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.     

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.     

Randomized comparison of progenitor-cell mobilization using chemotherapy, stem-cell factor, and filgrastim or chemotherapy plus filgrastim alone in patients with ovarian cancer

PURPOSE: This was the first randomized study to investigate the efficacy of peripheral-blood progenitor cell (PBPC) mobilization using stem-cell factor (SCF) in combination with filgrastim (G-CSF) following chemotherapy compared with filgrastim alone following chemotherapy. PATIENTS AND METHODS: Forty-eight patients with ovarian cancer were treated with cyclophosphamide and randomized to receive filgrastim 5 microg/kg alone or filgrastim 5 microg/kg plus SCF. The dose of SCF was cohort-dependent (5, 10, 15, and 20 microg/kg), with 12 patients in each cohort, nine of whom received SCF plus filgrastim and the remaining three patients who received filgrastim alone. On recovery from the WBC nadir, patients underwent a single apheresis. RESULTS: SCF in combination with filgrastim following chemotherapy enhanced the mobilization of progenitor cells compared with that produced by filgrastim alone following chemotherapy. This enhancement was dose-dependent for colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and CD34+ cells in both the peripheral blood and apheresis product. In the apheresis product, threefold to fivefold increases in median CD34+ and progenitor cell yields were obtained in patients treated with SCF 20 microg/kg plus filgrastim compared with yields obtained in patients treated with filgrastim alone. Peripheral blood values of CFU-GM, BFU-E, and CD34+ cells per milliliter remained above defined threshold levels longer with higher doses of SCF. The higher doses of SCF offer a greater window of opportunity in which to perform the apheresis to achieve high yields. CONCLUSION: SCF (15 or 20 microg/kg) in combination with filgrastim following chemotherapy is an effective way of increasing progenitor cell yields compared with filgrastim alone following chemotherapy.     

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.     

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.     

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.     

Detection of neuroblastoma cells in CD34+ selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry

A sensitive assay was developed for the detection of neuroblastoma cell contamination in CD34+ selected and unseparated peripheral blood stem cells (PBSC) used for autologous transplantation in stage 4 neuroblastoma patients. Specifically, we established a non-radioactive nested cDNA-PCR (nPCR) for detection of tyrosine hydroxylase (TH) gene expression combined with anti-disialoganglioside GD2 immunocytochemistry with the murine monoclonal antibody (MAb) 14G2a. Sensitivities of TH nPCR determined with a number of neuroblastoma cell lines and PBSCs correlated to cell line dependent basal TH gene expression levels and ranged from 1:10(4) to 1:10(6). The sensitivity obtained by immunocytochemistry was 1:10(5). We observed the highest PBSC contamination rate of 47% (18/38) among 38 PBSC specimens exclusively obtained from stage 4 neuroblastoma patients by using TH nPCR and GD2 immunocytochemistry in combination. Furthermore, a clinically applied purging method, CD34+ selection by immunoabsorption (CD34+ purity 42.4%), was used on 16 PBSCs. 10/16 (63%) preparations were contaminated prior to CD34+ selection and 56% (9/16) remained contaminated. A significant reduction of neuroblastoma cell contamination by CD34+ selection was not detectable, but the absolute amount of re-infused tumour cells was decreased due to 100-fold smaller cell counts of CD34+ selected grafts used for transplantation. 22 PBSC preparations were used for transplantation. A Kaplan-Meier analysis showed an event-free survival probability of 0.56 +/- 0.22 (n = 9) in the group with contaminated PBSCs versus 0.88 +/- 0.12 (n = 8) with no detectable neuroblastoma-cell contamination. Our data suggest that the combined use of TH nPCR and GD2 immunocytochemistry is optimal to detect contamination and monitor purging strategies.     

