Ifosfamide in combination with paclitaxel or doxorubicin: regimens which effectively mobilize peripheral blood progenitor cells while demonstrating anti-tumor activity in patients with metastatic breast cancer

For patients with metastatic breast cancer (MBC) who undergo high-dose therapy with autologous peripheral blood progenitor cell (PBPC) transplantation, an important prerequisite is a mobilization regimen that efficiently mobilizes PBPCs while producing an effective anti-tumor effect. We prospectively evaluated ifosfamide-based chemotherapy for mobilization efficiency, toxicity and disease response in 37 patients. Patients received two cycles of the ifosfamide-based regimen; ifosfamide (5 g/m2 with conventional-dose cycle and 6 g/m2 with mobilization cycle) with either 50 mg/m2 doxorubicin (if limited prior anthracycline and/or progression more than 12 months after an anthracycline-based regimen) or 175 mg/m2 paclitaxel. For the mobilization cycle, all patients received additional G-CSF (10 microg/kg SC, daily) commencing 24 h after completion of chemotherapy. The target yield was >6x10(6) CD34+ cells/kg, sufficient to support the subsequent three cycles of high-dose therapy. The mobilization therapy was well tolerated and the peak days for peripheral blood (PB) CD34+ cells were days 10-13 with no significant differences in the PB CD34+ cells mobilization kinetics between the ifosfamide-doxorubicin vs. ifosfamide-paclitaxel regimens. The median PBPC CD34+ cell content ranged from 2.9 to 4.0x10(6)/kg per day during days 9-14. After a median of 3 (range 1-5) collection days, the median total CD34+ cell, CFU-GM and MNC for all 44 individual sets of collections was 9.2x10(6)/kg (range 0.16-54.9), 37x10(4)/kg (range 5.7-247) and 7.3x10(8)/kg (range 2.1-26.1), respectively. The PBPC target yield was achieved in 35 of the 37 patients. The overall response rate for the 31 evaluable patients was 68% with 10% having progressive disease. Thirty-three patients have subsequently received high-dose therapy consisting of three planned cycles of high-dose ifosfamide, thiotepa and paclitaxel with each cycle supported with PBPCs. Rapid neutrophil and platelet recovery has been observed. Ifosfamide with G-CSF in combination with doxorubicin or paclitaxel achieves effective mobilization of PBPC and anti-tumor activity with minimal toxicity.     

High-dose therapy in patients with Hodgkin's disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells.

Register data suggest that patients with Hodgkin's disease (HD) given high-dose therapy (HDT) with peripheral blood progenitor cells (PBPC) have a less favourable prognosis as compared to those given bone marrow as stem cell support. Since this can be due to infusion of tumour cells contaminating the PBPC grafts, we initiated a feasibility study in which PBPC grafts from HD patients were purged by CD34(+) cell enrichment. Controversy exists about whether the use of CD34(+) enriched stem cells leads to a delayed haematological and immune reconstitution. We compared these parameters, including risk of infections and clinical outcome after HDT, in patients with HD given either selected CD34(+) cells or unmanipulated PBPC as stem cell support. From October 1994 to May 2000, 40 HD patients with primary refractory disease or relapse were treated with HDT and supported with either selected CD34(+) cells (n = 21) or unmanipulated PBPC (n = 19) as stem cell support. All patients had chemosensitive disease at the time of transplantation. A median of 5.8 (range 2.7-20.0) vs 4.5 (range 2.3-17.6) x 10(6) CD34(+) cells per kilo were reinfused in the CD34(+) group and PBPC group, respectively. No difference was observed between the two groups with regard to time to haematological engraftment, reconstitution of B cells, CD56(+) cells and T cells at 3 and 12 months and infectious episodes after HDT. Two (5%) treatment-related deaths, one in each group, were observed. The overall survival at 4 years was 86% for the CD34(+)group and 74% for the PBPC group with a median follow-up of 37 months (range 1-61) and 46 months (range 4-82), respectively (P = 0.9). The results of this study demonstrate that the use of CD34(+) cells is safe and has no adverse effects either with respect to haematological, immune reconstitution or to infections after HDT.     

