A Phase I trial was conducted to determine the safety, biological activity, and hematopoietic recovery by the combination of interleukin 6 (IL-6) and granulocyte-colony stimulating factor (G-CSF) after myelosuppressive chemotherapy in children. Patients <22 years of age at diagnosis with either recurrent or refractory solid tumors received ifosfamide 1,800 mg/m2/day x 5 days, carboplatin 400 mg/m2/ day x 2 days, and etoposide 100 mg/m2/day x 5 days, followed by daily s.c. G-CSF (5 microg/kg/day) and IL-6 (2.5, 3.75, or 5.0 microg/kg/day). Pharmacokinetic, proinflammatory mediator levels, hematopoietic colony assays, and cytokine receptor expression studies were performed during course one. Nineteen patients were evaluable for toxicity and received IL-6 at doses of 2.5 (n = 8), 3.75 (n = 5), or 5.0 (n = 6) microg/kg/day. Dose-limiting constitutional toxicity occurred in two of six patients at 5.0 microg/kg/day, two of five patients at 3.75 microg/kg/day, and two of eight patients at 2.5 microg/kg/day. The maximum tolerated dose (MTD) exceeded the lowest dose tested. Because of lack of drug availability, an MTD was not established. The maximum concentration of IL-6 (2.5 microg/kg/day) was 0.799 +/- 1.055 ng/ml (mean +/- SD). During the first course, the median time to absolute neutrophil count > or = 1,000/mm3 and platelets > or = 100,000 mm3 was estimated at 19 and 23 days, respectively. Peripheral blood progenitor cells expressing receptors to IL-3, IL-6, and G-CSF increased significantly over baseline (P < 0.05). After the first dose of IL-6, IFN-gamma levels were abnormal in 13 patients, and IL-1beta levels were abnormal in 10 patients. IL-6 has a high incidence of constitutional toxicity and a lower MTD in children compared with adults. In vivo use of IL-6 in children after chemotherapy remains limited. However, IL-6 may be more optimally investigated in children under ex vivo conditions.

Hematopoietic stem cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling as the donor is not available, an alternative donor stem cell source is needed. In primary T-cell immunodeficiencies, T-cell-depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the immune system in many but not all of the severe T-cell immune deficiency disorders. This study reports the use of umbilical cord blood (UCB) stem cell transplantation in severe T-cell immune deficiency. Umbilical cord blood was evaluated as a stem cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome (SCID), reticular dysgenesis, thymic dysplasia, combined immunodeficiency disease (CID), and Wiskott-Aldrich syndrome (WAS) when a matched sibling donor was unavailable. From 1/96 through 5/98, eight children received unrelated cord blood stem cell transplantation following a preparative regimen for the treatment of combined immunodeficiency diseases. The patients ranged in age from 2 weeks to 8 years. The cord blood units were 3/6 HLA antigen matches in two children. 4/6 in four children, and 5/6 in two child, with molecular HLA-DR mismatch in three of the children. The average time for neutrophil engraftment (absolute neutrophil count >500/mm3) was 12 days (range 10-15 days) and the average time for platelet engraftment (platelet count >20,000/mm3) was 36 days (range 24-50 days). A patient with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated donor cord blood transplantation. Five of six patients exhibited grade I graft-versus-host disease (GvHD). while one child had grade IV skin and gut GvHD. Immunologic reconstitution demonstrated that cord blood stem cell transplantation resulted in consistent and stable T-, B- and natural killer (NK) cell development. The kinetics of development were such that T-cell development occurred between 60 to 100 days. Initial T-cell engraftment consisted predominantly of CD45RO+, CD3+, and CD4+ T cells, and at 12 to 24 months changed to CD45RA+, CD3+, and CD4+ T cells, indicating de novo maturation of T cells. NK cell development occurred at approximately 180 days. B cells engrafted early, and study of functional B-cell antibody responses revealed that five of six patients in whom intravenous immune globulin has been discontinued have low detectable antibody responses to tetanus and diphtheria toxoid immunizations at 18 to 24 months posttransplantation. Unrelated umbilical donor cord blood is an alternative source of stem cells for transplantation in children with severe T-cell immune deficiency disorders when a suitable HLA-matched donor is not available and when a T-depleted haploidentical preparation is not beneficial. Benefits of UCB include rapid and reliable recovery of immune function, low risk of GvHD, and low viral transmission rate.

