Differentiation induction therapy is used in myelodysplastic syndromes (MDS) to improve maturation defects and to restore impaired function of malignant cells. To this end, 18 patients with MDS received either a combination therapy consisting in study 1 of all-trans retinoic acid (ATRA) and granulocyte-colony stimulating factor (G-CSF), or in study 2 of a combination with ATRA, G-CSF, erythropoietin (Epo) and tocopherol. The ANC increased in 19/20 patients in both studies, whereas an increase in haemoglobin concentration, platelet counts or reduction of transfusion requirement was seen in only 8/20 patients, correlating strongly with good BFU-E growth (P < 0.001). To assess the role of accessory cells in the modulation of the haemopoietic response to treatment, we analysed the capacity of peripheral blood monocytes to secrete cytokines (IL-1 beta, IL-6, IL-8, TNF alpha). Secretion of all cytokines was significantly reduced before therapy when compared with healthy controls, but increased during therapy, reaching normal levels for IL-8. These data indicate that a combination therapy with ATRA and cytokines improves impaired cytokine secretion from monocytes and induces a multilineage clinical response in a subgroup of MDS patients characterized by an almost intact erythroid compartment. In contrast, induction of TNF alpha might be responsible for treatment failure.

(1) To study the ability of mobilized peripheral-blood progenitor cells (PBPC) collected in a single large-volume leukapheresis performed on a predetermined date to accelerate engraftment after high-dose cyclophosphamide and thiotepa; (2) to establish the minimum dose of PBPC associated with early engraftment; and (3) to identify parameters predictive of collection of large numbers of PBPC.

The effect of priming on occult tumor cell involvement of peripheral blood (PB) and PB progenitor cell (PBPC) collections is poorly characterized. Using sensitive immunocytochemistry (ICC) and tumor clonogenic assays (TCA) specific for epithelial-derived tumor cells, hematopoietic specimens were analyzed for PBPC and occult tumor cell involvement in 28 patients with chemotherapy-sensitive stage IIIB or IV breast cancer. Before PBPC priming, tumor was detected by ICC in PB of 1 of 23 (4%) patients and in bone marrow (BM) harvests of 4 of 27 (15%) patients. Fifteen days after cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF) priming, 2 of 28 (7%) patients had ICC-positive PBPC collections. The median amplification of CD34+ PBPC during this time was over 19-fold (range, < 1 to 199). One patient had pretreatment tumor involvement of both PB and BM. One patient grew tumor colonies in TCA; the PB and BM were ICC- and TCA-positive, but the PBPC collection was ICC-positive and TCA-negative. After cytoreduction with conventional-dose chemotherapy, patients with advanced breast cancer and histologically negative BM biopsy specimens have rare tumor cell involvement of PB and BM. Despite effective PBPC priming with cyclophosphamide and GM-CSF, clonogenic breast cancer cells were not found in the PBPC collection performed on day 15.

No previously published studies have described double-lumen hemodialysis/apheresis catheters for use with continuous-flow apheresis collection of peripheral stem cell (PSC). We prospectively evaluated experiences with these catheters during both PSC collection and transplantation. Because of previously-described successful experiences with single-lumen apheresis catheters placed in the inferior vena cava, all catheters evaluated in this study were placed in this anatomic location. Our experience demonstrated high rates of thrombotic occlusion (65%) and catheter-related infections (15%). This method of access should not be considered optimal in its present state of use. Further investigation into preferred catheter design, anatomic location, and thrombosis prophylaxis during continuous-flow apheresis is warranted.