High-dose chemotherapy and CD34-selected peripheral blood progenitor cell transplantation for patients with breast cancer metastatic to bone and/or bone marrow.

Fifty women with breast cancer metastatic to bone or bone marrow involvement on light microscopy at the time of initial evaluation were treated with high-dose chemotherapy (HDC) and peripheral blood progenitor cell (PBPC) transplantation with CD34(+) cell selection using the Isolex 300i system. All patients received induction chemotherapy. PBPC were mobilized with chemotherapy and granulocyte colony-stimulating factor. The median CD34(+) progenitor purity was 94.7% (range 72-98.7%) and recovery 38.4% (range 21-60%). Forty-eight hours after HDC with cyclophosphamide, cisplatin and carmustine, PBPC were reinfused. Median time to neutrophil count >0.5 x 10(9)/l was 9 (range 9-12) days and to platelet transfusion independence 11 (4-30) days. These data demonstrate that selected CD34(+) PBPCs allow rapid hematologic reconstitution after HDC. During follow-up, 23% of patients developed herpes zoster. Two patients developed cytomegalovirus infections. Three patients developed fungal infections. The development of these infections was not associated with steroid use but appeared more frequently in patients with diabetes mellitus. Seventy-four per cent of patients received steroids for pulmonary toxicity. Treatment-related mortality was 4%. Progression-free survival and overall survival at 35 months was 22.4% and 40.5%, with a median of 11.4 months and 15.4 months, respectively.     

Influence of amifostine on reconstitution of lymphocyte subpopulations after conventional- and high-dose chemotherapy in patients with germ cell tumor

We assessed the influence of amifostine on immune reconstitution after conventional-dose paclitaxel, ifosfamide, cisplatin and high-dose carboplatin, etoposide and thiotepa followed by autologous peripheral blood progenitor cell (PBPC) rescue in patients with germ cell tumor (GCT). A total of 40 patients were treated with one cycle of paclitaxel and ifosfamide (TI) followed by granulocyte-colony stimulating factor (G-CSF) to mobilize PBPC, three cycles of paclitaxel, ifosfamide and cisplatin (TIP) and one course of high-dose carboplatin, etoposide and thiotepa (CET) plus PBPC rescue. Patients were randomized to receive an absolute dose of 500 mg amifostine (group A, n=20) on each day of chemotherapy or no amifostine (group B, n=20). Prior to each cycle of chemotherapy, after hematologic engraftment from CET, 6 weeks and 3 months after transplantation the subpopulations of lymphocytes were phenotyped. Between the two study groups no statistically significant differences were observed concerning reconstitution of lymphocyte subpopulations. Throughout treatment with TIP or CET lymphocyte counts and their subpopulations remained low without severe clinical complications. Delayed reconstitution of the CD4(+) cell compartment after PBPC rescue was observed in both study groups, but did not result in any severe or atypical infections. Treatment with amifostine administered at this dose did not significantly influence the reconstitution of lymphocyte subpopulations. Low numbers of lymphocytes during chemotherapy and delayed reconstitution of CD4(+) cells and other lymphocyte subpopulations after PBPC rescue had no clinical relevance for patients with GCT.     

Cytotoxic chemotherapy preceding apheresis of peripheral blood progenitor cells can affect the early reconstitution phase of naive T cells after autologous transplantation

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy.     

Keratinocyte growth factor augments immune reconstitution after autologous hematopoietic progenitor cell transplantation in rhesus macaques

Opportunistic infections contribute to morbidity and mortality after peripheral blood progenitor cell (PBPC) transplantation and are related to a deficient T-cell compartment. Accelerated T-cell reconstitution may therefore be clinically beneficent. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells in mice. Here, we evaluated immune reconstitution after autologous CD34(+) PBPC transplantation in rhesus macaques conditioned with myeloablative total body irradiation in the absence or presence of single pretotal body irradiation or repeated peritransplant KGF administration. All KGF-treated animals exhibited a well-preserved thymic architecture 12 months after graft. In contrast, thymic atrophy was observed in the majority of animals in the control group. The KGF-treated animals showed higher frequencies of naive T cells in lymph nodes after transplantation compared with the control animals. The animals given repeated doses of KGF showed the highest levels of T-cell receptor excision circles (TRECs) and the lowest frequencies of Ki67(+) T cells, which suggest increased thymic-dependent reconstitution in these animals. Of note, the humoral response to a T-cell-dependent neo-antigen was significantly higher in the KGF-treated animals compared with the control animals. Thus, our findings suggest that KGF may be a useful adjuvant therapy to augment T-cell reconstitution after human PBPC transplantation.     

