We investigated the feasibility of mobilizing peripheral blood stem cells (PBSC) with G-CSF alone in 24 patients with multiple myeloma. The median age was 53 years (range 33-62). All patients had stage II/III disease and responded to standard first-line (n = 6) or salvage chemotherapy (n = 18). The median number of previous chemotherapy cycles was 7 (4-18) and the median number of prior melphalan-cycles was 6 (0-14). Nine (35%) patients had experienced prior radiation therapy. The patients received either 10 microg/kg G-CSF (n = 18) or 24 microg/kg G-CSF (n = 7, including one patient with previous 10 microg/kg G-CSF stimulation) daily s.c. for 5 or more consecutive days until completion of harvesting, starting apheresis on the fifth day. G-CSF treatment was well tolerated, with only slight bone pain in half of the patients (51%). After a median of three (range 1-7) apheresis procedures, medians of 3.8 (0.3-17) x 10(6) CD34+ cells/kg, 8.5 (4.5-24) x 10(8) MNC/kg, 2.9 (0.6-39.4) x 10(4) CFU-GM/kg, and 5.6 (0.9-49) x 10(4) BFU-E/kg were harvested. Three patients (12%) with extensive melphalan pretreatment failed the target collection of at least 2.0 x 10(6) CD34+ cell/kg. Pretreatment with six or more cycles of melphalan yielded a smaller number of CD34+ cells than pretreatment with fewer than six cycles (2.5 vs 5.3 x 10(6)/kg; p = 0.001). Nineteen patients underwent high-dose chemotherapy consisting of either total marrow irradiation (9 Gy)/busulfan (12 mg/kg) and cyclophosphamide (120 mg/kg) (n = 10), or busulfan (14 mg/kg)/cyclophosphamide (120 mg/kg) (n = 5), or tandem melphalan (200 mg/m2). The median time for granulocyte (> 1.0/nl) and platelet (> 50/nl) recovery was 10 and 14 days (ranges 7-12 and 8-40), respectively. G-CSF alone is a safe, alternative approach to mobilizing sufficient PBSC in patients with multiple myeloma and allows an exact prediction of harvest time. G-CSF-mobilized PBSCs ensure rapid engraftment after myeloablative therapy. Melphalan treatment should be avoided in patients who are candidates for high-dose chemotherapy.

This study was designed to identify and quantify concentrations of growth factors/cytokines released by Vero cells during the co-culture interval. The factors screened for in this preliminary investigation, namely platelet-derived growth factor (PDGF), transforming growth factor beta (TGFbeta), interleukin-6 (IL-6), leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) have each been identified to impact on early embryo development or are secreted by embryos themselves, suggesting an autocrine regulatory role. Vero cell culture supernatants were collected at 2, 3, 4, 5 and 6 days after seeding. Samples were assessed by enzyme-linked immunoassay for growth factor/cytokine secretion at each designated time interval. Conditioned medium from all days contained IL-6, PDGF and LIF. The concentration of IL-6 increased from 294 pg/well on day 2 to almost 1600 pg/well on day 6. PDGF also accumulated rapidly in co-culture wells, rising from 19-40 pg/well early in the culture period to around 500 pg/ well by day 6. In the second half of this study, medium supernatants from patients enrolled in our co-culture programme were analysed. Retrospective evaluation of medium supernatants collected at the time of transfer from co-cultures from 11 randomly selected patients showed considerable patient-to-patient variation in concentrations of secreted growth factors and cytokines. These findings indicate that during the co-culture interval embryos are exposed to a dynamic environment, with increasing concentrations of growth factors and cytokines. The positive effects of co-culture on embryo quality and in-vitro blastulation need to be balanced against the variation that this technique can potentially introduce into the embryo culture system.

Circulating myeloid (CFU-GM) and erythroid (BFU-E) progenitor levels were evaluated weekly throughout 3 courses of treatment with vinorelbine (VNB) ifosfamide (IFO) and filgrastim (G-CSF) with or without addition of cisplatin (DDP) in 20 stage IIIB or IV non small-cell lung cancer patients. In IFO, VNB, DDP-treated patients, BFU-E mobilization in peripheral blood following chemotherapy and G-CSF was completely lacking, in contrast with the patients treated with IFO, VNB plus G-CSF. CFU-GM release, however, was of the same order in the 2 groups of patients. Further investigations are needed to explain why presence of DDP in this chemotherapeutic protocol hinders erythroid progenitor release in peripheral blood.