Pruritus is a major clinical problem in patients with polycythaemia vera (PV). Conventional symptomatic treatment is unsatisfactory. Recently, a favourable effect of interferon-alpha on pruritus in patients with PV has been reported. Also, interferon-alpha suppresses the increased haemopoiesis in PV. However, long-term treatment with interferon-alpha may be hampered by side-effects and the inconvenience of chronic subcutaneous injection therapy. We conducted a long-term study (median follow-up 13 months) of the efficacy and tolerability of interferon-alpha in 15 patients (mean age 68 years) with PV and severe pruritus. Six patients were evaluable after 1 year. Pruritus significantly improved in 12/15 patients. Haematological control improved, as evidenced by a decreased number of phlebotomies from a mean of 4.3 in the year before the study to 1.8 while on interferon-alpha. Leucocyte and platelet numbers also decreased significantly. Five patients (33%) did not tolerate interferon-alpha. The effects of interferon-alpha could not be ascribed to an inhibitive effect on histamine production or to the disappearance of the abnormal erythroid progenitor clone, because erythropoietin-independent erythroid colony formation persisted during interferon-alpha treatment. We conclude that long-term interferon-alpha treatment is feasible and effectively relieves pruritus in patients with PV, but side-effects are an important concern. The optimal dose regimen that is well tolerated, relieves pruritus, and offers satisfactory haematological control at the same time remains to be established.

A phase I dose-escalation study of ifosfamide, carboplatin, and etoposide (ICE) with autologous stem-cell rescue (ASCR) was conducted to determine the maximum-tolerated dose (MTD) of ICE given over 6 days.

A preliminary study and related clinical trial were performed to evaluate the effects of granulosa-lutein cell co-culture on human embryo development and pregnancy rates for in-vitro fertilization (IVF). In the study, sibling two-pronuclear zygotes were randomly allocated to culture with (co-culture) or without (control) autologous granulosa-lutein cells. After 24 h, embryos were examined for blastomere number and degree of fragmentation. Co-culture had no effect on the average number of blastomeres per embryo at 24 h; however, fragmentation was significantly decreased in co-cultured embryos (0.7 +/- 0.1) compared with controls (1.3 +/- 0.2; P < 0.05). In the subsequent clinical trial, all two-pronuclear zygotes were co-cultured for 48 h prior to embryo transfer. The live birth rate per embryo transfer was 43.4% with an implantation rate per embryo of 17.6%. Of the untransferred embryos, 68% developed to the blastocyst stage and were cryopreserved. We conclude that the simple system of autologous granulosa-lutein cell co-culture improves embryo development, implantation and subsequent pregnancy rates for IVF.

Twenty patients with advanced (stage III-IV), previously untreated ovarian carcinoma were treated by: (a) induction chemotherapy (40 mg/m2 cisplatin, days 1-4; 1.5 g/m2 cyclophosphamide, day 4; every 4 weeks for two cycles) followed by (b) intensification chemotherapy (100 mg/m2 cisplatin, day 1; 650 mg/m2 etoposide, day 2; 1.8 g/m2 carboplatin, day 3). Eligibility criteria further included: age less than 55 years, moderately good to poor tumour grade, macroscopic (> 0.5 cm) residual tumour. Autologous peripheral stem cells were recruited after the induction cycles and, to ensure haematological support, autologous bone marrow harvesting was routinely performed in the first 14 cases. Haematological support consisted of autologous peripheral stem cells and autologous bone marrow transplant in 16 and four patients, respectively. All patients are evaluable for toxicity and 19 for pathological response, one being dead of systemic mycosis 35 days after the autologous bone marrow transplant. Severe extra-haematological toxicities were the following: gastrointestinal (100%), neurological (10%), hepatic (10%). Pathological response was detected in 84% of cases (CR 37%, microscopic PR 26%, macroscopic PR 21%). Median follow-up times of 48 and 41 months have been reached respectively from enrolment and second-look. Four-year 62% overall and 57% progression-free survivals have been reached. Ten patients are still alive with NED (six of seven with CR, three of five with microscopic PR, and one of four with macroscopic PR). Autologous peripheral stem cell transplant significantly reduced the duration of aplasia compared with autologous bone marrow transplant, and toxicity was proved to be manageable in those patients undergoing autologous peripheral stem cell transplant. The prolonged disease-free survival in patients showing CR and microscopic PR suggests that further investigation on this new approach is worthwhile.

