It is important to optimize methods to mobilize hematopoietic stem cells into peripheral blood (PB) for successful allogeneic peripheral blood stem cell (PBSC) transplantation. Our primary intent was to investigate the role of GM-CSF for mobilization in normal healthy donors and to compare its efficacy in mobilizing stem cells alone, in concurrent combination and in sequential combination with G-CSF in this study. We analyzed the results of the PBSC harvest through large volume leukapheresis from 48 normal healthy donors mobilized by three different regimens including GM-CSF. Donors were assigned sequentially to one of the following regimens for mobilization: GM-CSF 10 microg/kg/day alone (group 1, n = 9); concurrent combination (group 2, n = 20) of G-CSF 5 microg/kg/day and GM-CSF 5 microg/kg/day; sequential combination (group 3, n = 19) of GM-CSF alone 10 microg/kg/day for 3 days followed by G-CSF alone 10 microg/kg/day for 2-3 days. The harvested CD34(+) cell count (P < 0.05) was statistically higher in group 3 than in group 1 or 2. Pre-collection WBC count in donors (P < 0.05), harvested MNC (P < 0.05) and CD3(+) cell count (P < 0.05) of group 2 or 3 were significantly higher than those of group 1. Recipients who received stem cells mobilized with combination regimens showed an earlier recovery of WBC and platelets count than those with GM-CSF alone. The incidence of acute graft-versus-host disease was not statistically different among three recipient groups. GM-CSF-based mobilization was well tolerated in normal healthy donors. The sequential combination regimen appears to be an excellent mobilization strategy and might be preferred as the optimal method in some clinical situations that need a higher number of stem cells.

Allogeneic mobilized peripheral blood progenitor cells instead of bone marrow are increasingly used to restore hematopoiesis after myeloablative therapy. Data supporting this important change of clinical practice are scarce. We therefore assigned patients with early leukemias to peripheral blood or bone marrow transplantation; the occurrence of acute and chronic graft versus host disease, survival, transplantation-related mortality, and relapse rates were compared. A total of 350 patients between 18 and 55 years of age with acute leukemias in remission or chronic myelogenous leukemia in first chronic phase were randomized to receive either filgrastim-mobilized peripheral blood progenitor cells or bone marrow cells from HLA-identical sibling donors after standard high-dose chemoradiotherapy. Neutrophil and platelet recovery occurred significantly faster after transplantation of peripheral blood progenitor cells than after bone marrow transplantation. Acute graft versus host disease of grades II-IV was significantly more frequent in recipients of peripheral blood progenitor cells than in recipients of marrow cells (52% vs 39%, odds ratio 1.74, 95% confidence interval 1.12-2.69, P =.013). The cumulative incidence of chronic graft versus host disease was 67% with peripheral blood progenitor cells and 54% with bone marrow cells (hazard ratio 1.67, 95% confidence interval 1.15-2.42, P =.0066). The estimated overall probability of survival at 2 years was 65% with either source of stem cells (hazard ratio 1.15, 95% confidence interval 0.79-1.67, P =.46). Disease-free survival, transplantation-related mortality at day 100, and relapse rates did not significantly differ between treatment arms. Peripheral blood is an equivalent source of hematopoietic stem cells compared with bone marrow if administered to patients with standard-risk leukemias. Long-term observation of patients with different diseases and stages of disease is necessary to ultimately define the role of both sources of stem cells.

Allogeneic haemopoietic stem-cell transplantation (HSCT) is the treatment of choice for many haematological malignancies and inherited disorders. When stem cells for transplantation come from a human leucocyte antigen matched unrelated donor, or from a partly mismatched related donor, ex-vivo T-cell depletion of the graft can prevent development of graft-versus-host disease, but lead in turn to a delay in immune reconstitution and a concordant increase in incidence of opportunistic infections and leukaemic relapses. We aimed to infuse T cells selectively depleted in allogeneic T cells that cause graft-versus-host disease using an ex-vivo procedure designed to eliminate alloactivated donor T cells, with an immunotoxin that reacts with a cell surface activation antigen, CD25.

