Peripheral blood progenitor cell mobilization using stem cell factor in combination with filgrastim in breast cancer patients

The safety and optimal dose and schedule of stem cell factor (SCF) administered in combination with filgrastim for the mobilization of peripheral blood progenitor cells (PBPCs) was determined in 215 patients with high-risk breast cancer. Patients received either filgrastim alone (10 microg/kg/d for 7 days) or the combination of 10 microg/kg/d filgrastim and 5 to 30 microg/kg/d SCF for either 7, 10, or 13 days. SCF patients were premedicated with antiallergy prophylaxis. Leukapheresis was performed on the final 3 days of cytokine therapy and, after high-dose chemotherapy and infusion of PBPCs, patients received 10 microg/kg/d filgrastim until absolute neutrophil count recovery. The median number of CD34+ cells collected was greater for patients receiving the combination of filgrastim and SCF, at doses greater than 10 microg/kg/d, than for those receiving filgrastim alone (7.7 v 3.2 x 10(6)/kg, P < .05). There were significantly (P < .05) more CD34+ cells harvested for the 20 microg/kg/d SCF (median, 7.9 x 10(6)/kg) and 25 microg/kg/d SCF (median, 13.6 x 10(6)/kg) 7-day combination groups than for the filgrastim alone patients (median, 3.2 x 10(6)/kg). The duration of administration of SCF and filgrastim (7, 10, or 13 days) did not significantly affect CD34+ cell yield. Treatment groups mobilized with filgrastim alone or with the cytokine combination had similar hematopoietic engraftment and overall survival after PBPC infusion. In conclusion, the results of this study indicate that SCF therapy enhances CD34+ cell yield and is associated with manageable levels of toxicity when combined with filgrastim for PBPC mobilization. The combination of 20 microg/kg/d SCF and 10 microg/kg/d filgrastim with daily apheresis beginning on day 5 was selected as the optimal dose and schedule for the mobilization of PBPCs.     

Granulocyte colony-stimulating factor (G-CSF) dose-dependent efficacy in peripheral blood stem cell mobilization in patients who had failed initial mobilization with chemotherapy and G-CSF

For 10 consecutive patients in our unit who did not show a significant rise in blood progenitor cells within 14 days following chemotherapy and G-CSF, we increased the G-CSF dose from 5 to 10 microg/kg/day (n = 9) or from 10 to 15 microg/kg/day (n = 1). As a result, there were significant increases in total yield as well as yield per apheresis of mononuclear cells, CD34+ cells and CFU-GM (P < 0.025, <0.01 and <0.005, respectively). After G-CSF dose escalation, six of the 10 patients had sufficient CD34+ cells for performing transplantation. These results demonstrate a dose-dependent response of progenitor cell mobilization by G-CSF when used in combination with chemotherapy. Moreover, increasing the dose of G-CSF as late as the third week of mobilization may still provide sufficient cell yield even with patients who did not show a significant mobilization with conventional doses of G-CSF.     

Hematopoietic progenitor cell collection and neoplastic cell contamination in breast cancer patients receiving chemotherapy plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization

BACKGROUND: We compared hematopoietic progenitor cell (HPC) collection and neoplastic cell contamination in breast cancer patients given cyclophosphamide (CTX) plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization. PATIENTS AND METHODS: In 57 stage II-III breast cancer patients, CD34+ cells, colony-forming units-granulocyte macrophage (CFU-GM), early HPC and breast cancer cells were counted in HPC collections obtained after CTX plus G-CSF (n = 27) or G-CSF-alone mobilization (n = 30). RESULTS: The CD34+ cell collection was about two-fold greater after CTX plus G-CSF mobilization (11.0 +/- 7.9 vs. 5.8 +/- 3.5 x 10(6)/kg, P < 0.001). Similarly, the total number of CFU-GM, CD34+CD38- cells and of week-5 cobblestone area forming cells (CAFC) collected was significantly higher in patients mobilized with CTX plus G-CSF. Breast cancer cells were found in the apheresis products of 22% of patients mobilized with CTX plus G-CSF and in 10% of patients mobilized with G-CSF alone (P = 0.36). Of seven patients who failed G-CSF-alone mobilization and eventually underwent chemotherapy plus G-CSF mobilization, none had cytokeratin-positive cells after G-CSF mobilization, whereas four out of seven had cytokeratin-positive cells after chemotherapy plus G-CSF (P = 0.07 by chi 2 test). CONCLUSION: The CTX plus G-CSF mobilization protocol was associated with a significantly higher HPC collection. However, this benefit was not accompanied by a reduction in the incidence of tumor-contaminated HPC graft.     

