Cell cycle distribution of cord blood-derived haematopoietic progenitor cells and their recruitment into the S-phase of the cell cycle.

The objective of this study was to evaluate the cycling status of cord blood (CB)-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle. By using the cytosine arabinoside (Ara-C) suicide approach, we found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96 +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6 +/- 0.4% in the S-phase. Staining of CD34+ cells with an antistatin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase 68 +/- 7% of cells were in the G0 phase of the cell cycle. Incubation (24 h) with interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in numbers. Flow cytometric DNA analysis also showed an increase in CD34+ cells in the S-phase upon continuous exposure to these cytokines. Our findings indicate that: (i) very few CB-derived CFC or LTC-IC were in the S-phase of the cell cycle; (ii) a substantial amount of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction was cycling, as found in the G1 phase of the cell cycle; and (iii) 24-h incubation with IL-3, SCF and G-CSF could drive a proportion of progenitor cells into the S-phase without reducing their number. These data might be useful for gene transfer protocols and the ex vivo expansion of CB-derived progenitor cells.

Efficacy of epirubicin/paclitaxel combination in mobilizing large amounts of hematopoietic progenitor cells in patients with metastatic breast cancer showing optimal response to the same chemotherapy regimen.

BACKGROUND AND OBJECTIVE: Based on our preliminary experience, we have further evaluated the capacity of the paclitaxel/epirubicin combination (at the dose of 175 and 90 mg/m(2), respectively) plus G-CSF to mobilize hematopoietic progenitors into the circulation. DESIGN AND METHODS: The study was conducted in a homogeneous cohort of 25 stage IV breast cancer patients showing response to three cycles of the same chemotherapy regimen and who were included in a high-dose chemotherapy program. RESULTS: In most cases (68%) more than 5_10(6) CD34+ cells/kg b.w. (the threshold fixed in our study) were collected by a single leukapheresis, 28% and 4% of patients requiring 2 and 3 procedures, respectively. Based on the CD34+ cell count in the peripheral blood, most of the leukaphereses (53%) were performed on day 11 after chemotherapy. More than 50 CD34+ cells/mL along with a preleukapheresis WBC count between 10 and 20_10(9)/L predicted that only a single harvest would be required in 100% of cases. The evaluation of the clonogenic potential of collected cells showed that a large number of committed colony-forming cells (CFCs) and more primitive long-term culture-initiating cell (LTC-IC) hematopoietic progenitors were present in 20 harvests studied. INTERPRETATION AND CONCLUSIONS: These data demonstrate that the epirubicin/paclitaxel combination followed by G-CSF, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into the circulation which can then be safely employed to support myeloablative regimens.

Mobilization and selection of peripheral blood hematopoietic progenitors in children with systemic sclerosis.

BACKGROUND AND OBJECTIVE: Autologous transplant of lymphocyte-depleted peripheral blood stem cells has been proposed for treatment of patients with severe autoimmune disease. However, until now, no data are available on the safety and feasibility of both stem cell collection and selection in pediatric patients with these disorders. We report on three children affected by systemic sclerosis with lung involvement, who received chemotherapy and granulocyte colony-stimulating factor (G-CSF) to mobilize autologous peripheral blood progenitors. DESIGN AND METHODS: The priming regimen consisted of cyclophosphamide (CY, 4 g/m(2)) and G-CSF (lenograstim, 10 microg/kg/day starting 2 days after cyclophosphamide administration until stem cell collection). Leukapheresis was performed when WBC and CD34+ cell count were at least 2 x 10(9)/L and 0.03 x 10(9)/L, respectively. In the first patient, positive selection of CD34+ cells was performed through the Ceprate SC stem cell concentrator (CellPro, Bothell, WA, USA). In the remaining 2 children, progenitor cells were also purged with negative selection of CD4+ and CD8+ lymphocytes performed by means of the Isolex 300i device (Baxter). RESULTS: All patients tolerated the priming regimen well and did not present any sign of autoimmune disease exacerbation. Collection was successful in all children and the number of CD34+ cells before selection ranged between 10.7 x 10(6) and 17.6 x 10(6)/kg of patient body weight. The selection of haematopoietic stem cells in the 3 patients resulted in at least 2. 6-log T-cell depletion of the cell content, with a recovery of the initial value of CD34+ cells comprised between 21 and 44%. After, a preparative regimen consisting of CY (200 mg/kg over 4 days) and Campath-1 G in vivo (10 mg/day for 2 consecutive days), patients were transplanted using cryopreserved lymphocyte-depleted progenitor cells. In all cases, a prompt hematopoietic engraftment was observed. INTERPRETATION AND CONCLUSIONS: Taken together these data suggest that mobilization, collection and selection of hematopoietic progenitors are safe and feasible in children with autoimmune disease.