Virus reactivation in high-risk non-Hodgkin’s lymphoma patients after autologous CD34+-selected peripheral blood progenitor cell transplantation

CD34⁺-selected peripheral blood progenitor cells (PBPCs) may not only reduce contaminated tumor cells but also compromise immunologic reconstitution and increase incidence of infections after transplantation. We analyzed the incidence of virus reactivation in CD34⁺-selected PBPCs autologous transplantation. From December 2001 to December 2004, ten high-risk aggressive non-Hodgkin’s lymphoma (NHL) patients were enrolled in a program of high-dose chemotherapy plus autologous CD34⁺-selected PBPCs support. Viral screening studies, including clinical symptoms, physical examinations, hepatitis B virus (HBV)-DNA, cytomegalovirus (CMV)-polymerase chain reaction (PCR), rapid diagnosis of fluorescent antibody stain for herpes-simplex virus (HSV), and viral culture from blood, fluid or tissue were performed weekly during the first 3 months and then monthly for 1 year. Two of four patients (50%) who were HBV carriers developed HBV reactivation. The other two HBV carriers who received prophylactic lamivudine therapy did not develop HBV reactivation. Two patients (20%) developed cytomegalovirus (CMV) infection, and three patients (30%) developed HSV infection in total ten serum-positive patients. The possibility of virus reactivation might increase in NHL patients undergoing autologous CD34⁺-selected PBPC transplantation. Administering prophylactic antivirus therapy and closely following patient’s clinical viral complications should be considered.     

Ex vivo expanded unselected peripheral blood: progenitor cells reduce posttransplantation neutropenia, thrombocytopenia, and anemia in patients with breast cancer

The safety and efficacy of administering ex vivo expanded peripheral blood progenitor cells (PBPC) to patients with breast cancer who undergo high-dose chemotherapy and PBPC transplantation was investigated. Unselected PBPC were cultured in gas-permeable bags containing 1-L serum-free media, granulocyte colony-stimulating factor, stem cell factor, and pegylated megakaryocyte growth and development factor for 9 days. Cell dose cohorts were assigned to have between 2 and 24 x 10(9) PBPC cultured at 1, 2, or 3 x 10(6) cells/mL. Twenty-four patients received high-dose chemotherapy followed by infusion of the cultured PBPC and at least 5 x 10(6) CD34(+) uncultured cryopreserved PBPC per kilogram. No toxicities resulted from infusions of the ex vivo expanded PBPC. The study patients had shorter times to neutrophil (P =.0001) and platelet (P =.01) recovery and fewer red cell transfusions (P =.02) than 48 historical controls who received the same conditioning regimen and posttransplantation care and at least 5 x 10(6) CD34(+) PBPC per kilogram. Improvements in all these endpoints were significantly correlated with the expanded cell dose. Nine of 24 (38%) patients recovered neutrophil counts above 500/microL by day 5 or 6 after transplantation, whereas none of the controls had neutrophil recovery before the eighth day. Seven (29%) patients had neutropenia for 3 or fewer days, and 9 (38%) patients did not experience neutropenic fevers or require broad-spectrum antibiotics. Therefore, ex vivo expanded PBPC are capable of ameliorating posttransplantation neutropenia, thrombocytopenia, and anemia in patients receiving high-dose chemotherapy.     