Structures of the van der Waals Isomers of Halosulfuric Acids: Microwave Spectra of HX-SO3 (X = F, Cl, Br)

The effects of different doses of filgrastim on yields of CD34+ peripheral blood stem cells were evaluated in patients with breast cancer. 55 were randomized to receive filgrastim 10, 20, 30 or 40 microg/kg/d with more CD34+ cells/kg/apheresis harvested after the three highest dose levels. 35 additional patients were randomized to receive 10 or 30 microg/ kg. The median number of CD34+ cells collected after 10 microg/ kg (n = 31) was 0.7 x 10(6)/kg/apheresis (range 0.1-4.4) as compared to 1.2 (range 0.1-6.8) after 30 microg/kg (n = 32) (P = 0.04). Among patients randomized to 10 v 30 microg/kg, more (50%) achieved > or = 5.0 x 10(6) CD34+ cells/kg and less aphereses were required to achieve > or = 2.5 x 10(6) CD34+ cells/kg after the higher dose (P = 0.04). In multivariate analyses, patients receiving 10 microg/kg (n = 31) had lower yields of CD34+ cells (P = 0.026) and had a 3.3-fold increase in the probability of not achieving > or = 5.0 x 10(6) CD34+ cells/kg as compared to patients receiving 20-40 microg/kg (n = 59). Patients who had received radiation had a 2.9-fold probability of not achieving > or = 2.5 x 10(6) CD34+ cells/kg. These data suggest that, in patients with good marrow reserves, doses of filgrastim > 10 microg/kg/d mobilized more CD34+ cells and may be useful when high numbers of CD34+ cells are desired.     

Mobilization of peripheral blood stem cells in children using G-CSF after carboplatin containing myelosuppressive chemotherapy

PURPOSE: Peripheral blood stem cell (PBSC) apheresis provides an alternative to autologous marrow harvest as a source of hematologic stem cells for transplantation in children with solid tumors. PATIENTS AND METHODS: Eight children with metastatic or recurrent solid tumors underwent 27 apheresis procedures. Recovery from myelosuppressive chemotherapy occurred without continuous daily growth factor support prior to mobilization. Granulocyte colony stimulating factor (G-CSF) at 16 microgs/kg/day was used to increase stem cells in the peripheral circulation. CD 34 positive cells, mononuclear cells (MNC), and CFU-GM were measured in the apheresis products. Prior chemotherapy was examined as a clinical factor that affected PBSC yield. RESULTS: A significant correlation was found between CD 34+/kg and CFU-GM/kg of the products (r = 0.758, P < 0.001). Patients receiving cumulative doses of carboplatin over 1,600 mg/m2 produced adequate MNC (1 x 10(8)/kg) but yielded significantly less CD 34+ cells or CFU-GM than those patients receiving less carboplatin. Prior doses of etoposide and ifosfamide did not effect PBSC yield. CONCLUSIONS: The mobilization technique was well tolerated, and the products obtained produced trilineage engraftment in the patients that underwent peripheral blood stem cell transplantation. Peripheral blood stem cell apheresis in children can be optimized by selection of appropriate candidates and mobilization with G-CSF after an absence of hematopoietic growth factor support.     