This study was conducted to determine the impact of immune activation, cytokine production and apoptosis on the naive CD4+ cell count and the function of hematopoietic progenitor cells during the initial phase of highly active antiretroviral therapy (HAART). Blood samples from 11 HIV-infected patients were collected prior to HAART and after 4 and 12 weeks of therapy. Flow cytometry was used to determine the naive CD4+ count and activated T cells. The cloning efficiency of progenitor cells was determined using a colony-forming cells assay. Finally, apoptosis and cytokine production were determined. During the study period, the naive CD4+ count and the cloning efficiency increased significantly. Immune activation was found in HIV-infected patients and decreased during HAART. The level of immune activation correlated negatively with both the naive CD4+ count and the function of progenitor cells. A negative correlation was found between apoptosis and the naive CD4+ count. Alterations in cytokine production during HAART or correlation between cytokine production and the naive CD4+ count or the cloning efficiency of progenitor cells were not detected. In conclusion, immune activation in HIV-infected patients treated with HAART is inversely correlated with the function of progenitor cells and the naive CD4+ count.

Granulocyte-colony stimulating factor (G-CSF) is being increasingly used in healthy volunteers to harvest haemopoietic stem cells. A possible role of G-CSF in the development of clonal disorders or leukaemia has been suggested. We analysed 144 patients with aplastic anaemia treated with immunosuppression protocols with or without G-CSF, with normal cytogenetics at diagnosis or immediately after immunosuppression. Our findings indicated that the risk of developing myelodysplasia or leukaemia was similar in patients with aplastic anaemia on immunosuppressive treatment with or without G-CSF. Therefore, it seems unlikely that G-CSF causes leukaemia in healthy volunteers.

In chronic myelogenous leukemia (CML), autologous stem cell transplantation could be a promising new approach for patients with no cytogenetic response after interferon alpha (IFN-alpha) therapy. We report data on 28 CML patients autotransplanted in chronic phase with peripheral blood progenitor cells mobilized with G-CSF (5 microg/kg/day x 5 days) given subcutaneously while continuing IFN-alpha therapy. At mobilization, 23 patients (82%) were in complete hematological remission (CHR), 16 (57%) achieved a minor cytogenetic response (mcr). We obtained, after stimulation, a median of 37.4 x 10(9)/l (6.9-108) white blood cells, 7.2 x 10(8)/kg (2.2-16.6) mononuclear cells, 39 x 10(4)/kg (4.8-403.5) CFU-GM and 4.2 x 10(6)/kg (0-58.6) CD34+ cells. Six patients received GM-CSF after transplantation. All patients engrafted, with no significant influence stemming from the Sokal index score and pretransplantation IFN-alpha therapy duration. The first cytogenetic evaluation after transplantation showed 11 (39%) major cytogenetic response (Mcr), and nine (32%) mcr with no significant correlation between these responses, the Sokal index score, and pretransplantation IFN-alpha therapy duration, although there was a significant impact from GM-CSF administration (P=0.01). After transplantation, 26 patients received IFN-alpha alone or associated with hydroxyurea. The median follow-up was 12 months after transplantation and 57 months after diagnosis. At the time of follow-up, nine patients were in CHR, six remained stable in chronic phase, three presented an mcr and one remained in Mcr. At the last follow-up, 22 patients were alive. We conclude that the results of this strategy are encouraging in poor IFN-alpha responders but that other prospective studies that try to maintain the cytogenetic responses obtained immediately after transplantation are needed.