Multiple cycles of PBPC-supported high-dose carboplatin and paclitaxel following mobilization with epirubicin and cisplatin are feasible but ineffective in treating patients with advanced non-small cell lung cancer

We verified the feasibility of a multi-cycle peripheral blood progenitor cell (PBPC)-supported high-dose chemotherapy (HDC) regimen in patients with non-small cell lung cancer (NSCLC). The HDC regimen consisted of a single course of high-dose epirubicin given in combination with cisplatin plus filgrastim, followed by three courses of high doses of carboplatin and paclitaxel with PBPC reinfusion and filgrastim. Of the 16 enrolled patients, 13 provided an adequate number of PBPCs by a single leukapheresis, while in the three needed two procedures, with a median number of CD34+, CD34+/CD33- and CD34+/CD38- cells collected per patient was 13.5 x 10(6), 10.9 x 10(6) and 0.9 x 10(6)/kg, respectively. No toxic death occurred, and the collected PBPCs supported a rapid hematopoietic reconstitution after HDC; however, seven patients early interrupted the treatment early due to early progressive disease (n=4) or prolonged grade 3 peripheral neurotoxicity (n=3). Despite an overall response rate of 42%, the median survival for stage IV patients has been 5 months (range: 1-25+). Of two patients with stage IIIB NSCLC, one is continuously disease-free at 71+ months, while of 14 with stage IV disease, one is currently alive with disease at 25+ months. In conclusion, the combination of high-dose epirubicin with cisplatin plus filgrastim is an effective regimen in releasing large amounts of PBPCs, which can then be safely employed to support multiple courses of HDC. Multiple cycles of PBPC-supported high-dose carboplatin and paclitaxel are ineffective in treating patients with advanced NSCLC.     

Persistence of clonal T-cell expansions following high-dose chemotherapy and autologous peripheral blood progenitor cell rescue

Analysing the regeneration of T lymphocytes after high-dose chemotherapy with autologous peripheral blood progenitor cell rescue (PBPCR) may help elucidate the mechanisms of immune recovery. The T-cell receptor variable beta chain (TCRBV) repertoire of adult patients undergoing high-dose chemotherapy was analysed by flow cytometry, before and after treatment. Four patients were found to have a stable expansion present (TCRBV3, 17, 21 and 22) ranging from 8% to 42% of the CD4(+) or CD8(+) repertoire. We demonstrated that, in these patients, following high-dose chemotherapy and autologous stem cell transplantation, the clonal expansions reappeared in peripheral blood and returned to pretransplant levels. Three expansions (CD3(+)CD8(+)TCRBV3(+), CD3(+)CD4(+)TCRBV21(+) and CD3(+)CD8(+)TCRBV22(+)) were further defined by sequence analysis of the complementarity-determining region (CDR)3 portion within the TCR rearrangements. These were shown to be predominantly clonal, with the same sequences being identified in peripheral blood before and after PBPCR, providing evidence that the overwhelming majority of T cells in these expansions arise from mature lymphocytes. This study demonstrated that patients undergoing autologous PBPCR for high-dose chemotherapy regenerate clonal expansions, consistent with pretreatment levels. They also regenerate T-cell repertoires with each TCRBV family represented to a similar level as that prior to high-dose chemotherapy.     

Positively selected autologous blood CD34+ cells and unseparated peripheral blood progenitor cells mediate identical hematopoietic engraftment after high-dose VP16, ifosfamide, carboplatin, and epirubicin

To investigate the feasibility of peripheral blood CD34+ cell selection and to analyze CD34+ cell-mediated engraftment after high-dose chemotherapy, we performed a phase I/II trial in 21 patients with advanced malignancies. The rationale for the selection of CD34+ cells from peripheral blood progenitor cell (PBPC) collections is based on the observation that contaminating tumor cells can be depleted approximately 3 logs using this procedure. CD34+ cells from chemotherapy+granulocyte colony-stimulating factor-mobilized PBPCs were positively selected with an avidin-biotin immunoadsorption column (CEPRATE SC system). One leukapheresis product with a median number of 2.8 x 10(6) CD34+ cells/kg was labeled with a biotinylated anti-CD34 monoclonal antibody and subsequently processed over the column. The yield of selected CD34+ cells was 73% +/- 24.6%. The purity of the CD34+ cell fraction was 61.4% +/- 19.7%. CD34+ cells were shown to represent predominantly committed progenitors coexpressing CD33, CD38, and HLA-DR molecules (lin+). They gave rise to myeloid as well as erythroid and multilineage colonies in vitro. In addition, positively selected CD34+ cells also comprised early hematopoietic progenitor cells, as shown by the presence of CD34+/lin- cells. Transfusion of positively selected CD34+ cells (2.5 x 10(6) CD34+/kg; range, 0.45 to 5.1) after high-dose VP16 (1,500 mg/m2), ifosfamide (12 g/m2), carboplatin (750 mg/m2), and epirubicin (150 mg/m2) (VIC-E) in 15 patients resulted in a rapid and stable engraftment of hematopoiesis without any adverse events. As compared with 13 historical control patients reconstituted with a comparable number of unseparated PBPCs, time to neutrophil and platelet recovery was identical in both groups (absolute neutrophil count > 500/microL, day + 12; platelet count > 50,000/microL, day + 15). These data indicate that autologous peripheral blood CD34+ cells and unseparated PBPCs mediate identical reconstitution of hematopoiesis after high-dose VIC-E chemotherapy. Because positive selection of CD34+ cells from mobilized blood results in a median 403-fold depletion of T cells, allogeneic CD34+ cells from mobilized blood should be investigated as an alternative to bone marrow cells for allotransplantation.     