During the last decade, molecular genetic techniques have been used increasingly to transfer human genes into mammalian cells, to correct and enhance cell function, and finally to treat human disease. Despite the current obstacles to developing even the simplest therapeutic strategy, gene therapy promises to have an almost unlimited future. The ability to collect specific blood cells in large numbers, to manipulate their expansion, growth, and differentiation in vitro, and also to cryopreserve these cells for later use has been central to the early developments in gene therapy. This article reviews the major concepts involved in blood cell-based gene therapy, a model for all somatic cell gene therapy.

Hematopoietic progenitor cells mobilized to peripheral blood by a chemotherapy combined or not with hematopoietic growth factors and harvested with cyto-apheresis (CTA) provide a rapid hematological recovery when infused as a support step after intensive chemotherapy (IC).

Bone marrow (BM) aspirate and biopsy specimens from seven female patients with advanced or metastatic breast cancer and preserved marrow function treated on a phase I trial of recombinant methionyl human stem cell factor (r-metHuSCF; SCF) were evaluated by immunohistochemical staining before and after treatment with SCF. Doses of SCF included 10 g/kg/day in 2 patients, 25 micrograms/kg/day in 2 patients, and 50 micrograms/kg/day in 3 patients administered as subcutaneous bolus injections for 14 days. Following treatment, bone marrow cellularity increased up to 1.6-fold (P = NS), with an increased frequency of promyelocytes (P < .002), but an unchanged relative frequency of other marrow hematopoietic cells. The mean relative frequency of BM CD34+ progenitor cells increased from 0.9% to 1.8% (P < .001). The mean proportion of BM cells stained by Ki-67/MIB 1 and PCNA/PC10, monoclonal antibodies (MoAb) recognizing proliferation-associated nuclear proteins, increased from 18.6% to 35.4% (P < .003) and from 32.4% to 49.4% (P < .01), respectively. Most of the Ki-67 and PCNA positive cells were represented by promyelocytes, proerythroblasts, and myeloblasts. SCF therapy was not associated with marrow fibrosis or increases in the number of macrophages. Peripheral white blood cell counts increased 1.3- to 3.6-fold following SCF. The mean absolute neutrophil counts increased from 3.9 x 10(9)/L (range 2.6-5.3) to 7.2 x 10(9)/L (range 4.7-12.3), and reticulocyte counts increased by a mean of 1.5 fold (range 1.2-fold to 2.0-fold). No consistent difference in platelet counts was seen. These results suggest that SCF given in vivo is effective in increasing the frequency of CD34+ BM progenitor cells, and has the capacity to increase the proliferation and differentiation rate of hematopoietic precursor cells. These effects indicate that SCF may represent a cytokine capable of affecting multiple hematopoietic lineages.

We studied the feasibility of in vitro expansion of CD34+ cells from patients with multiple myeloma (MM) or follicular non Hodgkin lymphoma (NHL). CD34+ cells were selected from peripheral blood (PB) using avidinbiotin immunoadsorption columns: purified CD34+ cells from three MM and five NHL patients were expanded. First, CD34+ cells (2 MM, 4 NHL) were grown for 14 days in 5 ml of IMDM plus 12.5% horse serum (HS), 12.5% fetal calf serum (FCS) and a commonly used combination of cytokines: IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (10 ng/ml each) and EP (4 UI/ml). In these conditions, at day 14, average increase in CD34+, CFU-GM and total cell numbers were, respectively: x 6.0 x 23 and x 2,113 fold with 20 to 35% of granulocytic cells. In terms of CD34+ cell, CFU-GM and total cell outputs, MM cultures were comparable to NHL cultures, but MM cultures seemed to produce less granulocytic cells than NHL cultures. Next, in vitro expansion of PB CD34+ cells was tested in culture media suitable for clinical use. Two cultures (1 MM, 1 NHL) were carried out for 14 days in 20 ml of X-Vivo 10 medium, 2% human serum, IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (6 ng/ml each) and EP (2 UI/ml). Increase in CD34+, CFU-GM and total cell numbers in these conditions were, respectively: x 5.7 and x 19.7, x 11.9 and x 40.9, x 424 and x 408 fold, with at least 75% of granulocytic cells in both cultures. We conclude that, although further improvements are necessary, in vitro expansion of PB CD34+ cells can presumably be carried out successfully for MM patients as well as for NHL patients, including in conditions suitable for clinical use.