We describe a single-center experience of 23 consecutive patients (median age, 35 years) with hematologic malignancies who received allogeneic peripheral blood stem cell transplants (alloPBSCTs) from HLA-identical siblings. Ten patients had standard-risk disease and 13 had high-risk disease. Twenty-one patients received alloPBSCT as a primary transplant, and the remaining 2, with high-risk disease, as a second transplant after posttransplantation relapse. All donors received daily subcutaneous injections of granulocyte colony-stimulating factor at a dose of 10 microg/kg, and peripheral blood stem cells were collected by 1 to 3 aphereses. Median numbers of CD34+ and CD3+ cells infused were 5.8 x 10(6)/kg (range, 1.3-19.7 x 10(6)/kg) and 4.9 x 10(8)/kg (range, 1.9-8.6 x 10(8)/kg), respectively. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and methotrexate (18 patients) or CyA and methylprednisolone (5 patients). Rapid hematologic engraftment was observed in 20 of the 23 patients. Median days to absolute neutrophil counts >0.5 x 10(9)/L and platelet counts >20 x 10(9)/L were 12 (range, 9-18 days) and 14 (range, 10-128 days), respectively. Acute GVHD of grade 2-4 was observed in 6 of 20 evaluable patients (30%) and extensive chronic GVHD in 8 of 15 evaluable patients (53%). Ten of the 23 patients (44%) were surviving in continuous complete remission 191 to 1492 days (median, 643 days) posttransplantation. Treatment-related death within 100 days posttransplantation was observed in 6 of the 23 patients (26%). Six of the 23 patients (26%) developed relapse at a median 81 days (range, 38-160 days) posttransplantation. Further study is needed to assess the precise benefits of alloPB-SCT compared with allogeneic bone marrow transplantation.

Chemotherapy, in addition to recombinant growth factors, has been effective in mobilizing stem cells. Unfortunately, the use of chemotherapy for this purpose has resulted in profound myelosuppression and increased morbidity. Docetaxel, the single most active agent in the treatment of advanced breast cancer, was evaluated for its potential to mobilize stem cells when given at conventional doses followed by granulocyte colony-stimulating factor (G-CSF). Sixteen high-risk breast cancer patients were mobilized with a regimen consisting of docetaxel (100 mg/m2) followed by daily G-CSF (10 microg/kg), beginning 72 h after the docetaxel, and continuing until completion of the apheresis. The median white blood cell count (WBC) nadir was 1,000/microl (range 500 to 4000/microl ) occurring a median of 6 days (range 4 to 9 days) after the docetaxel. No patient experienced a neutropenic febrile episode due to the mobilization regimen. The median time interval for initiating the apheresis was 8 days (range 6 to 11 days) following the docetaxel. The median number of apheresis was 2 (range 1 to 3) in each patient. Stem cell recovery as measured by the CD34 cell count x 10(6)/kg was a median of 5.2 (range 1.4 to 15.1). A significant correlation was found between CFU-GM, BFU-E, and CFU-GEMM/kg and CD34 cells/kg (r = 0.891, 0.945, and 0.749, respectively, p < 0.001). When our results were compared to a matched cohort receiving G-CSF alone, the docetaxel group demonstrated a superior CD34 cells/kg yield (p = <0.001). Following myeloablative chemotherapy consisting of thiotepa and cyclophosphamide with or without carboplatinum, the hematopoetic recovery determined by an absolute neutrophil count (ANC) of greater than 500/microl and an unsupported platelet count of > or =20,000/microl for 48 h, was a median of 10 days (range 9 to 14 days) and 10 days (range 8 to 30 days), respectively. The results demonstrate that conventional dose docetaxel, combined with G-CSF, is an effective mobilization regimen with minimal toxicity in high-risk breast cancer patients.