Flow cytometry was used to enumerate tumour cells in longitudinal studies of peripheral blood from small cell lung cancer (SCLC) patients, together with magnetic bead selection to isolate and identify these cells. As part of a trial, 11 patients received either standard (four weekly) chemotherapy with ifosfamide, carboplatin, and etoposide (ICE) or accelerated (two weekly) ICE with filgrastim (granulocyte colony-stimulating factor [G-CSF]) and autologous stem cell support.

We have reported that treatment of melanoma patients with a vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP) and preceded by low-dose cyclophosphamide induces delayed-type hypersensitivity (DTH) to autologous, unmodified tumor cells and that this response is a significant predictor of survival. We analyzed the vaccines prepared for 284 patients who were treated following resection of regional or distant metastases to find out whether the dose and composition determined the immunological response. A positive DTH response (> or =5 mm induration) to unmodified autologous tumor cells was induced in 57% of the patients (median: 5 mm; range: 0-22 mm). Regression analysis showed no significant association between the magnitude of DTH and the number of live (trypan blue exclusion) melanoma cells per dose over a dosage range of 0.5-25.0 x 10(6). Surprisingly, there was a small but significant positive relationship between the mean number of dead cells in the vaccines of a given patient and that patient's maximum DTH to unmodified melanoma cells. Only 37% of patients whose vaccines contained >50% live cells developed DTH, as compared with 69% and 65% of patients whose vaccines contained 26% to 50% or < or =25% live cells, respectively. Thus, it appears that dead tumor cells contribute to the immunogenicity of the DNP vaccine, but other factors such as the administration schedule may be more important determinants of immunological and clinical outcome.

Treatment for extensive-stage small cell lung cancer (ES SCLC) or extrapulmonary small cell carcinoma (EPSC) is typically palliative. We set out to determine progression-free survival (PFS) and overall long-term survival of ES SCLC and EPSC patients, physiologically aged < or = 60 years, responding to first-line chemotherapy followed by high-dose combination alkylating agents with hematologic stem cell support. Patients in first-line chemotherapy response underwent stem cell collection (marrow, peripheral blood progenitor cells, or both) followed by high-dose therapy with 1 of 2 regimens: CBP (cyclophosphamide, cisplatin, and carmustine) or ICE (ifosfamide, carboplatin, and etoposide) with or without etanidazole. Involved-field radiotherapy was given to selected patients with oligometastatic disease distributed in sites allowing for reasonable radiation ports, and prophylactic cranial radiotherapy was given upon recovery to patients in complete response (CR) or near-CR. A total of 36 patients were treated. Of 29 patients with ES SCLC, 6 (21%) had achieved CR, 18 near-CR, and 5 partial response prior to high-dose therapy. Of 7 patients with EPSC, 3 (43%) had achieved CR, 3 had achieved near-CR, and 1 had progression of disease prior to high-dose therapy. Thirteen ES SCLC patients received high-dose CBP. Of the remaining 23 patients with SCLC or EPSC, 17 were treated with ICE and 6 with ICE plus etanidazole, a hypoxic cell sensitizer. Treatment-related mortality was 11% (4 of 36 patients). For all patients, the median event-free survival (EFS) was 5 months. The 2- and 5-year survivals after intensification were 12% (95% confidence interval [CI], 5%-31%) and 9% (95% CI, 3%-27%), respectively. Of the 30 patients in or near CR prior to high-dose therapy, 5 remain continuously progression-free (2 ES SCLC, 3 EPSC) for a median of 55 months (range, 1-96 months) after high-dose therapy. By multivariate analysis, factors associated with more favorable EFS were the use of a more aggressive induction regimen (ICE), and the EPSC histology. These factors were also associated with more favorable overall survival. Other factors associated with more favorable overall survival were the use of short induction therapy (< or = 4 cycles) and younger age (<50 years). Except for high-dose ICE with etanidazole, the use of high-dose systemic therapy in ES SCLC and EPSC was associated with low treatment-related morbidity and mortality over the past 5 years. Late complications were infrequent, and most patients returned to full-time work and activity, barring disease recurrence. Nonetheless, few patients with ES SCLC have progression-free long-term survival. We conclude that high-dose therapy is not indicated as an approach for ES SCLC, except as part of an investigative trial. Conversely, 3 of the 7 patients with EPSC remain relapse-free (range, 1-96 months), warranting further phase II evaluation of this approach in this population.