Correlations between different peripheral blood parameters and harvesting yields of stem cells were analysed in 230 leukaphereses performed on 61 patients. According to multivariate analysis, the number of circulating CD34 positive cells was found to provide the best preleukapheresis parameter for predicting the quality of peripheral blood progenitor cell harvest. A high correlation coefficient and small 95% confidence and prediction intervals would indicate the assay of CD34 positive cells in peripheral blood to be a unique tool essential for monitoring the collection of circulating haematopoietic progenitors.

Double hemibody irradiation (DHBI) is an alternative treatment of stage III multiple myeloma (MM) in patients aged over 55 years. Toxic side-effects such as myelosuppression are a severe limiting factor to its use. We performed DHBI associated with human recombinant granulocyte-macrophage colony stimulating factor (hrGM-CSF) as support therapy in 10 patients with stage III MM to improve the tolerance to this treatment. Ten patients received subcutaneously 5 micrograms/kg/d of hrGM-CSF during 2 weeks after each course of hemibody irradiation. All these patients had stage III MM: eight previously received chemotherapy, six of them were regarded as patients with refractory MM and two with relapse. Two patients received DHBI as first-line treatment. hrGM-CSF increased safety and tolerance of DHBI. GM-CSF support reduced the mean time between upper body irradiation (UBI) and lower body irradiation (LBI): 41 v 108 d in a cohort of 32 patients previously treated without growth factor support. Overall there was no lethal infection with hrGM-CSF or granulocytopenia (5.0 x 10(9)/l v 0.4 x 10(9)/l at day 15 in patients without growth factor). hrGM-CSF also reduced stomatitis grading and thrombocytopenia (90 x 10(9)/l v 45 x 10(9)/l at day 15). Furthermore, hrGM-CSF increased blood colony forming unit-granulocyte macrophage (CFU-GM) and was well tolerated in all but one patient. hrGM-CSF reduces toxic side-effects of DHBI, thus providing an effective treatment in patients with advanced and resistant MM.

Normal volunteers received subcutaneous injections of recombinant human interleukin-3 (rhIL-3) on 4 consecutive days to characterize toxicity, pharmacokinetics, and hematopoietic effects. Dosages were 2.5, 5.0, and 7.5 micrograms/kg/day (n = 6 subjects per group). Adverse effects consisted predominantly of flu-like symptoms such as fever and headache. Mean area under the serum concentration-time curve and maximum serum concentration were linearly related to dose. Serum clearance was not apparently related to dose. Clearance increased slightly but significantly between days 1 and 4. Rapid but modest elevations in neutrophil and eosinophil counts were observed during treatment. Mean platelet counts rose modestly, peaking on day 10. Increases of CD34+ cell counts were correlated with increases of colony-forming unit-granulocyte macrophage (peak, day 7).