Therapeutic vaccination of tumor patients with cytokine gene-transfected tumor cells leads to tumor regression in animal models but has so far not resulted in significant clinical benefit. We and others demonstrated that tumor cells transfected to mediate overexpression of a cytokine gene activate immunologic effector cells for an improved proliferation rate and significantly higher antitumoral cytotoxic activity. Here, we performed a pilot study of therapeutic vaccination in patients with metastatic disease. Autologous tumor cells were simultaneously transfected with novel minimalistic, immunogenically defined, gene expression constructs (MIDGE) for overexpression of the two cytokines interleukin 7 (IL-7) and GM-CSF and newly designed double stem-loop immunomodulating oligodeoxyribonucleotides (d-SLIM) as a Th1-promoting and NK cell-stimulating adjuvant. Transfection was performed ex vivo by ballistomagnetic gene transfer. Patients received four subcutaneous injections of at least 1 x 10(6) of their expression-modulated and immunomodified autologous tumor cells. Ten patients have been enrolled in the study protocol. In all patients no adverse effects could be detected. IL-7 and interferon gamma levels were elevated in the serum of the patients after treatment. Interestingly, cytotoxicity of patient-derived PBLs increased significantly during treatment. All 10 patients had progressive disease when entering our protocol. One complete, one partial, and one mixed response with progression of abdominal metastases and regression of lung metastases were observed. Two patients showed a stable disease after treatment and five patients remained in progressive disease. Our observations confirm the capability of autologous expression-modified and immunomodulated tumor cell vaccines to stimulate a strong immune response in patients with metastatic cancer even in the presence of a large tumor burden.

In recipients of allogeneic hematopoietic-cell transplants, peripheral-blood cells mobilized with the use of filgrastim (recombinant granulocyte colony-stimulating factor) engraft more rapidly than bone marrow. However, the relative effects of these techniques on the rates of acute and chronic graft-versus-host disease, overall survival, and disease-free survival have not been determined in randomized studies.

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPs) are thought to play a role in postnatal angiogenesis. Emerging evidence suggests that angiogenic stress of vascular trauma may induce mobilization of CEPs to the peripheral circulation. In this regard, we studied the kinetics of CEP mobilization in two groups of patients who experienced acute vascular insult secondary to burns or coronary artery bypass grafting (CABG). In both burn and CABG patients, there was a consistent, rapid increase in the number of CEPs, determined by their surface expression pattern of vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial cadherin (VE-cadherin), and AC133. Within the first 6 to 12 hours after injury, the percentage of CEPs in the peripheral blood of burn or CABG patients increased almost 50-fold, returning to basal levels within 48 to 72 hours. Mobilized cells also formed late-outgrowth endothelial colonies (CFU-ECs) in culture, indicating that a small, but significant, number of circulating endothelial cells were BM-derived CEPs. In parallel to the mobilization of CEPs, there was also a rapid elevation of VEGF plasma levels. Maximum VEGF levels were detected within 6 to 12 hours of vascular trauma and decreased to baseline levels after 48 to 72 hours. Acute elevation of VEGF in the mice plasma resulted in a similar kinetics of mobilization of VEGFR2(+) cells. On the basis of these results, we propose that vascular trauma may induce release of chemokines, such as VEGF, that promotes rapid mobilization of CEPs to the peripheral circulation. Strategies to improve the mobilization and incorporation of CEPs may contribute to the acceleration of vascularization of the injured vascular tissue.

A reduced-intensity preparative regimen consisting of melphalan and a purine analog was evaluated for allogeneic transplantation in 86 patients who had a variety of hematologic malignancies and were considered poor candidates for conventional myeloablative therapies because of age or comorbidity. Seventy-eight patients received fludarabine 25 mg/m(2) daily for 5 days in combination with melphalan 180 mg/m(2) (n = 66) or 140 mg/m(2) (n = 12). Eight patients received cladribine 12 mg/m(2) continuous infusion for 5 days with melphalan 180 mg/m(2). The median age was 52 years (range, 22-70 years). Disease status at transplantation was either first remission or first chronic phase in 7 patients, untreated first relapse or subsequent remission in 16 patients, and refractory leukemia or transformed chronic myelogenous leukemia in 63 patients. Nonrelapse mortality rates on day 100 were 37.4% for the fludarabine/melphalan combination and 87.5% for the cladribine/melphalan combination. The median percentage of donor cells at 1 month in 75 patients was 100% (range, 0%-100%). The probability of grade 2-4 and 3-4 acute graft-versus-host disease was 0.49 (95% CI, 0.38-0.60) and 0.29 (95% CI, 0.18-0.41), respectively. Disease-free survival at 1 year was 57% for patients in first remission or chronic phase and 49% for patients with untreated first relapse or in a second or later remission. On multivariate analysis the strongest predictor for disease-free survival was a good or intermediate risk category. In summary, fludarabine/melphalan combinations are feasible in older patients with associated comorbidities, and long-term disease control can be achieved with reduced-intensity conditioning in this population.