Giant cell tumor of bone with selective metastases to mediastinal lymph nodes

We examined the efficiency of disease-specific "standard" chemotherapies epirubicin, cyclophosphamide (EC); cyclophosphamide, vincristine, doxorubicin, etoposide, prednisolone (CHOEP); epirubicin, ifosfamide (EPI/IFOS) for peripheral blood progenitor cell (PBPC) mobilization in comparison to well-characterized mobilization protocols, i.e. etoposide, ifosfamide, cisplatin, epirubicin (VIPE) and dexamethasone, carmustine, etoposide, cytarabine, melphalan (DexaBEAM). Twenty-seven patients with various malignancies underwent 75 apheresis procedures for PBPC collection. Median cell yields from all 75 aphereses were 1.18 x 10(5) mononuclear cells/kg [range (0.28-3.7) x 10)8)], 1.4 x 10(5) granulocyte/macrophage-colony-forming units (CFU-GM)/kg [range (0.2-11) x 10(5)] and 3.3 x 10(6) CD34+cells/kg [range (0.35-17.7) x 10(6). CD34+/ CD90+ cells could be mobilized by all mobilization regimens used. The difference observed in the mobilization of CD34+ cells was only of low significance when the mobilization regimens were compared, whereas the mobilizations of MNC and CFU-GM were significantly different between the groups. Breast cancer patients treated with the VIPE regimen (including pretreated women) had a significantly higher CFU-GM rate than patients treated with EC (P=0.0005). Mobilized CD34+ PBPC were correlated with CFU-GM in all apheresis products. The linear correlation coefficients differed for the various mobilization groups: DexaBEAM (r=0.9, P < 0.0001), VIPE (r=0.68, P=0.0024), CHOEP (r=0.52, P=0.022), EPI/ IFOS (r=0.34, P=0.11) and EC (r=0.23, P=0.2). We conclude that clonogenic assays can provide additional information about the autotransplant quality, particularly when alternative or new mobilization regimens are being investigated.     

Mobilization of peripheral blood progenitor cells by disease-specific chemotherapy in patients with soft tissue sarcoma

We investigated peripheral blood progenitor cell (PBPC) mobilization by disease-specific chemotherapy in patients with metastatic soft tissue sarcoma (STS). Nine patients, five females and four males, aged 12-51 years, pretreated by one to nine courses of cytotoxic chemotherapy, underwent STS-specific mobilization followed by G-CSF at 5 microg/kg/day. PBPC were collected by 19 conventional-volume aphereses (8-12 l) with one to four procedures in individual patients. Leukaphereses started on median day 15 (range 13-18) from the first day of mobilization chemotherapy at medians of 25.8 x 10(3) WBC/microl (6.8-46.9), 3.5 x 10(3) MNC/microl (1.1-8.8), 122 x 10(3) platelets/microl (72-293) and 30.7 CD34+ cells/microl (6.7-207.8). Cumulative harvests resulted in medians of 4.6 x 10(8) MNC/kg (3.0-6.4), 2.9 x 10(6) CD34+ cells/kg (1.1-11.1) and 12.0 x 10(4) CFU-GM/kg (2.0-37.8). Eight patients underwent high-dose chemotherapy (HDCT) followed by PBPC rescue. Seven patients recovered hematopoiesis at medians of 12 days (8-15) for ANC >0.5 x 10(3)/microl and 14 days (8-27) for platelets >20 x 10(3)/microl. One patient, who received 1.6 x 10(6) CD34+ cells/kg, exhibited delayed ANC recovery on day +37 and failed to recover platelets until hospital discharge on day +55. We conclude that in patients with metastatic STS, who are pretreated by standard chemotherapy, PBPC can be mobilized by a further course of STS-specific chemotherapy plus G-CSF. One to four conventional-volume aphereses result in PBPC autografts that can serve as hematopoietic rescue for patients scheduled for HDCT.     

Composition and function of peripheral blood stem and progenitor cell harvests from patients with severe active rheumatoid arthritis