Circumstantial evidence has implicated tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of anemia of chronic disease (ACD) in rheumatoid arthritis (RA). We investigated the role of TNF-alpha in erythropoiesis of patients with active RA (n = 40) and the effect of anti-TNF-alpha antibody administration (cA2). Patients with RA had lower numbers of CD34+/CD71+ and CD36-/glycophorin A+ (glycoA+) bone marrow (BM) cells and increased proportions of apoptotic cells within the CD34+/CD71+ and CD36+/glycoA+ cell compartments, compared to healthy controls (n = 24). Erythroid burst-forming units (BFU-Es) obtained by BM mononuclear or purified CD34+ cells were significantly lower in RA patients compared to controls. These abnormalities were more pronounced among patients with ACD. Increased TNF-alpha levels in patient long-term BM culture supernatants inversely correlated with BFU-Es and hemoglobin levels and positively with the percentage of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells. Following cA2 therapy, a normalization was documented in the number of CD34+/CD71+ and CD36-/glycoA+ cells, the number of BFU-Es, and the proportion of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells, which was associated with a significant increase in hemoglobin levels compared to baseline. Recovery from anemia was more prominent in patients with ACD. The exogenous addition of an anti-TNF-alpha antibody in the cultures increased BFU-E number in patients prior to cA2 treatment but not after treatment, further substantiating the inhibitory role of TNF-alpha on patients' erythropoiesis. We conclude that TNF-alpha-mediated apoptotic depletion of BM erythroid cells may account for ACD in RA and that cA2 administration may ameliorate ACD in these patients by down-regulating the apoptotic mechanisms involved in erythropoiesis.

Allogeneic stem cell transplantation is curative for certain cancers, but the high doses of chemotherapy/radiotherapy lead to toxicity. Here, we treat patients with refractory cancer with 100 cGy total body irradiation (TBI) followed by infusion of nonmobilized pheresed allogeneic peripheral blood cells. Twenty-five patients, with a median age of 47 years, with refractory cancers were enrolled. Eighteen patients received sibling and 7 received unrelated cord blood cells. Donor chimerism was assessed at weeks 1, 2, 3, 4, and 8 after transplantation. Seven patients with solid tumors received a sibling transplant and 6 received a cord blood transplant; none achieved donor chimerism, but 1 treated at the higher dose level of 1 x 10(8) CD3+ cells/kg had a transient nodal response. Twelve patients with hematologic malignancies were treated; 1 received a cord blood transplant and 11 received sibling donor cells. Nine of these 11 patients achieved donor chimerism, ranging from 5% to 100%. Four patients had sustained complete remission of their cancers, including one patient with transient 5% donor chimerism. The development of chimerism correlated with hematologic malignancy (P <.001), total previous myelotoxic chemotherapy (P <.001), T-cell dose (P =.03), and graft-versus-host disease (P =.01). Tumor response correlated with donor chimerism (P =.01). Engraftment was achieved in patients with hematologic malignancies who had been heavily pretreated, suggesting the degree of immunosuppression may be a determinant of engraftment. Low-dose TBI and allogeneic lymphocyte infusion may induce remission in patients with refractory hematologic malignancy.