Collection of peripheral blood progenitor cells after the administration of cyclophosphamide, etoposide, and granulocyte-colony-stimulating factor: an analysis of 497 patients

BACKGROUND: There is great interpatient variability in the number of peripheral blood stem cells collected, as measured by CD34+ cell content, after the administration of chemotherapy and a growth factor. The ability to predict patients who fail to yield adequate quantities of CD34+ cells would be of value. However, very few reports include large numbers of patients treated in an identical fashion. STUDY DESIGN AND METHODS: Between 1991 and 1995, 497 consecutive patients with a variety of malignant diseases received cyclophosphamide (4 g/m2), etoposide (600 mg/m2), and granulocyte-colony-stimulating factor (6 micrograms/kg/day) for mobilization and collection of a target dose > or = 2.5 x 10(8) CD34+ cells per kg. Multivariate analyses were performed to determine the factors associated with failure to achieve this target harvest. RESULTS: A median of 14.71 x 10(6) CD34+ cells per kg (range, 0.08-137.55) was harvested with a median of 2 (range, 1-11) apheresis procedures. Ninety-one percent of patients yielded > or = 2.5 x 10(5) CD34+ cells per kg. Patients with Stage II-III breast cancer, who had pretreatment platelet counts > or = 150 x 10(9) per L and patients who underwent < or = 1 prior chemotherapy regimen had improved CD34+ cell yields. However, most patients with adverse risk factors yielded > or = 2.5 x 10(6) CD34+ cells per kg. CONCLUSION: A regimen of cyclophosphamide, etoposide, and granulocyte-colony-stimulating factor led to the successful collection of adequate numbers of CD34+ cells in most patients without excessive toxicity. These observations confirm previous reports that intense prior therapy adversely affects the quantity of CD34+ cells harvested. Pretreatment and posttreatment variables did not predict with any certainty the small fraction of patients who fail to yield > or = 2.5 x 10(6) CD34+ cells per kg via multiple apheresis procedures.     

Successful collection of peripheral blood progenitor cells in patients with acute myeloid leukaemia following early consolidation therapy with granulocyte colony-stimulating factor-supported high-dose cytarabine and mitoxantrone

We evaluated the feasibility of collecting peripheral blood progenitor cells (PBPC) in patients with acute myeloid leukaemia (AML) following two cycles of induction chemotherapy with idarubicin, cytarabine and etoposide (ICE), and one cycle of consolidation therapy with high-dose cytarabine and mitoxantrone (HAM). Thirty-six patients of the multicentre treatment trial AML HD93 were enrolled in this study, and a sufficient number of PBPC was harvested in 30 (83%). Individual peak concentrations of CD34+ cells in the blood varied (range 13.1-291.5/microl; median 20.0/microl). To reach the target quantity of 2.5 x 10(6) CD34+ cells/kg, between one and six (median two) leukaphereses (LP) were performed. The LP products contained between 0.2 x 10(6) and 18.9 x 10(6) CD34+ cells/kg (median 1.2 x 10(6)/kg). Multivariate analysis showed that the white blood cell count prior to HAM and the time interval from the start of HAM therapy to reach an unsupported platelet count > 20 x 10(9)/l were predictive for the peak value of CD34+ cells in the blood during the G-CSF stimulated haematological recovery. In 16 patients an intraindividual comparison was made between bone marrow (BM) and PBPC grafts. Compared to BM grafts, PBPC grafts contained 14-fold more MNC, 5-fold more CD34+ cells and 36-fold more CFU-GM. A CD34+ subset analysis showed that blood-derived CD34+ cells had a more immature phenotype as indicated by a lower mean fluorescence intensity for HLA-DR and CD38. In addition, the proportion of CD34+/Thy-1+ cells tended to be greater in the PBPC grafts. The data indicate that sufficient PBPC can be collected in the majority of patients with AML following intensive double induction and first consolidation therapy with high-dose cytarabine and mitoxantrone.     

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.     