Evidence for naive T-cell repopulation despite thymus irradiation after autologous transplantation in adults with multiple myeloma: role of ex vivo CD34+ selection and age

Immunodeficiency following autologous CD34+-purified peripheral blood stem cell (PBSC) transplantation could be related to T-cell depletion of the graft or impaired T-cell reconstitution due to thymus irradiation. Aiming to assess the role of irradiated thymus in T-cell repopulation, we studied 32 adults with multiple myeloma, randomly assigned to receive high-dose therapy including total body irradiation (TBI) followed by autologous transplantation with either unselected or CD34+-selected PBSCs. The median number of reinfused CD3+ cells was lower in the selected group (0.03 versus 14 x 10(6)/kg; P =.002). Lymphocyte subset counts were evaluated from month 3 to 24 after grafting. Naive CD4+ T cells were characterized both by phenotype and by quantification of T-cell receptor rearrangement excision circles (TRECs). The reconstitution of CD3+ and CD4+ T cells was significantly delayed in the CD34+-selected group, but eventually led to counts similar to those found in the unselected group after month 12. Mechanism of reconstitution differed, however, between both groups. Indeed, a marked increase in the naive CD62L+CD45RA+CD4+ subset was observed in the selected group, but not in the unselected group in which half of the CD45RA+CD4+ T cells appear to be CD62L-. Age was identified as an independent adverse factor for CD4+ and CD62L+CD45RA+CD4+ T-cell reconstitution. Our results provide evidence that infusing PBSCs depleted of T cells after TBI in adults delays T-cell reconstitution but accelerates thymic regeneration.     

Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.     

High-dose ara-C with autologous peripheral blood progenitor cell support induces a marked progenitor cell mobilization: an indication for patients at risk for low mobilization

A high-dose (HD) chemotherapy scheme was designed for the collection of large numbers of peripheral blood progenitor cells (PBPC) in lymphoma patients who were candidates for myeloablative therapy with autograft. The scheme included the sequential administration of HD cyclophosphamide (CY) (7 g/m(2)) and HD ara-C (2 g/m(2) twice a day for 6 consecutive days), followed by final consolidation with PBPC autograft. PBPC harvests were scheduled following both HD CY and HD ara-C. To minimize hematologic toxicity, small aliquots of PBPC (20 circulating CD34(+) cells/microl, whereas the remaining 19 'low-mobilizer' patients did not reach this cut-off value. In spite of poor mobilization after HD CY, 16 out of 19 low mobilizers provided good harvests following HD ara-C; overall, median collected CD34(+) cells x 10(6)/kg were 1.4 (0-3.1) and 10.2 (0-37) after HD CY and HD ara-C, respectively (P = 0.00007). Similar patterns were observed when PBPC were evaluated by CFU-GM/kg. Complete and durable hemopoietic reconstitution followed autograft with post HD ara-C PBPC. Within the high-mobilizer group, 88 patients received HD ara-C and 79 (90%) still showed high mobilization; overall, median collected CD34(+)cells x 10(6)/kg were 17.8 (range 3-94) and 19 (range 0-107) after HD CY and HD ara-C respectively (P = NS). Thus, the scheme allowed sufficient PBPC collections for autografting in low mobilizer patients; in addition, the scheme could be considered whenever extensive chemotherapy debulking is needed prior to PBPC collection.     