We have studied the kinetics of plasma levels of circulating (c)selectins in 8 patients undergoing bone marrow or stem cell transplantation to gain estimates for the distribution and half-life of (c)selectins and to potentially identify an endothelial source of cP-selectin in patients who are deprived of platelets and megakaryocytes. Blood was sampled just before conditioning treatment and immediately after, at 2 occasions under bone marrow aplasia (1 and 2 wk after BMT), on 2 separate days (3-8 d apart) before and at 60 min after platelet transfusion, and 30-72 d after BMT. All (c)selectins showed a strikingly parallel decrease during bone marrow aplasia: cP-selectin decreased by a median of 70% (range: 59-95%), cE-selectin by 63% (range: 27-78%), and cL-selectin by 75% (range: 66-90%) compared to baseline (p=0.012 for all comparisons). Estimates for plasma half-lives of all (c)selectins were obtained from individual time vs. concentration profiles of the maximal decreases and ranged from 2 to 4 d. cP-selectin increased by 23% (p=0.003), cE-selectin by 5% (p=0.041) and cL-selectin by 19% (p=0.009) 1 h after the platelet transfusions. Based on these results the calculated volume of distribution (Vd) of transfused (c)selectins was 2.5-fold higher than the plasma volume, which supports the concept of the in-vivo expression of binding sites, i.e. ligands, for cE-selectin and cL-selectin. In summary, while our study cannot provide any evidence for endothelial cells as a source of cP-selectin, we have found for the first time evidence for the existence of ligands for soluble cE-selectin and cL-selectin in humans.

Adjuvant interleukin-2 (IL-2) therapy after stem cell transplantation can improve the prognosis of patients with Ewing tumors. This has been attributed to stimulation of the immune system and its antineoplastic activity, thus eliminating minimal residual disease. As the side effects of systemic IL-2 limit the dosage, attempts have been made to locally augment the concentration of IL-2 in the proximity of the tumor. To achieve this, fibroblasts and/or tumor cells can be genetically modified to secrete IL-2 and then be injected to generate tumor immunogen.

The importance of coexpression of myeloid antigens in childhood acute lymphoblastic leukemia (ALL) has long been debated; results are conflicting. We studied children with ALL treated at Italian Association for Pediatric Hematology-Oncology (AIEOP) institutions over 6 years with Berlin-Frankfurt-Muenster (BFM)-based protocols and have analyzed the incidence of coexpression of six MyAg (CD11b, CD13, CD14, CD15, CD33, CD65w) to determine its prognostic impact. Criteria for MyAg coexpression (MyAg+ALL) included positivity to one or more MyAg on at least 20% of blasts and confirmation of coexpression at double-fluorescence analysis. A total of 291 of 908 cases were MyAg+ALL (32%). Incidence was similar in B-ALL and T-ALL; among common, pre-B, and pre-pre-B-ALL. CD13 and CD33 were most common. Patients with MyAg+ALL had presenting features similar to MyAg-ALL. They entered standard or intermediate risk protocols more frequently and had better prednisone response, but similar complete remission rates. Six-year event-free survival (EFS) was 69.0% in 291 MyAg+ALL cases and 65.3% in 617 MyAg-ALL cases, without significant difference. Cases expressing two or more MyAg presented similar clinical features and treatment response. MyAg+ALL had worse EFS only in infants (0% v 47%) (P = .01). Therefore, in this series of homogeneously diagnosed and treated ALL, coexpression of MyAg was not associated with prognostic significance, without relevance for clinical purposes or for patient stratification, except for infants.

Despite the wide use of G-CSF for mobilization of PBPC the best dose and schedule of G-CSF has not been definitively established. In this study we have compared three different schedules of G-CSF for mobilization of PBPC in normal donors including a single daily dose of 10 microg/kg/day for 5 days (21 donors) and doses of 6 (21 donors) or 8 microg/kg/12 h (6 donors) for 5 days. We demonstrate that G-CSF at doses of 6 and 8 microg/kg/12 h mobilizes significantly more CD34+ cells/ml of blood (83.3 +/- 6.7 and 121 +/- 6.9, respectively) than 10 microg/kg/day (71.6 +/- 6.5). Mobilization with 6 or 8 microg/kg/12 h of G-CSF was also associated with collection of significantly more CD34+ cells in comparison with 10 microg/kg/24 h (2.24 +/- 1.2 and 2.46 +/- 1.22 vs 1.15 +/- 0.8 CD34+ cells/kg of donor/blood volume). PBPC collection was associated with a significant decrease in platelet count which was not significantly different between the three groups. Ten days after the last PBPC collection platelet counts were within normal limits while there was a decrease in WBC and ANC. We conclude that G-CSF administered every 12 h at doses of 6 microg/kg provides better CD34+ cell yield than 10 microg/kg once a day in normal donors which may translate into a decrease in the number of aphereses required to obtain enough numbers of CD34+ cells for allogeneic PBPC transplant.