Granulocyte colony-stimulating factor given in addition to interferon-alpha to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia

In order to potentially mobilize and harvest the Ph cells observed in most patients with chronic myeloid leukaemia (CML) during interferon-alpha (IF-alpha) therapy, G-CSF (filgrastim), 5 microg/kg/d, was administered subcutaneously together with IF-alpha to 30 CML patients in haematological remission but with various degrees of cytogenetic remission, after IF-alpha therapy. Peripheral blood stem cells (PBSC) were harvested using standard aphereses from day 5 of G-CSF Patients underwent one to four (median three) aphereses. Median total yields/kg were 7.6 (range 3.8-25) x 10(8) MNC, 3.4 (0-140) x 10(6) CD34+ cells, and 17 (1.1-107) x 10(4) CFU-GM. No patient had a significant increase in the percentage of Ph+ cells in the bone marrow under G-CSF therapy. The percentage of Ph+ cells in apheresis products tended to decrease between the first and the last apheresis (P = 0.05). 14 patients who were not responsive to IF-alpha were transplanted after conditioning with busulphan 16 mg/kg and melphalan 140 mg/m2. Median time to neutrophils > 0.5 x 10(9)/l was 20 d (16-114 d) and to platelets > 50 x 10(9)/l 18 d (12-149 d). Nine patients had a major cytogenetic response post graft, which correlated with the amount of Ph+ cells reinfused with the graft (P = 0.02). We conclude that this procedure is feasible, allowing the harvest of enough PBSC, some of them Ph- in patients who responded to IF-alpha, to allow autologous transplantation.     

Does eradication of Helicobacter pylori impair healing of nonsteroidal anti-inflammatory drug associated bleeding peptic ulcers? A prospective randomized study

High-dose therapy with peripheral blood stem cell (PBSC) support is a frequently used treatment option in younger patients with poor prognosis histologically indolent (low-grade) non-Hodgkin's lymphoma (NHL), usually at the time of second or subsequent response to conventional-dose therapy. We have undertaken PBSC collection in 57 patients with histologically indolent NHL mobilized with either cyclophosphamide 1.5 g/m2 or the ESHAP regimen, followed by daily G-CSF. Progenitor cell yields were determined by quantification of CD34+ cells and GM-CFC. Twelve patients (21%) failed to achieve the minimum progenitor cell requirements of 1 x 10(6)/kg CD34+ cells or 1 x 10(5)/kg GM-CFC in their pooled harvests and 40 patients (70%) failed to achieve the optimal harvest thresholds of 3.5 x 10(6)/kg CD34+ cells or 3.5 x 10(5)/kg GM-CFC. This high failure rate is significantly higher than that in patients with histologically aggressive NHL or Hodgkin's disease. A multivariate analysis was performed to identify factors contributing to the low stem cell yields in this group. This identified the time interval from the last chemotherapy to the priming chemotherapy as the most important predictive factor. With respect to CD34 and GM-CFC numbers, on the single harvest on the day the white cell count first exceeded 5 x 10(9)/l the P values were 0.0078 and 0.0065, respectively, and for the progenitor cell values on the pooled harvests the P values were 0.004 for CD34+ cells and 0.015 for GM-CFC. Progenitor cell yields may therefore be improved in patients with low grade lymphoma by harvesting at diagnosis if no marrow disease is present, or by delaying mobilization for 6 months post-chemotherapy in patients in first or subsequent remission.     

