, Germany). The gated percentage of CD34+ cells was multiplied by the absolute MNC of the apheresis product to yield and absolute CD34+ cell count for each apheresis.
High-Dose Chemotherapy Regimens
Conditioning regimens included: CNV for breast cancer (n=13): Cyclophosphamide 2.4 g/m2, mitoxantrone 35 mg/m2, etoposide 250 mg/m2/d ×6 days; BEAM for Hodgkin’s and non-Hodgkin’s lymphoma (n=9): BCNU 300 mg/m2, etoposide 200 mg/m2/d ×4 days, Ara-C 200 mg/m2/d ×4 days, melphalan 140 mg/m2; ICE for testicular cancer, osteosarcoma, lung cancer (n=8): Ifosfamide 2.5 g/m2/d ×6 days, carboplatin 250 mg/m2/d ×6 days, etoposide 250 mg/m2/d x6 days; CEP for breast cancer with lung metastasis (n=1): Cyclophosphamide 60 mg/kg/d ×2 days, etoposide 200 mg/m2/d ×6 days, carboplatin 200 mg/m2/d
×6 days; L-PAM for multiple myeloma: Melphalan 140 mg/m2.
Stem Cell Transplantation and GM-CSF Administration
At the time of transplantation (day 0), stem cell bags were quickly thawed at bedside and immediately infused intravenously through a central catheter. After transplantation the patients were randomized in cytokine or no cytokine group. In the cytokine group, the patients received 5 µg/kg/day recombinant human GM-CSF (Molgramostim, Leucomax; Sandoz-Schering-Plough Laboratories, Paris, France) by intravenous route from day 7 until leukocyte counts exceeded 1×109/L for 3 consecutive days.
Evaluation of Posttransplant Reconstitution of Hematopoiesis
Leukocyte count greater than 1×109/L was documented as leukocyte engraftment, neutrophil count greater than 0.5×109/L as neutrophil engraftment, and platelet count grater than 50×109/L as platelet engraftment.
Posttransplant Supportive Therapy
Single donor thrombopheresis was performed as needed to keep the platelet number above 20×109/L. Erythrocyte transfusion was performed to keep the hemoglobin level above 8 g/dL. All blood products were irradiated (2500 cGy) and transfused via leukocyte filter. Fever was defined as any temperature elevation over 38.1 ºC or fever over 38.0 ºC lasting at least one hour. Posttransplant hospitalization duration was documented as the period elapsed from reinfusion to discharge.
Statistical Analysis
Statistical analysis was performed using a statistical software (SPSS for Windows, version 9.0, SPSS Inc., USA). Mann-Whitney U test was used for comparison of distribution of values for unpaired series such as age, number of patients enrolled, time to transplantation, number of previous chemotherapy cycles, number of total nucleated cells, number of CD34+ cells, time to leukocyte and platelet engraftment, number of febrile days, number of days with parenteral antibiotherapy, number of erythrocyte and platelet units transfused. Chi-square test was used for comparison between groups regarding gender, history of previous radiotherapy, preparative regimens and type of malignity. If the expected frequency in table cells was under 5 or total sample size was under 20, Fisher’s exact test was used. The p value was considered statistically significant if <0.05.
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