The triazinoaminopiperidine derivative S 9788 is a new multidrug-resistance modulator that is currently being evaluated in phase I clinical trials. In this study, the reversal effect of S 9788 in comparison with verapamil was shown in vitro in human T-leukemic CCRF-CEM/VLB cells expressing the multidrug-resistance (MDR) phenotype. S 9788 increased in a dose-dependent manner the cytotoxic activity of doxorubicin or vinblastine, with complete reversal of resistance occurring at 2 microM for a concomitant continuous exposure (96 h) to the cytotoxic drugs. At respective concentrations equivalent to the IC10 value (the concentration inhibiting 10% of cell growth), S 9788 was 44 times more potent than verapamil in CCRF-CEM/VLB cells. S 9788 at 2 microM did not enhance the in vitro toxicity of doxorubicin or vinblastine in the human normal bone-marrow erythroid (BFU-E) and myeloid (CFU-GM) progenitors. The effect of exposure duration and concentrations on the synergistic action of modulator and cytotoxic agent closely depended on the cytotoxic agent studied. Post-incubations with S 9788 alone after a 1-h coadministration with vinblastine and S 9788 dramatically increased the reversal effect (4-41 times) in proportion to both the duration of postincubation and the concentration of S 9788. In contrast, for doxorubicin resistance, post-incubation with S 9788 alone induced a maximal 2-fold increase in the reversal effect that was not proportional to the post-incubation duration. In patients treated with S 9788 as a 30-min intravenous infusion during phase I trials, a good correlation was found between the serum levels of S 9788 and the ability to reverse MDR in CCRF-CEM/VLB cells. The reversal effect was dose-dependent and was effective beginning at a plasma concentration of 0.25 microM. These data form a basis for the design of phase II trials using a combination of a loading dose of S 9788 given before vinblastine or doxorubicin administration followed by a maintenance infusion of S 9788 alone for a period of 2-24 h.

The effect of methylprednisolone on fresh cells from patients with chronic lymphocytic leukaemia (CLL) has been studied using the differential staining cytotoxicity (DiSC) assay resulting in LC90s of < or = 0.2 to 2,000 micrograms/ml. Cells from previously treated patients were, on average, significantly more sensitive to methylprednisolone than those from untreated patients (mean LC90 = 5.7 micrograms/ml, n = 61 vs 31.0 micrograms/ml, n = 17, respectively; p < 0.05). Twelve patients with advanced disease were given high-dose methylprednisolone (1 g/m2/day i.v. x 5 days). In 7 cases, > or = 3 courses were given; 3 patients did not respond (2 achieved palliation) and 4 (57%) achieved a good partial response. These latter 4 patients were all clinically resistant to chlorambucil and anthracyclines and 2 were resistant to fludarabine. In 5 cases, 1 or 2 courses were given but no patients responded. The 8 nonresponders survived a median of 3.5 months whilst the responders have survived a median of 28.5+ months (3 of 4 still alive). This work suggests a rationale for why CLL patients resistant to standard chemotherapy may benefit from high-dose methylprednisolone therapy. Due to cost and toxicity associated with therapy, the decision to treat would be best made on the basis of a DiSC assay result. This pilot study requires confirmation with a well-designed controlled clinical trial.

We have tested the efficiency of GM-CSF to mobilize peripheral blood progenitor cells (PBPC) and evaluated the hematological reconstitution after GM-CSF primed-PBPC infusion following myeloablative therapy. Twenty three patients suffering from hematological malignancies were included in this study. Starting 24 hours after completion of a standard dose chemotherapy including vindesine, cyclophosphamide, adriblastine, prednisone, (VCAP), 5 micrograms/kg sub-cutaneous daily dose GM-CSF was given for a median time of 14 days followed by three consecutives cycles of leukapheresis. Fifteen of these 23 patients underwent GM-CSF primed-PBPC autotransplantation following high dosed intensification regimen. PBPC collection and hematopoietic recovery were compared with a 15 patients control group who did not receive GM-CSF. No marrow or growth factors were administered after PBPC reinfusion in the two groups. VCAP/GM-CSF mobilization induced significantly higher yields of CFU-GM (3.8 fold) than did VCAP mobilization alone, 19 x 10(4)/kg (2-73) vs 5 x 10(4)/kg (2-27), (p < 0.005). The median number of days to achieve 1.10(9)/l neutrophils, platelet count > 20.10(9)/l and > 50.10(9)/l was significantly lower in the GM-CSF group than in the control group, respectively 13 vs 19 days (p = 0.04), 15.5 vs 27 days (p < 0.02), 19 vs 51 days (p < 0.01). When compared with the control group, transfusion requirements and median of hospital stay were both significantly decreased for the patients receiving GM-CSF primed-PBPC. Our study confirms that infusion of GM-CSF primed-PBPC as a sole source of hematopoietic support improves hematopoietic reconstitution following myeloablative therapy.