The addition of stem cell factor (SCF) to G-CSF and chemotherapy results in a dose-dependent, significantly increased mobilisation of peripheral blood progenitor cells compared with the use of chemotherapy and G-CSF alone. The enhanced mobilisation may benefit patients in several ways. Firstly, in clinical settings where currently multiple aphereses are having to be performed to obtain a specified target number of cells, the addition of SCF may lead to a reduction in this number. Alternatively, when only a single apheresis is currently being performed to obtain sufficient cells then the addition of SCF to the mobilisation regimen would allow between 5- and 8-fold reduction in the volume of blood required to be processed during the apheresis procedure to obtain a specified target of GM-CFC, CD34+ cells and LTC-IC for those receiving the highest dose of SCF (20 microg/kg) plus G-CSF following chemotherapy compared with those patients receiving G-CSF alone following chemotherapy. The increased mobilisation resulting from the addition of SCF to the regimen makes feasible the use of whole blood aliquots to support dose-intensive therapy. We have calculated that in patients mobilised using cyclophosphamide 3 g/m2, SCF 20 microg/kg and G-CSF 5 microg/kg a median 512 ml aliquot of whole blood would contain 2 x 10(6)/kg CD34+ cells and over 3.7 x 10(5)/kg GM-CFC. This aliquot would be sufficient to rescue the patient following a myeloablative therapy. Significantly, because less individual variation was seen after the administration of SCF the patient who showed the worst mobilisation in that group would have the usually required content of 2 x 10(6) CD34+ cells/kg and 1 x 10(5) GM-CFC/kg in only 731 ml of blood.

In order to find out the effect of peripheral blood (PB) hematopoietic progenitor cells on immune reconstitution the present study compares, through a randomized trial, some lymphoid subsets after peripheral blood (PBT) or bone marrow (BMT) autologous transplantation.

Cytogenetic, interphase fluorescent in situ hybridization (FISH) and RT-PCR methods were used to study minimal residual disease in peripheral blood stem cells collected for autografting in three chronic myeloid leukemia (CML) patients in sustained complete cytogenetic remission after treatment with interferon alpha (IFNalpha). Karyotypic analysis failed to reveal Ph-positive metaphases. FISH detected 9-16% nuclei with a BCR-ABL fusion gene, contrasting with RT-PCR, performed in two cases, which was negative in one case and weakly positive in the other. RT-PCR was also subsequently weakly positive in the third patient. This discrepancy suggests that the BCR-ABL genomic rearrangement persists unexpressed in quiescent cells. These preliminary results, which need to be confirmed in larger series, suggest that monitoring residual disease in CML should be performed both at DNA and RNA levels. Moreover, autografting following IFNalpha therapy should be considered with caution because of the persistence of the BCR-ABL genomic rearrangement in a sizeable proportion of the cells.

We have prospectively performed peripheral blood stem cell autotransplants in six patients with hematological malignancies on an entirely outpatient basis. Patients were conditioned with high-dose melphalan and received a median of 4.2 x 10(8)/kg non-cryopreserved, non-purged mononuclear cells, containing a median of 3.9 x 10(6)/kg CD34 + cells. The median time to achieve > 500 granulocytes/microl was 21 days, with a range of 16 to 40, whereas the median time to achieve > 20,000 platelets/microl was 38 days, with a range of 21 to 48. Only three patients were transfused with platelets whereas packed red blood cells were transfused in two. All patients survived 60 days after the autograft and three are alive at 450, 690, and 1,950 days after the autotransplant. One patient was given an allogeneic bone marrow transplant when relapsing after the autotransplant. One patient had to be admitted to the hospital on day +10 because of fever. A median of 6,500.00 USD per patient was calculated as the total cost of each outpatient autotransplant. Since outpatient autologous transplants with non-frozen PBSC are feasible, restrictions to PBSC autotransplant programs may be overcome and costs may be diminished.