Feasibility and safety of a new technique of extracorporeal photochemotherapy: experience of 240 procedures.

BACKGROUND AND OBJECTIVE: Extracorporeal photochemotherapy (ECP) is a therapeutic approach based on the biological effects of ultraviolet light (UV) - A and psoralens on mononuclear cells collected by apheresis. Recently, ECP has been under investigation as an alternative treatment for various immune and autoimmune diseases. The aim of this study was to evaluate the safety and feasibility of a new three-step ECP technique, in terms of reproducibility, acceptance, tolerability, and short and long term side effects. DESIGN AND METHODS: Seventeen patients affected by acute or chronic graft-versus-host disease (GvHD), pemphigus vulgaris, or interferon-resistant chronic hepatitis C and one patient being treated for prevention of heart transplant rejection underwent 240 ECP procedures. MNC collection and processing parameters were recorded, biological effects of UV-A/8 methoxy-psoralen (8-MOP) were evaluated, and short and long term side effects were monitored. RESULTS: At a mean follow up of 7 months (range 2-19) 240 ECP had been completed, a mean of 7,136 mL (range 1,998-10,591) of whole blood having beenprocessed per procedure. The mean of total nucleated cells collected per procedure was 6.5x109 (range 0.65-23.8), with a mean MNC percentage of 85% (41. 4-98%) in a mean final volume of 115.5 mL (37-160). No severe side effects were documented and no infectious episodes occurred throughout the course of the treatment. INTERPRETATION AND CONCLUSIONS: The new ECP technique was highly reproducible as regards the collection and each processing step. Short and long term side effects were mild. No increase in infectious episodes was recorded. All patients willingly underwent ECP, demonstrating an excellent tolerability for the procedure even after several courses.

Autologous platelet collection and storage to support thrombocytopenia in patients undergoing high-dose chemotherapy and circulating progenitor cell transplantation for high-risk breast cancer.

OBJECTIVES: The use of circulating progenitor cell support following high-dose chemotherapy for malignancies decreases but does not entirely abolish platelet transfusion requirement. We investigated the feasibility of supporting the posttransplant thrombocytopenic phase exclusively with autologous platelets collected by apheresis and cryopreserved. METHODS: 25 patients underwent plateletpheresis during the platelet rebound occurring after high-dose cyclophosphamide. Autologous platelets were cryopreserved in 5% dimethylsulfoxide, thawed and transfused during the aplastic phase after the myeloablative regimen whenever clinically required. RESULTS: A single plateletpheresis was carried out in all patients, allowing the harvest of a platelet concentrate with a mean value of 7.7 x 10(11) platelets. No significant procedure- or transfusion-related side effects were recorded. Mean platelet recovery after freezing and thawing was 63% and the mean number of platelet reinfused was 4.8 x 10(11); 23 of 25 patients were fully supported with autologous platelets. CONCLUSION: Plateletpheresis performed in our selected group of patients was found to be a safe and effective procedure to collect large amounts of autologous platelets; the numbers obtained proved to be sufficient for the transfusion demand of almost all patients.

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.

Autologous platelet transfusion in patients receiving high-dose chemotherapy and circulating progenitor cell transplantation for stage II/III breast cancer.

BACKGROUND AND OBJECTIVE: Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC). DESIGN AND METHODS: PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells > 5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour. RESULTS: Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (> 38 degrees C) and had mucositis > G2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment > 7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion). INTERPRETATION AND CONCLUSIONS: Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.