High-dose chemotherapy with autologous stem cell rescue has been proposed as an intensive therapy for severe rheumatoid arthritis (RA). In view of previous observations of abnormal haemopoiesis in RA patients, the composition and function of peripheral blood stem cell harvests (PBSCH) was investigated. Compared with PBSCH from healthy allogeneic donors mobilized with the same dose of G-CSF (filgrastim; 10 microg/kg/d, n = 14), RA PBSCH (n = 9) contained significantly fewer mononuclear cells (375 v 569 x 10(6)/kg, P = 0.03) and CD34+ cells (2.7 v 5.8 x 10(6)/kg, P = 0.003). However, there were increased proportions of CD14+ cells (P = 0.006) and CD14+ CD15+ cells (the phenotype of previously described 'abnormal' myeloid cells, P = 0.002) in the RA PBSCH which translated into 3.5- and 7-fold increases respectively on a per CD34+ cell basis. There were no differences in T-cell activation status as judged by proportions of CD4+ and CD8+ expressing CD45RA, CD45RO, HLA-DR and CD28 (RA PBSCH, n = 7, donor PBSCH, n = 5, P = 0.2-0.7). Phytohaemagglutinin responses determined fluorocytometrically with induction of CD69 expression were reduced in CD4+ and CD8+ cells following filgrastim administration in 3/3 RA patients tested. Compared with bone marrow as a potential source of CD34+ cells, PBSCH contained 11-fold more T cells (P < 0.0005), 8-fold more B cells (P < 0.0005) and 4-fold more monocytes (P = 0.02). In short-term methylcellulose culture there were no differences in colony counts (CFU-GM, CFU-GEMM, BFU-E) per CD34+ cell from PBSCH from RA patients (n = 11) and healthy donors (n = 10). Long-term culture initiator cells were cultured successfully from cryopreserved PBSCH from RA patients (n = 9). In conclusion, PBSCH from RA patients differed significantly in composition from normal individuals, but in vitro studies support normal stem and progenitor cell function. Changes in T-cell function occur during mobilization in RA patients. This work provides reassurance for the use of PBSCH as haematological rescue and baseline data for clinical trials of graft manipulation strategies in patients with RA.     

2-Hydroxyisonicotinate dehydrogenase isolated from Mycobacterium sp. INA1

The objective of this study was to identify factors associated with poor mobilization of peripheral blood progenitor cells (PBPCs) or delayed platelet engraftment after high-dose therapy and autologous stem cell transplantation in patients with lymphoma. Fifty-eight patients with Hodgkin's disease or non-Hodgkin's lymphoma underwent PBPC transplantation as the "best available therapy" at Memorial Sloan-Kettering Cancer Center (New York, NY) between 1993 and 1995. PBPCs were mobilized with either granulocyte colony-stimulating factor (G-CSF) alone (n = 19) or G-CSF following combination chemotherapy (n = 39). Forty-eight of these patients underwent a PBPC transplant, receiving a conditioning regimen containing cyclophosphamide, etoposide, and either total body irradiation, total lymphoid irradiation, or carmustine. A median number of 4.6 x 10(6) CD34+ cells/kg were obtained with a median of three leukapheresis procedures. Mobilization of PBPCs using chemotherapy plus G-CSF was superior to G-CSF alone (6.7 x 10(6) versus 1.5 x 10(6) CD34+ cells/kg; P = 0.0002). Poorer mobilization of progenitor cells was observed in patients who had previously received stem cell-toxic chemotherapy, including (a) nitrogen mustard, procarbazine, melphalan, carmustine or > 7.5 g of cytarabine chemotherapy premobilization (2.0 x 10(6) versus 6.0 x 10(6) CD34+ cells/kg; P = 0.005), or (b) > or = 11 cycles of any previous chemotherapy (2.6 x 10(6) versus 6.7 x 10(6) CD34+ cells/kg; P = 0.02). Platelet recovery to > 20,000/microliter was delayed in patients who received < 2.0 x 10(6) CD34+ cells (median, 13 versus 22 days; P = 0.06). Patients who received > or = 11 cycles of chemotherapy prior to PBPC mobilization tended to have delayed platelet recovery to > 20,000/microliter and to require more platelet transfusions than less extensively pretreated patients (median, 13.5 versus 23.5 days; P = 0.15; median number of platelet transfusion episodes, 13 versus 9; P = 0.17). These data suggest that current strategies to mobilize PBPCs may be suboptimal in patients who have received either stem cell-toxic chemotherapy or > or = 11 cycles of chemotherapy prior to PBPC mobilization. Alternative approaches, such as ex vivo expansion or the use of other growth factors in addition to G-CSF, may improve mobilization of progenitor cells for PBPC transplantation.     