To determine if multinucleation in normally fertilized embryos is indicative of poor developmental or clinical pregnancy prognosis and to examine the ovulation induction characteristics associated with multinucleation.

The combination of high or intermediate-dose cyclophosphamide (CY) plus granulocyte colony-stimulating factor (G-CSF) is useful to mobilize hematopoietic progenitor cells to peripheral blood, but the patients require hospitalization. The aim of this study was to evaluate the efficiency of low-dose CY plus G-CSF (5 ug/kg/day s.c.) as an outpatient treatment in order to collect enough progenitor cells for hematopoietic rescue in autologous peripheral blood transplantation (APBSCT).

To explore the effect of cytokine therapy on the NADPH oxidase in mature myeloid cells, we isolated neutrophils from patients receiving recombinant human granulocyte colony stimulating factor (G-CSF) and recombinant human stem cell factor (SCF) and evaluated oxidase activity. All patients had relapsed neoplastic disease and were at least 3 three weeks since the last course of chemotherapy or cytokine therapy.

We investigated the reconstitutive potential of haematopoietic progenitor cells collected in autologous whole blood during multicycle dose-intensified chemotherapy. Forty patients with metastatic solid tumours were treated with up to six cycles of cisplatin and escalating doses of ifosfamide every 14 days. Cisplatin was administered in 3% sodium chloride over 3 h, followed by ifosfamide over 24 h and mesna over 36 h. The first cohort of patients received granulocyte colony-stimulating factor (G-CSF) days 4-14. Once dose-limiting toxicity was reached in cohort 1, the study continued with a second cohort of patients, in whom, in addition to G-CSF on days 4-14, 500 ml of G-CSF and chemotherapy-'primed' whole blood was collected on day 15, i.e. on day 1 of treatment cycles two to six, before cisplatin administration. This volume of blood was kept unprocessed at 4 degrees C and reinfused 20-24 h after the completion of ifosfamide. In cohort 1, dose-limiting toxicity (DLT) was reached at ifosfamide 6.0 g m(-2) with two out of six of the patients developing neutropenic fever. Although in cohort 2 no neutropenic fever was encountered, neither the frequency nor the duration of grade 4 neutropenia and thrombocytopenia were reduced. Cumulative asthenia resulted in DLT at 7.0 g m(-2). The median number of CD34+ cells in 500 ml of whole blood after the first cycle (i.e. at start of cycle 2) was 1.15 x 10(6) kg(-1). This number was significantly greater after the second cycle (2.06 x 10(6) kg(-1), P = 0.01) and then gradually decreased after cycles three to six. After storing whole blood, the number of CD34+ cells had not decreased (median + 10%). We conclude that the method of combined bone marrow support by G-CSF and haematopoietic progenitor cells in autologous whole blood collected before each cycle of a 2-weekly regimen of cisplatin-ifosfamide does not result in clinically measurable reduced bone marrow toxicity compared with what can be expected by the use of G-CSF alone.

B-cell chronic lymphocytic leukaemia (CLL) cannot be cured by conventional therapy. To improve the prognosis of patients with CLL, we have designed a sequential treatment strategy that comprises intensive chemotherapy for mobilization of peripheral blood progenitor cells (PBPCs) and induction of minimal disease, followed by high-dose radiochemotherapy with stem cell reinfusion and post-transplant molecular monitoring by polymerase chain reaction (PCR) amplification of the complementary determining region III (CDRIII) gene. In a prospective study, we have evaluated this protocol in 18 patients with CLL, also including early stages of the disease. The median age was 49 (29-61) years; Binet stages were A, six; B, nine; and C, three. Adverse prognostic factors [high lymphocyte count and/or diffuse bone marrow (BM) infiltration] were present in 16 out of 18 patients. All patients showed a clone-specific molecular marker as demonstrated by PCR amplification of CDRIII rearrangements. For stem cell mobilization and reduction of tumour load, one to two cycles of Dexa-BEAM chemotherapy were administered, resulting in minimal disease (circulating lymphoma cells <1 x 10(9) l(-1); BM infiltration <20%; lymphomas <2 cm) in 16 out of 18 patients, including four patients who already had minimal disease before Dexa-BEAM. Stem cell harvesting was successful in 14 patients. All grafts [three BM, 11 peripheral blood (PB)] were purged from leukaemic cells using immunomagnetic methods. Thirteen patients having achieved minimal disease were reinfused with purged autologous stem cells (ASC) after preparation with total body irradiation and cyclophosphamide. Engraftment was delayed in patients receiving BM (n = 3) but prompt [neutrophils >0.5 x 10(9) l(-1) after 10 (9-13) days, platelets >20 x 10(9) l(-1) after 11 (9-214) days] in patients restored with PBPCs (n = 10). Procedure-related deaths did not occur. Although the results of CDRIII PCR suggest persistence or recurrence of the leukaemic clone in at least three cases, to date only one patient has relapsed, whereas all others survive without clinical evidence of disease with a maximum follow-up of 48 months. We conclude that sequential high-dose therapy using Dexa-BEAM and autologous stem cell transplantation is a safe and highly effective treatment for patients with CLL. However, a longer follow-up is needed to assess whether definite cures can be achieved using this strategy.