Multicyclic dose-intensive chemotherapy with circulating progenitor cell support for high-risk primary breast cancer.

The feasibility and safety of the administration of multiple cycles of dose-intensive chemotherapy (CT) supported with repeated reinfusions of circulating progenitor cells (CPCs) were evaluated in a prospective study of adjuvant initial therapy of poor-prognosis breast cancer. Eighteen patients with resectable breast cancer involving >/= 10 axillary nodes or >/= 5 axillary nodes and negativity of the estrogen receptor status received a cycle of standard FEC regimen (5-FU 600 mg/m2, epirubicin 60 mg/m2, CTX 600 mg/m2, i.v. on day 1) followed by G-CSF as CPC mobilization technique. Collected CPCs were fractionated and reinfused, with G-CSF, after each of the 4 subsequent cycles of high-dose FEC (HD-FEC) (5-FU 750 mg/m2, epirubicin 120 mg/m2, CTX 3 g/m2, i.v.) planned at 21 day intervals. The median numbers of CD34+ cells and CFU-GM collected (with one or two leukaphereses per patient) were 9.7x10(6)/kg (range: 2.5-22.9) and 9.9x10(4)/kg (range: 1.9-27.3), respectively, and day 9 was the median first day of procedure (range: 8-12) after FEC. All patients received the 4 courses of HD-FEC (for a total of 72 cycles). Hemopoietic recovery was rapid after each cycle and there was no treatment-related delays in CT administration. Mucositis was the major non-hematological toxicity. There were 2, 3, 7 and 9 episodes of WHO grade 3/4 mucositis in cycles 1, 2, 3 and 4, respectively. These severe episodes lasted a median of 4 days (range: 2-6) but no patient required parenteral nutrition. The mean +/- SD total hospital stay lasted 10 +/- 2 days. The delivery of 4 cycles of dose-intensive FEC CT supported by CPCs (mobilized with a single course of standard-dose FEC + G-CSF) is feasible and safe. It could represent an effective alternative strategy to other more aggressive programs for the adjuvant therapy of high-risk early breast cancer.

Both cycling and noncycling primitive progenitors continue to be mobilized into the circulation during the leukapheresis of patients pretreated with chemotherapy and G-CSF.

Colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) include a spectrum of progenitor types whose potential contributions to the haemopoietic recovery seen in patients transplanted with mobilized peripheral blood progenitor cells (PBPC) remains unclear. We evaluated both the number and cycling status of the circulating LTC-IC and CFC harvested from 12 patients treated with chemotherapy and G-CSF using a modified 6-week LTC-IC assay. The frequency of the LTC-IC and CFC in the mobilized PB samples were increased 45- and 750-fold, respectively. Interestingly, comparison of these values for PB samples, taken just prior to the start of the leukapheresis, with the progenitor content of the 3 h harvest, showed that, on average, the leukapheresis product contained 19 times more LTC-IC (P < 0.01) than had been detectable in the entire blood volume of the patients at the start of the collection, whereas the number of CFC collected was approximately the same as the number in the initial circulating pool of PBPC. Cycling studies showed many of the LTC-IC in the apheresis collections to be proliferating although not more so than in the steady-state marrow LTC-IC compartment (i.e. per cent kill of mobilized LTC-IC after 16 h in 3H-Tdr = 70 +/- 8%, n = 9). On the other hand, the majority of the CFC in the apheresis collections were initially quiescent (per cent kill after 16 h in 3H-Tdr = 37 +/- 6%, n = 12). These findings demonstrate the rapidity with which a primitive subset of LTC-IC may enter the circulation during the early phase of rebound haemopoiesis induced by chemotherapy plus G-CSF and provide evidence of differences in the mechanisms regulating LTC-IC and CFC mobilization.

Collection of circulating progenitor cells after epirubicin, paclitaxel and filgrastim in patients with metastatic breast cancer.