Synergy of growth factors during mobilization of peripheral blood precursor cells with recombinant human Flt3-ligand and granulocyte colony-stimulating factor in rabbits

The potential of recombinant human (rh)Flt3 ligand (FL), alone or in combination with other recombinant growth factors, to mobilize peripheral blood precursor cells (PBPCs) was examined in an animal model. Adult outbred New Zealand White rabbits received subcutaneous injections daily for 14 days in a standardized protocol; whole blood cell counts and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies were measured 3 times weekly during the injection period and for an additional observation period of 14 days. Two animals in each group were treated as follows: 200 or 500 microg/kg FL, 10 microg/kg granulocyte colony-stimulating factor (G-CSF), 10 or 75 microg/kg stem cell factor (SCF), 10 microg/kg G-CSF + 500 microg/kg FL, 10 microg/kg G-CSF + 75 microg/kg SCF + 500 microg/kg FL. Both G-CSF and FL induced a sustained and dose-dependent increase in the leukocyte count to a maximum of 5-fold. They were additive in combination, leading to a tenfold increase in white blood cell counts. No consistent pattern was observed for platelet counts or red blood cells. No toxic side effects were seen. Both G-CSF and FL mobilized CFU-GM in a dose-dependent fashion to a 59-fold increase for G-CSF and 116-fold for FL. Maximum mobilization occurred on day 4 with G-CSF and on day 11 with FL. G-CSF + FL in combination acted synergistically, inducing a 503-fold increase of CFU-GM over baseline. The addition of SCF to this combination did not alter leukocyte counts or CFU-GM mobilization. Our results indicate that FL is a potent and safe agent for the mobilization of PB-PCs and is synergistic with G-CSF.     

Combination chemotherapy with mitoguazon, ifosfamide, MTX, etoposide (MIME) and G-CSF can efficiently mobilize PBPC in patients with Hodgkin's and non-Hodgkin's lymphoma

Many centers use CY and G-CSF to mobilize PBPC. In this study we explored whether a standard chemotherapy regimen consisting of mitoguazon, ifosfamide, MTX and etoposide (MIME) combined with G-CSF was capable of mobilizing PBPC in lymphoma patients. Twelve patients with Hodgkin's disease (HD) and 38 patients with non-Hodgkin's lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3 to 20) of chemotherapy prior to mobilization. It was found that the optimal time of PBPC harvest was at days 12 and 13 after initiating the mobilization regimen. The median number of collected CD34+ cells per kg body weight was 7.1 x 10(6) (range 0.5-26.2). More than 2.0 x 10(6) CD34+ cells/kg were achieved in 69% of the patients after one apheresis. When additional cycles of apheresis were done, only 6% failed to harvest this number of CD34+ cells. There was a statistically significant inverse correlation between the number of prior chemotherapy cycles and CD34+ cell yield (P = 0.003). No such association was found between CD34+ cell yield and prior radiotherapy. When MIME/G-CSF was compared with Dexa-BEAM/G-CSF, it was found that MIME/G-CSF tended to be more efficient in mobilizing PBPC in spite of being less myelotoxic. All patients transplanted with MIME/G-CSF mobilized PBPC had fast and sustained engraftment. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF can be successfully used to mobilize PBPC.     

Efficiency of high-dose FEC chemotherapy for the mobilization of hematopoietic stem cells into peripheral blood

The best regimen for mobilizing hematopoietic stem cells (HSC) into peripheral blood is not yet defined. The efficiency of FEC chemotherapy in the treatment of breast cancer is well established and the effects of FEC on HSC mobilization have been characterized. We tested the feasibility and the toxicity of a high-dose FEC regimen which may improve the mobilizing capacity of conventional FEC. Twenty patients with poor prognosis breast cancer received high-dose FEC and filgrastim 5 micrograms/kg. Three leukaphereses were performed on each patient for 3 consecutive days. Total numbers of CFU-GM and CD34+ cells were assessed, and a retrospective analysis was made. The numbers of CFU-GM/kg and CD34+ cells/kg collected (mean +/- standard error) were respectively 12.2 x 10(5) (+/- 2.4) and 14.6 x 10(6) (+/- 2.5). Extra-hematologic toxicity was negligible. Hematologic recovery after CTCb high-dose chemotherapy and HSC infusion was rapid. High-dose FEC is efficient for collecting HSC in peripheral blood. Extra-hematologic toxicity is weak and hematologic recovery after autograft is normal. Increased dosage of epirubicin and cyclophosphamide could allow a single leukapheresis collection of sufficient HSC from peripheral blood.     