In a previous multicenter phase III trial comparing peripheral blood stem cell transplantation (PBSCT) to bone marrow transplantation (BMT) from HLA-matched related donors, we found no statistically significant difference in the cumulative incidence of clinical extensive chronic graft-versus-host disease (GVHD) in the 2 groups. We have analyzed the results in more detail to determine whether the clinical characteristics of chronic GVHD after PBSCT might be distinct from those that occur after BMT. Clinical extensive chronic GVHD developed in 39 of 63 recipients of PBSCs and in 32 of 63 BM recipients who were alive and free of malignancy at day 100 after the transplantation. No significant differences were found in the time and type of onset of clinical extensive chronic GVHD or in the frequency of complications associated with severe morbidity. Involvement of skin and female genital tract was more frequent in PBSC recipients than in BM recipients. The cumulative incidence of chronic GVHD at 3 years was similar in the 2 groups, but the number of successive treatments needed to control chronic GVHD was higher after PBSCT than after BMT (P =.03), and the duration of glucocorticoid treatment was longer after PBSCT compared to BMT (P =.03). These results suggest that chronic GVHD after PBSCT may be more protracted and less responsive to current treatment than chronic GVHD after BMT. Assessment of the overall benefits of PBSCT compared to BMT will require continued long-term follow up of morbidity associated with chronic GVHD.

Recent observations suggested that targeted monoclonal antibodies might be best employed in lymphoid malignancies under conditions of minimal residual disease. This prompted us to investigate the role of Campath-1H as treatment for patients with chronic lymphocytic leukemia (CLL) in whom fludarabine (FAMP) had produced a marked disease debulking with persistence of bone marrow (BM) infiltration or a complete remission (CR) without the disappearance of the molecular aberration (IgH monoclonal expression). As intravenous Campath-1H is almost invariably associated with reactions, sometimes of WHO grade 3-4, we adopted the subcutaneous route of administration, which proved to induce rare and mild adverse reactions but had comparable efficacy. DESIGN AND METHODS. Nine patients (7 males, 2 females) with a median age of 55 years (range 41-61) who responded to FAMP (1 had a CR, 5 a nodular partial remission [PRN], and 3 a partial remission [PR]), according to NCI Working Group Criteria, received subcutaneous Campath-1H, three times a week for 6 weeks in escalating doses up to 10 mg. Monoclonal rearrangement of IgH was present in all patients before immunotherapy. Patients received acyclovir and cotrimoxazole as infection prophylaxis. Granulocyte colony-stimulating factor (G-CSF), at the dosage of 5-10 microg/kg/die, or intermediate-dose Ara-C (800 mg/m(2)/q 12h x 6 doses), was administered to obtain peripheral blood stem cell (PBSC) mobilization.

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

We tested the feasibility and toxicity of high activities Rhenium-186 hydroxyethylidene diphosphonate, with peripheral blood stem cell rescue in patients with progressive hormone refractory prostate cancer metastatic to bone. Twenty-five patients received between 2500 and 5000 MBq of Rhenium-186 hydroxyethylidene diphosphonate followed 14 days later by the return of peripheral blood peripheral blood stem cells. Activity limiting toxicity was defined as grade III haematological toxicity, lasting at least 7 days, or grade IV haematological toxicity of any duration or any serious unexpected toxicity. Activity limiting toxicity occurred in two of six who received activities of 5000 MBq and maximum tolerated activity was defined at this activity level. Prostate specific antigen reductions of 50% or more lasting at least 4 weeks were seen in five of the 25 patients (20%) all of whom received more than 3500 MBq of Rhenium-186 hydroxyethylidene diphosphonate. The actuarial survival at 1 year is 54%. Administered activities of 5000 MBq of Rhenium-186 hydroxyethylidene diphosphonate are feasible using autologous peripheral blood peripheral blood stem cell rescue in patients with progressive hormone refractory prostate cancer metastatic to bone. The main toxicity is thrombocytopaenia, which is short lasting. A statistically significant activity/prostate specific antigen response was seen. We have now commenced a Phase II trial to further evaluate response rates.