Effects of prior therapy on the in vitro proliferative potential of stem cell factor plus filgrastim-mobilized CD34-positive progenitor cells

The quantity of hematopoietic progenitors in an apheresis collection is defined by the number of CD34(+) cells or granulocyte macrophage colony-forming units present. These parameters are believed to give roughly equivalent information on graft quality. We here report that the in vitro proliferative potential of r-metHuSCF (stem cell factor) plus filgrastim (granulocyte colony-stimulating factor; r-metHuG-CSF) mobilized peripheral blood (PB) CD34(+) cells obtained from previously heavily treated non-Hodgkin's lymphoma patients inversely correlates with extent of prior therapy. CD34(+) cells were enriched using the CellPro Ceprate system and placed in liquid culture for 4 weeks in the presence of either r-metHuSCF, IL-3, IL-6, filgrastim (S36G), or S36G plus erythropoietin (S36GE) with a weekly exchange of media and cytokines with reestablishment of culture at the starting cell concentration (Delta assay) and enumeration of progenitors. Starting with 4 x 10(4) CD34(+) cells from apheresis samples from patients who had received <10 cycles of prior chemotherapy, progenitors were detectable in culture at 4 weeks 81% of the time as compared to 14% with CD34(+) cells from patients who had received >10 cycles and 5% for >10 cycles plus radiotherapy. The total number of progenitors generated over the duration of culture (area under the curve) was calculated using the trapezoidal rule as a novel measure of the proliferative potential of the enriched PB CD34(+) cell population. The median area under the curve of CD34(+) cells from patients receiving <10 cycles of prior chemotherapy was 7.4 and 5.7 (x10(5)) using S36G or S36GE, respectively, 1.8 and 1.9 if the patients received >10 cycles of prior chemotherapy, and 1.4 and 1.2 if the patients received >10 cycles of prior chemotherapy plus radiotherapy (P < 0.001). These data show that prior therapy impacts on the quality of PB CD34(+) cells as measured by their ability to generate committed progenitors over a number of weeks in liquid culture.     

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.     

Extensive phenotypic analysis of CD34 subsets in successive collections of mobilized peripheral blood progenitors

The transplantation of mobilized progenitor cells after high-dose chemotherapy shortens haemopoietic engraftment. CD34 cell subsets were examined in 20 consecutive mobilized progenitor cell collections obtained from patients with solid tumours that had not been previously treated. The analysis of CD34 cells was based on the expression of intracellular antigens, surface antigens including CD38, and cell size using multi-dimensional flow cytometry. We also correlated the numbers of stem cell subsets reinfused to haemopoietic recovery. The majority of CD34+ cells expressed CD13 and CD33. A significant proportion was cytoplasmic myeloperoxidase (cMPO) positive. CD34+ MPO+ cells increased significantly in late collections. MPO expression was related to cell size. Cells expressing CD13 also increased in late collections in parallel to CFU-GM count. Small subpopulations of CD34+ CD38+ were committed to B cells, T cells and erythroid cell lineages. A small population expressing the megakaryocytic antigen had a small size and were predominantly CD38-. A minor subpopulation expressed stem cells antigens. These were significantly higher in late collections (CD34+ Thy-1+ and CD34+ CD33-). After mobilization, patients received three cycles of intensive chemotherapy followed by reinfusion of mobilized progenitors (5.45 x 10(6)/kg CD34+ cells, range 3.4-11.88). The numbers of reinfused CD34 cells or the individual subsets did not influence recovery of leucocytes (9 d) or platelets (9 d). In conclusion, the numbers of stem cells and their subsets differed between collections and, in unpretreated patients receiving intensive chemotherapy, there was no delayed engraftment when sufficient numbers of stem cells were reinfused. The recovery period was short and not correlated to any stem cell subsets.     

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.     

Engraftment and outcomes of patients receiving myeloablative therapy followed by autologous peripheral blood stem cells with a low CD34+ cell content