Factors affecting progenitor cell yields using three tandem leukaphereses in previously treated malignancies

BACKGROUND: Mobilized peripheral blood progenitor cells (PBPCs) have increasingly been used to replace autologous bone marrow to allow faster hematopoietic reconstitution after myeloablative therapy in various malignancies. There is a paucity of data concerning factors that affect the total yield of three tandem leukaphereses. METHODS: Factors affecting the yield of PBPCs were analyzed in a series of 121 consecutive patients including 36 with non-Hodgkin's lymphoma, two with Hodgkin's disease, four with multiple myeloma, 44 with acute leukemia, 20 with breast cancer and 15 with other solid tumors. PBPCs were mobilized using granulocyte-colony-stimulating factor (G-CSF) alone (group I, n = 15), or after conventional-dose (group II, n = 70) or high-dose (group III, n = 36) chemotherapy followed by G-CSF. The total yield of three tandem PBPC collections for each patient was assessed by the number of mononuclear cells (MNCs), CD34+ cells and colony-forming units of granulocyte macrophages (CFU-GM). The factors evaluated included age, sex, diagnosis, history of marrow involvement, previous radiotherapy, the number of prior chemotherapy cycles and mobilization method. The two -sample t-test and logistic regression analysis were performed for univariate and multivariate analysis, respectively. RESULTS: With univariate analysis, a diagnosis of acute leukemia, positive history of bone marrow involvement, more chemotherapy cycles and mobilization with high-dose chemotherapy adversely affected the yields of CD34+ cells. By multivariate analysis, Group II had higher yields of MNCs (p = 0.039), CFU-GM (p = 0.002) and CD34+ cells (p = 0.011) than Group III. Fewer cycles of prior chemotherapy is the common favorable factor for the yields of both CD34+ cells (p = 0.016) and CFU-GM (p = 0.017). CONCLUSIONS: The number of prior chemotherapy cycles adversely affects progenitor cell yield. Conventional-dose chemotherapy followed by G-CSF seems to be the mobilization methods of choice for heavily pretreated cancer patients with limited bone marrow reserve. PBPCs should be harvested early, when the tumor burden is less, to avoid cumulative marrow toxicity from chemotherapy.     

Inhibition of nitric oxide modulates the effect of oral arginine supplementation in acute liver injury

We investigated the efficacy of bone marrow (BM) processing by an automated large-volume apheresis procedure (6 x original BM volume) in 10 paediatric and adult patients undergoing BM harvesting before myeloablative therapy. Volume-dependent kinetics during apheresis were analyzed by sequential collection of processed cells into a six-fold collection bag system with consecutive analysis of the single bags. BM processing resulted in an 83.3% (+/- 21) recovery of mononuclear cells (MNC), a 97.9% (+/- 1.1) reduction of erythrocytes (RBC) and a 87.7% (+/- 2.9) volume reduction. To determine volume-dependent kinetics of haematopoietic progenitor cell (HPC) enrichment during apheresis, leukocytes (WBC), mononuclear cells (MNC), CD34 cells and colony-forming cells (CFU-GM) were serially quantitated in subsequent collection bags. Large-volume BM processing significantly enhanced absolute yields of CD34+ cells (mean: 4.01 (+/- 2.81) x 10(6)/kg bw) and CFU-GM (mean: 1.92 (+/- 1.47) x 10(4)/kg bw) compared with the standard procedure (3 x BM volume) by 26.9% (+/- 10.9) and 27.2% (+/- 11.6), respectively. We concluded that large-volume apheresis for BM processing is an efficient technique significantly improving the yields of haematopoietic progenitor cells (HPC) without any relevant changes in the purity of the final product. Moreover, sequential collection and analysis of HPC represents a good model to investigate the volume-dependent kinetics and efficacy of BM processing.     

Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34+ hematopoietic progenitor cells

PURPOSE: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. METHODS AND MATERIALS: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34+ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34+ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. RESULTS: The radiobiological parameters (D[0] and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34+ cells compared to optimal two-factor combinations. The D[0] values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n=1 for all tested GF(s). DNA repair capacity of CD34+ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34+ cells was similar. CONCLUSION: Our data indicate that increased survival of irradiated CB CD34+ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.     