High-dose chemotherapy followed by transplantation of autologous haemopoietic stem cells (BEAM-HSCT) is frequently used to treat patients with relapsed Hodgkin's disease. We aimed to compare this treatment with conventional aggressive chemotherapy without stem-cell transplantation (Dexa-BEAM).

It is logical to expect that large-volume leukapheresis may be able to collect adequate numbers of PBSC with fewer procedures. To date, there is no agreement on the optimal volume of leukapheresis. Therefore, in this study we compared 8 l volume with 12 l and assessed whether a 50% increase in the blood volume processed would decrease the number of leukaphereses each patient needed to collect > or =2.5 x 10(6) CD34(+) cells/kg in normal mobilizers. PBSC mobilization was done with cyclophosphamide etoposide followed by rhG-CSF in all patients. Forty patients were randomized to undergo 8 l leukaphereses (n = 20 patients) or 12 l leukaphereses (n = 20). The median numbers of leukaphereses required in order to collect > or =2.5 x 10(6) CD34(+) cells/kg in patients processed with 8 l and 12 l were 1 (range 1-5) and 1 (1-4), respectively (P = 0.50). The median number of total nucleated cells (TNC) collected per patient was greater for the 12 l group (7.47 x 10(8)/kg vs 3.90 x 10(8)/kg, P < 0.001), as was the median number of total mononuclear cells (TMNC) (4.26 x 10(8)/kg vs 2.16 x 10(8)/kg, P < 0.001), whereas there was no difference between the two groups for the median number of CD34(+)cells collected per patient (8.94 x 10(6)/kg vs 8.60 x 10(6)/kg, P = 0.85). The TNCs and TMNCs collected per leukapheresis were again greater for the 12 l group (3.64 x 10(8)/kg vs 1.91 x 10(8)/kg, P = 0.001 and 2.17 x 10(8)/kg vs 0.88 x 10(8)/kg, P < 0.001), whereas there was no difference between the two groups for the median number of CD34(+) cells collected per leukapheresis (3.98 x 10(6)/kg vs 3.26 x 10(6)/kg, P = 0.90). This study showed that there is no difference between 8 l and 12 l volumes in regard to collected CD34(+) cells/kg and also the use of a 12 l leukapheresis volume did not decrease the number of leukaphereses performed compared with a 8 l leukapheresis volume. In fact, the use of the larger leukapheresis volume had the disadvantage of adding 60 min to the time the patient was on the machine.

The aim of this study was to evaluate and to compare in terms of toxicity the modulations of dose intensity of cyclophosphamide and doxorubicin in adjuvant chemotherapy for high-risk breast cancer. Four cycles of sequential high-dose chemotherapy with doxorubicin and cyclophosphamide (AC), supported with G-CSF and peripheral blood stem cells (PBSC) were administered to 81 women. Three successive cohorts were studied: doxorubicin (75 mg/m(2)) + cyclophosphamide (3000 mg/m(2)) every 21 days (group 1), doxorubicin (75 mg/m(2)) + cyclophosphamide (3000 mg/m(2)) every 15 days (group 2), and doxorubicin (75 mg/m(2)) + cyclophosphamide (6000 mg/m(2)) every 21 days (group 3). Seventy-five patients received four cycles of treatment with a total of 310 cycles administered. The received dose intensity of doxorubicin was higher in group 2 and that of cyclophosphamide was lower in group 1 than in the other two groups. Hematological and extra-hematological toxicities, as well as the number and duration of hospitalizations for toxicity, were significantly higher in group 3. We conclude that the group 3 regimen is associated with toxicities comparable to autologous transplantation. Increasing dose intensity of doxorubicin and cyclophosphamide is feasible in an outpatient setting and safe in groups 1 and 2 with the support of hematopoietic factor and PBSC.