The efficacy of high-dose chemotherapy (HDC) and circulating progenitor cell (CPC) transplantation in metastatic breast cancer (MBC) relies mainly on giving this treatment after a response to conventional induction chemotherapy has been achieved. For this reason an optimal mobilization regimen should be therapeutically effective while minimizing the number of leucaphereses required to support the myeloablative therapy. The combination of an anthracycline and paclitaxel in chemotherapy-untreated MBC has produced impressive response rates. We evaluated the CPC-mobilizing capacity of the combination epirubicin (90 mg m(-2)) and paclitaxel (135 mg m(-2)) followed by filgrastim (5 microg kg(-1) day(-1)) starting 48 h after chemotherapy administration in ten patients with MBC who were eligible for an HDC and CPC transplantation programme. Leucaphereses were performed by processing at least two blood volumes per procedure at recovery from neutrophil nadir when CD34+ cells in the peripheral blood exceeded 20 microl(-1). In most patients (six out of 10) more than 2.5 x 10(6) CD34+ cells kg(-1), a threshold considered to be sufficient for haematopoietic reconstitution, were collected with a single apheresis. In the remaining four patients an additional procedure, performed the following day, was enough to reach the required number of progenitors. These data suggest that the epirubicin-paclitaxel combination, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into circulation.

Peripheral blood stem cell collection from healthy donors for allogeneic transplantation.

There is great interest in the use of peripheral blood stem cells (PBSC) for allogeneic transplantation, based on the good results seen with autologous PBSC infusion. Reasonable caution exists regarding the use of allogeneic PBSC for transplantation because of donor toxicities due to rhG-CSF administration and the risk of graft-versus-host-disease (GVHD) in the recipient because of the large number of T-cell infused. We present preliminary data on allogeneic PBSC collections and transplantation in ten patients affected by advanced leukemia (eight patients), severe aplastic anemia (one patient) and sickle cell anemia (one patient). Seven donors were HLA-identical siblings, while the other three were mismatched for three, two and one locus, respectively. All donors received rhG-CSF at a dose of 12 micrograms/kg for a mean of 5 days. Leukaphereses were performed with the aim of collecting a minimum of 5 x 10(6)/kg (recipient's weight) CD 34+ cells. Collection timing was determined by monitoring CD 34+ cells in the donor's peripheral blood from the second day of rhG-CSF therapy. The PBSC collections yielded a mean of 10.05 x 10(8) MNCs/kg and of 10.48 x 10(6) CD 34+ cells/kg (recipient's weight). PBSC were immediately infused after collection in patients given myeloablative therapy. Engraftment was observed in each patient at a mean of 13.2 days for an absolute neutrophil count (ANC) more than 0.5 x 10(9)/L and of 26.5 days for a platelet count of more than 20 x 10(9)/L. Eight patients experienced no or moderate acute GVHD, whereas two patients died of grade 4 GVHD, notwithstanding GVHD prophylaxis with cyclosporine and prednisone. Two other patients died of viral and fungal infections, respectively, despite prophylaxis. The remaining six patients are alive between 58 and 430 days after transplant. Our results document that allogeneic PBSC are capable of engraftment after a myeloablative regimen. Controlled trials are necessary to compare the potential benefits of this approach with the results obtained in allogeneic bone marrow transplantation.

Circulating progenitor cell collection: experience from 275 leukaphereses in various malignancies and in healthy donors.

BACKGROUND: Blood cell transplantation has become a new type of support in high-dose chemotherapy (HDC) for several oncologic and hematologic diseases. Over the last few years the demand for circulating progenitor cell (CPC) collection by blood cell separators has grown dramatically, and transfusion services must manage new CPC programs. MATERIALS AND METHODS: A protocol for optimizing the collection and clinical use of CPC is described. The results of 275 harvestings were studied: 128 patients were divided into 5 groups according to tumor type (A: breast cancer; B: Hodgkin's disease; C: non-Hodgkin lymphoma; D: multiple myeloma; E: various solid tumors). An additional group (F) consisted of 11 healthy donors. Factors affecting collection (mobilizing regimen or previous radiation therapy) and side effects were investigated. RESULTS: The mean values of mononuclear cells (MNC x 10(7)/kg) and CD34+ cells (x 10(6)/kg) collected per leukapheresis in the 6 respective groups were: 31.4 and 4.6 in group A; 26.4 and 3.4 in group B; 21.8 and 5.8 in group C; 24.6 and 2.4 in group D; 26.8 and 2.9 in group E; 60 and 6 in group F. Previous chemotherapy and/or radiation therapy were the main factors influencing CPC harvesting. The different chemotherapy regimens employed demonstrated no significant differences in their mobilizing efficacy. Side effects related to leukapheresis were few (2.3% of the procedures) and manageable. CONCLUSIONS: CPC collection is feasible in a wide range of clinical situations. Careful clinical evaluation of patients, accurate monitoring of progenitor cell release and collection timing are important for obtaining a sufficient number of CPC for hemopoietic recovery. Previous chemotherapy and radiotherapy are the main factors influencing CPC harvests. The mobilizing regimens employed showed no substantial differences in their efficacy.