Salvage of patients with relapsed and refractory Hodgkin disease (HD) with high-dose chemoradiotherapy (HDT) and autologous stem cell transplantation (ASCT) results in event-free survival (EFS) rates from 30% to 50%. Unfortunately, the reduction in toxicity associated with modern supportive care has improved EFS by only 5% to 10% and has not reduced the relapse rate. Results of a comprehensive 2-step protocol encompassing dose-dense and dose-intense second-line chemotherapy, followed by HDT and ASCT, are reported. Sixty-five consecutive patients, 22 with primary refractory HD and 43 with relapsed HD, were treated with 2 biweekly cycles of ifosfamide, carboplatin, and etoposide (ICE). Peripheral blood progenitor cells from responding patients were collected, and the patients were given accelerated fractionation involved field radiotherapy (IFRT) followed by cyclophosphamide-etoposide and either intensive accelerated fractionation total lymphoid irradiation or carmustine and ASCT. The EFS rate at a median follow-up of 43 months, as analyzed by intent to treat, was 58%. The response rate to ICE was 88%, and the EFS rate for patients who underwent transplantation was 68%. Cox regression analysis identified 3 factors before the initiation of ICE that predicted for outcome: B symptoms, extranodal disease, and complete remission duration of less than 1 year. EFS rates were 83% for patients with 0 to 1 adverse factors, 27% for patients with 2 factors, and 10% for patients with 3 factors (P <.001). These results compare favorably with other series and document the feasibility and efficacy of giving uniform dose-dense and dose-intense cytoreductive chemotherapy and integrating accelerated fractionation radiotherapy into an ASCT treatment program. This prognostic model provides a basis for risk-adapted HDT.

To evaluate whether administration of interleukin-2 (IL-2) with granulocyte colony-stimulating factor (G-CSF) improves mobilization of immune effector cells into the stem-cell graft of patients undergoing high-dose chemotherapy and autografting.

The consequences of using cyclosporine (CsA) therapy to modulate P-glycoprotein-mediated multidrug resistance include increased myelosuppression, hyperbilirubinemia, and altered disposition of the cytotoxin. The purpose of this study was to analyze further the relationship between the degree of leukopenia, and etoposide pharmacokinetic factors.

An 8-F 24-cm-long apheresis catheter was placed in the basilic vein with imaging-guided percutaneous technique in 15 children undergoing leukapheresis for collection of autologous peripheral blood stem cells. There were no immediate or long-term complications. This is a low-morbidity procedure requiring minimal sedation that results in successful collection of peripheral blood stem cells and allows flow rates comparable to those with surgically placed central catheters.