The objectives of this study were to evaluate the effect of the number of infused CD34+ cells on hematopoietic recovery and on the cost in autologous peripheral blood stem cell transplantation (PBSCT).

To evaluate therapeutic results and prognostic factors from a series of 44 patients affected by de novo acute myeloid leukemia with multilineage dysplasia (MD-AML), treated with the combination of fludarabine, cytarabine and G-CSF (FLAG).

To investigate the effect of granulocyte colony-stimulating factor (G-CSF) donor-marrow priming on hematopoietic recovery and clinical outcome after allogeneic hematopoietic stem cell transplantation, we compared HILA-matched related marrow transplantation with and without G-CSF donor priming in a prospective randomized study for a homogeneous group of chronic myelogenous leukemia (CML) patients. Fifty patients (aged 12-41 years) with CML were enrolled in the study. Thirty-two patients (study group) received the marrow grafts primed with G-CSF at 3 to 4 micro/kg per day for 7 days prior to the marrow harvest, and 18 patients (control group) received the marrow grafts without G-CSF priming. All patients received the same graft-versus-host disease (GVHD) prophylaxis (cyclosporine A and methotrexate) and postgraft G-CSF treatment, 3 to 4 micro/kg daily until the absolute neutrophil counts (ANCs) were >10(9)/L. The primary end points were engraftment and incidence of acute GVHD. The secondary end points were the incidence of chronic GVHD, relapse, and overall disease-free survival. The study and control groups were comparable for age, sex, donor selections, conditioning regimens, and disease status. The median times to both neutrophil and platelet engraftment (ANC > 0.5 x 10(9)/L; platelets > 20 x 10(9)/L) were significantly faster in the study group than in the control group, at 15 versus 21 days (P < .001) and 17.5 versus 24 days (P < .001), respectively. G-CSF donor printing yielded significantly higher numbers of total nuclear cells in the marrow grafts compared to the numbers in the control grafts (7.2 versus 2.9 x 10(8)/kg, P < .001). Similar results were seen for CD34+ (6.1versus 2.7 x 10(6)/kg, P < .001) and colony-forming unit-granulocyte/macrophage (CFU-GM) cells (68 versus 16 x 10(4)/kg, P < .001). The incidence of grades II to IV acute GVHD was surprisingly low in the study group: only 2 (6.3%) of 32 transplantation patients in the study group developed grade II acute GVHD, limited to the skin, whereas 5 (27.8%) of 18 patients in the control group developed grades II to IV acute GVHD (P = .032). G-CSF priming did not change the total numbers of CD3+ cells in the marrow grafts but lowered CD4+ cells and increased CD8+ cells, resulting in a significant reduction of CD4:CD8 ratio (P = .018). Six patients in the study group developed chronic GVHD either during or after cyclosporine taper. There were no significant differences in chronic GVHD (24% versus 33.3%), relapse rates (12.5% versus 11.1%), and overall survival rates (78.1% versus 66.7%, P = .32) between the study and control groups during a median follow-up period of 24 months (range, 6-50 months). There was, however, a trend in favor of improved chronic GVHD and disease-free survival in the study group. We conclude that G-CSF donor-marrow priming accelerates both neutrophil and platelet engraftment and is associated with a very low incidence of grades II to IV acute GVHD in CML patients after HLA-matched sibling marrow transplantation.

There have been several studies on the presence of circulating tumor cells in the peripheral blood of patients with malignant tumors including prostate cancer (PCa) using reverse transcription-PCR (RT-PCR). One of the aims of these studies was to obtain high sensitivity that would enable early-stage diagnosis. However, they varied in their detection rates, and the methods were rather complicated. We have improved the RT-PCR assay combining three prostate-associated molecules, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) to reveal patients with poor prognosis.

To examine whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres before embryo transfer.