Cryoprecipitate-poor plasma fraction (cryosupernatant) in the treatment of thrombotic thrombocytopenic purpura at onset. A report of four cases.

Treatment of thrombotic thrombocytopenic purpura (TTP) with cryoprecipitate-poor plasma (cryosupernatant) as substitution liquid has brought a portion of the relapsing forms of this syndrome under control. We experimented the use of plasma exchange (PE) with CPP as plasma substitute in the initial treatment of four patients with TTP at onset, in an attempt to elicit a faster response to therapy. We observed a clinical response in the four patients treated; however, laboratory indices of complete and stable response (platelet count, serum lactate dehydrogenase level) did not normalize in concert with clinical improvement.

Usefulness of plasma exchange in recurrent nephrotic syndrome following renal transplant.

Nephrotic syndrome due to focal segmental glomerulosclerosis (FSGS) frequently recurs even after transplantation and may cause failure of the renal allograft. We report a case in which plasma exchange (PE) was used to treat a 32-year-old patient with biopsy-proven recurrence of FSGS in a second renal transplant after the first allograft had failed. One year after the second renal graft, the patient presented with proteinuria of 5 g/day and a creatinine level of 1.46 mg/dl. A course of 9 PE was performed over a 15-day period. Proteinuria improved rapidly, and 20 days after the last PE, it was already down to 0.8 g/day. This level continued to decrease progressively (0.5 g/day after 3 months) and reached zero after approximately 6 months. Eleven months after PE treatment, the patient had no proteinuria and his plasma creatinine level was normal. In our opinion PE should be instituted early in recurrent nephrotic syndrome after renal transplantation. The optimum frequency of such treatment still has to be established, especially with regard to its use as long-term maintenance therapy.

[Autologous blood collection in gynecologic oncologic surgery]. / Il predeposito di sangue autologo in chirurgia oncologica ginecologica.

Autologous blood transfusion, as an alternative to homologous blood, must be considered for those patients who require blood. Transfusion in gynecologic oncology surgery is often required and autologous blood transfusion has been utilized. Between January 1988 and December 1992 a total of 162 surgical procedures for gynecologic cancers were performed in the same number of patients. Of these only 102 were eligible for autologous blood transfusion as predonation. The mean age of patients was 57.8 years (range 35-81). Forty-three patients were affected with endometrial carcinoma, 31 with carcinoma of the cervix, 21 with ovarian carcinoma and 7 with vulvar cancer. Collected autologous blood units were 138 (mean 1.35 every patient). Indications for predeposited blood transfusion was given by a hemorrhage greater than 100 cc intraoperatively or hemoglobin level less than 10 g/dl until 1988 or less than 8 g/dl since 1989. Forty-eight (34.8%) of the collected units were transfused to 39 autologous donors (mean 1.2 units every patient). There was a significant difference in transfusion rate in patients: endometrium 25.8%, ovary 28%, cervix 45%, vulva 72.7%. Unused autologous blood units were discarded at the expiration date: they were 90, 65% of collected ones. In 6 patients homologous blood was necessary other than autotransfusions. Our experience demonstrates that the transfusion requirement in gynecologic cancer surgery depends on pattern of neoplasm and consequently of surgical procedure. Patients with carcinoma of the cervix and vulva are at risk for transfusion and have appropriate indications for autologous donation.(ABSTRACT TRUNCATED AT 250 WORDS)