Between October 1993 and March 1999, 29 patients with CML who were ineligible for allogeneic BMT underwent PBSC harvest using idarubicin, cytarabine and G-CSF. In 9/29 (31%) patients all collected stem cells were Ph-negative, and 15/29 patients' (52%) collections were substantially (>95%) Ph-negative. The proportion of patients from whom Ph-negative stem cells were obtained was similar between patients who had, or had not, received prior alphaIFN. Fifteen patients in chronic phase (median age 45) proceeded to PBSCT following busulphan 16 mg/m2 and cyclophosphamide 120 mg/m2. Nine of the 13 patients who had failed to respond to prior alphaIFN proceeded to stem cell transplantation as soon as was feasible and six of the newly diagnosed patients were transplanted after failing to achieve a cytogenetic response after a minimum of 12 months on alphaIFN following progenitor cell harvest. The median number of days to neutrophils >0.5 and platelet >50 was 18 (range 13-69) and 28 (range 13-234), respectively. There was no procedure-related mortality. At median follow-up of 2.3 years post autograft 10 of 15 patients remain alive and in chronic phase. Overall survival for all 27 patients at 5 years after initial diagnosis is 70% and median survival from diagnosis 7.3 years. Survival for alphaIFN non-responders who were transplanted is 74% at 5 years from diagnosis and 75% at 3 years from transplant. Cytogenetic analysis performed 3 months post transplant demonstrated one patient with a complete cytogenetic response, seven with a partial response and three with no response. Six patients remain partially Ph-negative, with one major CR. Survival for all patients in the protocol is favourable compared with conventional therapy and is particularly encouraging following PBSCT for alphaIFN non-responsive patients. Patients not responding to alphaIFN can be induced into Ph-negativity with PBSCT but this may not always be sustainable. There seems to be no obvious disadvantage in harvesting stem cells after prior exposure to alphaIFN, providing an adequate alphaIFN-free rest period is used.

We have evaluated the outcome of allogeneic haemopoietic stem cell transplantation for multiple myeloma using a conditioning regimen comprising fractionated total body irradiation and high-dose melphalan (110 mg/m2). The study comprised 25 patients (median age 49 years) who had been transplanted by either bone marrow (n = 13) or G-CSF mobilized peripheral blood stem cells (n = 12). Overall transplant-related mortality was 30% but was lower for patients < 50 years of age at transplant (21%). The main cause of treatment-related mortality was viral infection. Of the 19 patients evaluable post-transplant, 17 have so far achieved complete remissions. Currently, with a median follow-up of 3.4 years, 18 out of 25 patients are alive, of whom 15 are in continuing complete remission (CR) and 2 in second remission after suffering localized relapses, which were treated with radiotherapy and donor leucocyte infusions. Patients transplanted after 1 line of previous therapy, < 50 years of age and receiving peripheral blood stem cells (PBSC) rather than bone marrow (BM) had a superior outcome, although there was no statistically significant factor. We conclude that allogeneic transplantation should be considered as a potentially curative option for younger patients with myeloma and that the regimen using fractionated total body irradiation and melphalan has a high CR rate and a relatively low risk of treatment-related mortality, particularly in younger patients.

High dose therapy (HDT) and stem cell transplantation (SCT) results in alterations in the immunologic network, thymic re-education and the induction of peripheral tolerance. The changes to the immunoregulatory cascade and tolerance induction associated with autotransplants have been investigated in a series of studies focused on leukocyte reconstitution and function following HDT and autologous SCT. In these studies, we observed a significant decrease in the CD4:CD8 T cell ratio post-transplantation compared to normal peripheral blood (PB) donors due to a decrease in CD4+ cells. In addition, T cell function (phytohemagglutinin (PHA) mitogenesis) was consistently depressed compared to samples obtained from normal PB donors. The loss of T cell function was associated with an increased frequency of circulating monocytes, their expression of Fas ligand (FasL) and a high frequency of apoptotic CD4+ T cells. Indeed, 28-51% of circulating CD4+ T cells were observed to be apoptotic during the first 100 days following HDT and SCT. These studies suggest that 'primed' or activated Fas+ CD4+ lymphocytes interact with FasL+ monocytes, resulting in apoptosis, leading to the preferential deletion of CD4+ T cells, a decrease in the CD4:CD8 T cell ratio and depressed T cell function. Further, as discussed herein, the T cells are activated with a predominantly type 2 phenotype, which may also contribute to the maintenance of the immunosuppressive condition. Therefore, there is the potential to regulate immune recovery by stem cell product manipulation or post-transplantation cytokine administration.