The potential role of FLT3 ligand in progenitor and primitive hematopoietic cell expansion

We have previously defined the experimental conditions for hematopoietic cell expansion. CD34+ human marrow cells were maintained in a serum-free, stroma-free liquid culture system, at a concentration of 10(3) cells/ml, for 10 days at 37 degrees C, in the presence of various cytokine combinations. The basic combination of early cytokines SCF (100 ng/ml), IL3 (5 ng/ml), IL6 (10 ng/ml), has a modest stimulating effect on all compartments: the number of total cells increased 56-fold and CD34+ cells 1-fold; CFU-GM, BFU-E and CFU-MK, increased 6-fold, 5-fold and 3-fold respectively. As far as CD34+ cells are concerned, the subpopulation CD34+/CD38- was only maintained. Interestingly, the addition of 100 ng/ml of Flt3 ligand (FL) significantly enhanced the amplification of total cells (276-fold), CFU-GM (54-fold) and BFU-E (15-fold). The number of CD34+ cells and the subpopulation CD34+/38- increased to 7-fold and 22-fold respectively. Moreover, long term culture-initiating cells (LTC-ICs) in limiting dilution assay (LDA) were found to increase 3-fold. Further addition of MGDF (10 ng/ml), G-CSF (10 ng/ml) and Epo (0.5 U/ml), in various combinations, acted synergically with the previous cytokine combination to support the formation of multiple types of hematopoietic colonies. As expected, the addition of MGDF increased the number of CFU-MK up to 5-fold expansion. Interestingly, MGDF addition was synergistic also for BFU-E and CFU-GM expansion. In the combination of SCF+ IL3+ IL6+ FL + MGDF, CFU-GM expanded to 73-fold and BFU-E to 17-fold. G-CSF in SCF + IL3 + IL6 + FL conditions stressed the expansion of the granulopoietic compartment doubling the number of CFU-GM and CD33+ cells, with no consequence on LTC-IC or BFU-E. Surprisingly, G-CSF induced the expansion of the megakaryocytic lineage up to 6-fold, in a similar way as MGDF. Epo in presence of SCF+ IL3+ IL6+/-FL dramatically increased total cell expansion (2300-2800-fold), mainly erythroblastic (70% glycoA) without exhaustion of all other compartments. The simultaneous use of these three cytokines (MGDF + G-CSF + Epo) in presence of four early cytokines (SCF + IL3 + IL6 + FL) clearly allows a significant expansion of all hematopoietic compartments, precursors, progenitors, and primitive stem cells. In conclusion, these data show the ability of a stroma-free, serum-free liquid system to expand all myeloid lineages, including CFU-MK and LTC-IC which are critical for clinical application of ex vivo expanded cells.     

Recombinant human granulocyte and granulocyte-macrophage colony-stimulating factor (G-CSF and GM-CSF) administered following cytotoxic chemotherapy have a similar ability to mobilize peripheral blood stem cells

The availability of hematopoietic growth factors has greatly facilitated the mobilization and collection of peripheral blood stem cells (PBSC). It was the aim of this double-blind study to compare the PBSC-mobilizing efficacy of recombinant human G-CSF and GM-CSF when administered post-chemotherapy. Twenty-six patients with relapsed Hodgkin's disease were included in the study. Their median age was 31 years (range, 22-59) and 14 patients were males and 12 were females. Patients were pretreated with a median of eight cycles of cytotoxic chemotherapy, while 18 patients had undergone extended field irradiation. The patients received dexamethasone 24 mg days 1-7, melphalan 30 mg/m2 day 3, BCNU 60 mg/m2 day 3, etoposide 75 mg/m2 days 4-7, Ara-C 100 mg/m2 twice daily days 4-7 (Dexa-BEAM). Twelve patients were randomized to receive 5/microg/kg/day G-CSF and 14 patients to receive 5 microg/kg/day GM-CSF, both administered subcutaneously starting on day 1 after the end of Dexa-BEAM. Primary endpoints of the study were the number of CD34+ cells harvested per kg body weight on the occasion of six consecutive leukaphereses and the time needed for hematological reconstitution following autografting. Twenty-one patients completed PBSC collection, and six patients of the G-CSF group and nine of the GM-CSF group were autografted. No difference was observed with respect to the median yield of CFU-GM and CD34+ cells: 32.5 x 10(4)/kg vs 31.3 x 10(4)/kg CFU-GM, and 7.6 x 10(6)/kg vs 5.6 x 10(6)/kg CD34+ cells, for G-CSF and GM-CSF, respectively (U test, P= 0.837 and 0.696). High-dose chemotherapy consisted of cyclophosphamide 1.7 g/m2 days 1-4, BCNU 150 mg/m2 days 1-4, etoposide 400 mg/m2 days 1-4. All patients transplanted with more than 5 x 10(6) CD34+ cells/kg had a rapid platelet recovery (20 x 10(9)/l) between 6 and 11 days and neutrophil recovery (0.5 x 10(9)/1) between 9 and 16 days, while patients transplanted with less than 5 x 10(6)/kg had a delayed reconstitution, regardless of the kind of growth factor used for PBSC mobilization. In conclusion, our data indicate that in patients with Hodgkin's disease G-CSF and GM-CSF given after salvage chemotherapy appear to be not different in their ability to mobilize PBSC resulting in a similar time needed for hematological reconstitution when autografted following high-dose therapy.     