We performed an ultrastructural analysis of 10 skin biopsy specimens that had been obtained from three women who were undergoing daily subcutaneous dosing with recombinant methionyl-human stem cell factor (rhSCF) as part of a phase I clinical trial. The biopsy specimens were obtained at sites of subcutaneous administration of rhSCF, within approximately 1 to 2 hours of rhSCF injection, and, at the same time, at contralateral control sites that had not been directly injected with rhSCF. We previously reported that subcutaneous dosing with rhSCF in these subjects induced the local development of a wheal and flare response, which was associated with evidence of mast cell degranulation, as well as a systemic increase in numbers of cutaneous mast cells. The present electron microscopic analysis revealed that all biopsies of swollen, erythematous rhSCF-injected sites exhibited anaphylactic degranulation of both mature and immature mast cells, an acute inflammatory response characterized by the migration of neutrophils, basophils (some of which exhibited evidence of piecemeal degranulation), and eosinophils through blood vessel walls into the perivascular and extravascular spaces, and edema and fibrin deposition within the interstitium. By contrast, the control biopsies contained no evidence of mast cell degranulation or acute inflammation. However, both control and rhSCF-injected sites exhibited mast cells that were undergoing granule building and maturation. Thus at the doses tested in these subjects, subcutaneous injection of rhSCF induced anaphylactic-type degranulation of dermal mast cells at the injection site, with an acute inflammatory response that was associated with the recruitment of granulocytes. By contrast, mast cells at sites distant from those directly injected with rhSCF exhibited no evidence of enhanced secretion.

The purpose of this study was to evaluate the frequency of detecting occult tumor cells in peripheral blood stem cell (PBSC) harvests and to determine the impact of infusing such cells on relapses after high-dose chemotherapy (HDC). Peripheral blood stem cell harvests from 223 patients with breast cancer were examined by an immunocytochemistry (ICC) method for detection of occult tumor cells, and infused after HDC without consideration of test results. Two hundred and four patients, 114 with stage II-III and 90 with stage IV disease who received only PBSC, that were tested by ICC were evaluated for time to relapse. Five hundred and eighty-one of 619 PBSC harvests (94%) from 223 patients were tested. Fifty-three of 581 harvests (9%), 8% from stage II-III and 10% from stage IV patients, were positive by ICC (P = 0.68). Forty-one of 223 patients (18%), 17/122 (14%) with stage II-III and 24/101 (24%) with stage IV disease, had positive harvests (P = 0.06). Eleven percent of patients who had 1-2 harvests tested were positive as compared to 32% of patients who had > or =3 PBSC harvests tested (P < 0.001). Nineteen patients who were infused with a mixture of ICC negative and untested PBSC harvests were excluded from analyses of relapse. The probabilities of relapse at 18 months for the 97 patients with stage II-III disease infused with ICC-negative and the 17 with ICC-positive PBSC were 0.19 and 0.13, respectively (P = 0.48). The probabilities of relapse at 18 months for patients achieving a CR or a CR in non-bone sites and improvement in bone lesions were 0.55 for the ICC-negative group (n = 30) and 0.45 for the ICC-positive group (n = 11) (P = 0.60). It was concluded that occult tumor cells were detected by ICC in PBSC harvests from a relatively small fraction of women with breast cancer, but were not associated with a significant increase in the probability of early relapse or progression when infused after HDC.