Engraftment kinetics after high-dose chemotherapy (HDC) were evaluated in patients receiving autologous peripheral blood stem cell (PBSC) infusions with a low CD34+ cell content. Forty-eight patients were infused with < 2.5 x 10(6) CD34+ cells/kg; 36 because of poor harvests and 12 because they electively received only a fraction of their harvested cells. A median of 2.12 x 10(6) CD34+ cells/kg (range, 1.17-2.48) were infused following one of seven different HDC regimens. All patients achieved absolute neutrophil counts > or = 0.5 x 10(9)/l at a median of day 11 (range, 9-16). Forty-seven patients achieved platelet counts > or = 20 x 10(9)/l at a median of day 14 (range, 8-250). Nine of 47 (19%) had platelet recovery after day 21, 4/47 (9%) after day 100 and one died on day 240 without platelet recovery. Twenty-six patients (54%) died of progressive disease in 51-762 days; 22 (46%) are alive at a median of 450 days (range, 94-1844), 17 (35%) of whom are surviving disease-free at a median of 494 days (range, 55-1263). No patient died as a direct consequence of low blood cell counts. These data demonstrate that PBSC products containing 1.17-2.48 x 10(6) CD34+ cells/kg resulted in relatively prompt neutrophil recovery in all patients but approximately 10% had delayed platelet recovery.     

Comparison of chronic neutral endopeptidase inhibition and furosemide in an ovine model of heart failure

OBJECTIVE: Due to the elevated levels of hematopoietically active cytokines such as tumor necrosis factor (TNF) and granulocyte macrophage colony stimulating factor (GMCSF) in rheumatoid arthritis (RA) serum and synovium, the increased bone marrow activity in RA, and the effectiveness of GMCSF in mobilizing progenitor cell release from the bone marrow into the periphery, we hypothesized that hematopoietic progenitors are altered in the peripheral blood (PB) of patients with RA. METHODS: Flow cytometry assisted cell surface analysis was employed to compare the distribution of myeloid (CD34+CD33+), B lymphoid (CD34+CD10+), and erythroid (CD34+CD71+) committed progenitor cell subsets in the PB of healthy controls and patients with RA. Since RA and Sjogren's syndrome (SS) are related autoimmune disorders, primary SS PB was also investigated. RESULTS: Only those patients with RA exhibiting clinically active disease (RA-A) demonstrated increases in myeloid and B lymphoid progenitor cell subsets. Growth of RA-A progenitors in cytokines promoting myelopoiesis (GMCSF, TNF, stem cell factor) produced increased monocyte and dendritic cell progeny, in support of the flow cytometry data. Lineage committed (CD34+CD38+) progenitors were increased in SS PB (p <0.03). However, these did not correlate with either the myeloid, erythroid, or B lymphoid lineages. CONCLUSION: Distinct alterations in the distribution of PB progenitors are present in RA and primary SS. Since progenitor cells retain a proliferative capacity, their infiltration into the synovial/glandular environment may contribute to the accumulation of inflammatory cells within these sites. We propose that PB progenitors enter the diseased microenvironment through similar mechanisms as mature hematopoietic elements.     

供者外周血干细胞动员采集198例临床分析

目的 研究外周血造血干细胞移植( PBSCT)供者外周血干细胞动员采集的方法及其效 果。方法 198名健康供者每天皮下注射重组人粒细胞集落刺激因子(rhG-CSF)(5～10) μg/kg进行外周血干细胞动员,第5天开始采集。采用血细胞分析仪行单个核细胞( MNC)计数,流式细胞术(FCM)行CD34+细胞计数。分析供者性别、身高、年龄、采集当天外周血白细胞(WBC)计数对动员采集效果的影响。结果所有供者均成功动员采集,采集当天的MNC计数平均为(4.19±1.96)×108/kg,CD34+细胞计数平均为(2.98±1.40) ×106/kg; MNC和CD34+细胞计数与供者性别、身高、年龄无关;采集当天外周血WBC计数与MNC、CD34+细胞计数呈正相关(r= 0.9201,P=0.0035;r=0.8420,P= 0.0149);采集当天外周血WBC计数≥20.0×109/L的供者比＜20.0×109/L的供者采集效果更显著(F=4.688,P= 0.0013;F= 4.622,P=0.0006)。结论rhG-CSF动员的健康供者采集当天外周血WBC计数是一项预测CD34+细胞采集数量的简单、可行的指标。