Mobilization of autologous peripheral blood stem cells by mitoxantone, arabinosylcytosin and granulocyte colonystimulating factor regimen and harvesting by large-volume leukapheresis / 中国组织工程研究

ABSTRACT

BACKGROUND: The primary qualification of peripheral blood stem cell transplantation (PBSCT) is the effective mobilization and harvesting of hematopoietic stem cells. The mobilization efficacy is closely related to the selection of high-efficacy low-toxicity regimen, the timing of mobilization and harvesting as well.OBJECTIVE: To investigate the efficacy of mitoxantone (MIT) combined with high-dose arabinosylcytosin (Ara-C),followed by granulocyte colony-stimulating factor (G-CSF) alone or combination of G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF) on mobilizing PBSCs in patients with hematological malignancies and solid tumors.DESIGN: Controlled study with observation.SETTTNG Department of Hematology, the Affiliated Hospital of Xuzhou Medical College.PARTICIPANTS: Forty-two patients with hematological malignancies and solid tumors admitted to Department of Hematology, Xuzhou Medical College from September 1998 to December 2006 were involved in this study. They were diagnosed according to FAB classification criteria and new WHO proposals. The involved patients, 25 male and 17 female, averaged 29 years ranging from 7 to 54 years and weighted (52±18) kg. Among them, 12 were patients with acute myeloblastic leukemia, 6 were patients with acute lymphoblastic leukemia (ALL), 1 was patient with chronic granulocytic leukemia (CGL) at chronic phase, 15 were patients with non-Hodgkin lymphoma (NHL), 4 were patients with Hodgkin lymphoma (HL), 2 was patient with multiple myeloma (MM), 2 were patients with advanced breast cancer. All the patients apprcached to or got complete remission after conventional chemotherapy. No tumor cell infiltration was observed in bone marrow cytological examination. The functions of the main organs such as heart, lung, liver and kidney,and so on, were normal. The patients underwent an average of 8-course chemotherapy before the mobilization. Informed consents of all the patients were obtained.METHODS: MIT was intravenously injected at 10 mg/(m2·d)for 2 to 3 days, then Ara-C was also intravenously injected at 2 g/m2 every 12 hours for 1 to 2 days. When white blood cell (WBC) count recovered from the lowest value, 5 to 7.5 μg/ (kg·d)G-CSF was applied in 20 patients for 3 to 5 days successively. And 5 to 7.5 μg/ (kg·d)G-CSF and 5 to 7 μ g/(kg·d)GM-CSF were applied in another 22 patients at 600 in the morning and in the evening, respectively. PBSCs harvesting started when WBC ＞ 2.5×109 L-1, especially when CD34+ cells≥ 1%,WBC was doubly increased. Autologous peripheral blood mononuclear cells (MNCs) were collected with CS3000 plus blood cell separator for detecting the level of CD34+ cells and T lymphocyte subsets. CFU-GM assays were performed in a methyl-cellulose-based clonogenic assay.① MNCs mixed with FITC-labeled CD34+, CD3 and CD8 monoclonal antibodies as well as CD4 PE-labeled CD monoclonal antibody at 4 ℃ for 30 minutes. 5×105 cells were determined, and CD3 and CD34+ levels, CD4/CD8 were determined by flow cytometer.Colony forming unit-granulocyte macrophage (CFU-GM) was determined with methyl cellulose. ② Related adverse reactions were observed after operation. ③ Aiming to different types of diseases,autologous PBSCs were back infused 36 to 48 hours after pre-disposal treatment. MNCs count and trypan-blue drying were done. Levels of CFU-GM and CD34+ cells were determined after unfreezing.MATN OUTCOME MEASURES: ① Changes in CD34+ cells and T lymphocyte subsets before and after mobilization. ② Postoperative related adverse reactions. ③ Back perfusion volume of autologous PBSCs (MNCs count, the number of CFU-GM and CD34+ cells).RESULTS: Forty-two involved patients participated in the final analysis. ① Changes in CD34+ cells and T lymphocyte subsets before and after mobilization Without using hematopoietic growth factors (HGF), the percentage of CD34+ cells in peripheral blood of the patients was (0.054±0.032)%. After using G-CSF/GM-CSF treatment, it was (1.82±0.76)%,which was obviously increased compared with that of without using HGF (P ＜ 0.001). The CD34+ cells and CFU-GM yields of 22 patients in C-CSF plus GM-CSF combination group [(8.76±3.39)×106/kg, (3.52±1.33)×105/kg, respectively]were significantly higher than those of 20 patients in G-CSF alone group [(6.12±2.11)×106/kg, (2.03±1.07)×105/kg,respectively (P ＜ 0.05)]. There were no obvious changes of T lymphocyte subsets in the patients when using G-CSF/GM-CSF for some days except that CD34+ cells increased gradually (P ＞ 0.05). ② Postoperative related adverse reactions Ⅱ to Ⅲ degree hair-loss was seen in all the patients. Blood platelets dropped to (54.43±26.14)×109 L-1 at different degrees. Infective fevers (37.8 ℃ to 41.0 ℃) occurred in 21 patients. But they were controlled in short term after antibiotics treatment. All the side effects of G-CSF and GM-CSF were mild and reversible, easily controlled with paracetamol or steroids. Bone pain (mainly in lumbosacral region) occurred in 13 patients when WBC went up quickly. ③ Back perfusion volume of autologous PBSCs PBSCs were cryopreserved at -80 ℃ without program control for 2.0 to 6.5 months. The cell recovery rate was (88.7±7.4) %. Trypan blue exclusion rate was (92.1±5.5) %. The back perfusion volume of MNCs, CD34+ cells and CFU-GM yields were (5.21±2.44)×108/kg, (6.89±3.55)×106/kg, (2.58±2.33)×105/kg,respectively. ④Circulation blood volume were 10 to 16 L (end-point separation blood volume were all above trebling TBV). Hematopoiesis was well reconstituted in 40 patients received autologous PBSCT.CONCLUSTON MIT and high-dose Ara-C chemotherapy combined with both G-CSF alone and G-CSF plus GM-CSF can safely and effectively mobilize autologous PBSCs, while G-CSF plus GM-CSF is superior to G-CSF alone.Large-volume leukapheresis is an important method to enhance the productive rate of stem cells and decrease the times of harvesting.