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33-CD34+ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33-CD34+ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (Kir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31% of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCF. Kir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs+ or Ba2+ Antisense oligodeoxynucleotides directed against Kir blocked both mRNA and functional expression of Kir channels. Kir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33-CD34+ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs+ or Ba2+ induced a similar degree of inhibition (40-60%) of progenitor cell generation. These findings strongly suggest an essential role for Kir in the process of cytokine-induced primitive progenitor cell growth and differentiation.     

Pulsed field agarose gel electrophoresis in the study of morphogenesis: packaging of double-stranded DNA in the capsids of bacteriophages

Circulating hemopoietic progenitors were evaluated in 19 multiple myeloma patients at diagnosis. Eleven patients received either high-dose cyclophosphamide (7 g/m2, 8 patients) or etoposide (2 g/m2, 3 patients) followed by GM-CSF administration; the remaining 8 patients received intermediate-dose cyclophosphamide (1.2 g/m2 on days 1 and 3), 4 of them with GM-CSF support. The highest levels of circulating progenitor cells were observed among patients in the high-dose chemotherapy group (median CFU-GM peak value of 6432 per ml), while in patients receiving intermediate-dose, with or without GM-CSF, median peak values were 2588 and 462 per ml, respectively. In all groups a remarkable heterogeneity in the yield of circulating progenitors was observed; this was particularly pronounced in the high-dose group, where CFU-GM peak values ranged between 200 and 38,070 per ml. At variance with the effect observed in previously untreated patients with lymphoma or breast cancer, the degree of mobilization in myeloma patients was rather unpredictable. The only pre-treatment characteristic correlating to some extent with a poor expansion of the circulating progenitor pool was heavy BM infiltration with plasma cells. The mobilizing effect was not restricted to the myeloid lineage, as demonstrated by the rise of BFU-E; CD34+ cells were increased as well. Indeed, a simultaneous evaluation of CFU-GM and CD34+ cells was carried out and a highly significant correlation (r = 0.9) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of prechemotherapy filgrastim administration on late hematopoietic progenitor cells

Administration of filgrastim influences the proliferative kinetics of myeloid progenitor cells. Even after it is discontinued, increased levels of cycling of granulocyte precursors are sustained for approximately 4 days. Beginning chemotherapy during this period of enhanced marrow activity can cause great damage to late, and possibly early, progenitor cell pools. Peripheral blood samples were collected from two research study patients who were prospectively randomized to receive filgrastim by different schedules after chemotherapy. The mononuclear cell fraction was analyzed by clonogenic progenitor cell assay and flow cytometry. Ex vivo and clinical findings were correlated with filgrastim and chemotherapy administration times. Although quantitative recovery of circulating neutrophils occurred, a substantial decrease in peripheral blood progenitor cells was observed when chemotherapy was started 72 hours after cessation of filgrastim therapy. Neutrophil recovery alone is not a precise index of short-term marrow granulocyte progenitor status. Starting chemotherapy within 72 hours of filgrastim therapy is biologically, and possibly, clinically relevant.