Immune reconstitution after autologous hematopoietic stem cell transplantation in relation to underlying disease, type of high-dose therapy and infectious complications

BACKGROUND AND OBJECTIVES: Autologous peripheral stem cell transplantation (APSCT) is increasingly used for various hematologic malignancies and solid tumors. The objective of this study was to analyze the immune reconstitution after APSCT and see if there was any correlation with diagnosis, age, type of high-dose therapy, CD34(+) selection of the autograft and double vs single APSCT. DESIGN AND METHODS: Lymphocyte subset recovery was studied in 46 consecutive patients with hematologic malignancies and breast cancer, who underwent APSCT. Eleven patients with multiple myeloma received tandem autografts. Thirty-one patients were given total body irradiation (TBI) as part of the high-dose treatment. Eighteen patients received a CD34(+) selected graft. The percentage and absolute numbers of lymphocyte populations, T-cells (CD2(+), CD3(+)), B-cells (CD19(+)), NK cells (CD56(+ )CD3(-) and CD16(+)CD3(-)) and T-cell subpopulations (CD4(+), CD8(+), CD4(+)CD45RA(+), CD4(+ )CD45RO(+), CD4(+)DR(+), CD8(+ )CD45RO(+), CD8(+)DR(+)), were monitored with flow cytometry during the first year after APSCT. RESULTS: The total B-cell (CD19(+)) and T-cell (CD3(+)) counts were reconstituted to normal levels 2-4 months after APSCT. All patients had a low CD4/CD8 ratio during the observation period, related to both a low number of CD4(+) cells and elevated numbers of CD8(+) cells. The low number of CD4(+) cells was due to a persistently low level of naive CD4(+)CD45RA(+) cells. A high proportion of the CD8+ cells displayed a phenotype compatible with activated T-cells (CD8(+)DR(+)) up to 10 months after autografting. The number of NK cells (CD56(+)3(-) or CD16(+)3(-)) reached normal values within one month post-transplant. No single variable, such as diagnoses, age, TBI as part of the high-dose treatment, tandem autografting or CD34(+) selection of the graft, influenced the immune or hematopoietic reconstitution and no correlation with documented infectious complications was found. INTERPRETATION AND CONCLUSIONS: Despite heterogeneity of diseases, age, initial treatment and high-dose regimens, lymphocyte subset analysis did not reveal any differences in hematopoietic or immune reconstitution. All patients had a low CD4(+)/CD8(+) ratio during at least the first year post-transplant, caused by a persistent increase of CD8(+) lymphocytes and a constant reduction of CD4(+) lymphocytes, making the patients susceptible to infections for a prolonged period of time post-transplant.     

An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy

The CD34 antigen is expressed by committed and uncommitted hematopoietic progenitor cells and is increasingly used to assess stem cell content of peripheral blood progenitor cell (PBPC) collections. Quantitative CD34 expression in PBPC collections has been suggested to correlate with engraftment kinetics of PBPCs infused after myeloablative therapy. We analyzed the engraftment kinetics as a function of CD34 content in 692 patients treated with high-dose chemotherapy (HDC). Patients had PBPCs collected after cyclophosphamide based mobilization chemotherapy with or without recombinant human granulocyte colony-stimulating factor (rhG-CSF) until > or = 2.5 x 10(6) CD34+ cells/kg were harvested. Measurement of the CD34 content of PBPC collections was performed daily by a central reference laboratory using a single technique of CD34 analysis. Forty-five patients required a second mobilization procedure to achieve > or = 2.5 x 10(6) CD34+ cells/kg and 15 patients with less than 2.5 x 10(6) CD34+ cells/kg available for infusion received HDC. A median of 9.94 x 10(6) CD34+ cells/kg (range, 0.5 to 112.6 x 10(6) CD34+ cells/kg) contained in the PBPC collections was subsequently infused into patients after the administration of HDC. Engraftment was rapid with patients requiring a median of 9 days (range, 5 to 38 days) to achieve a neutrophil count of 0.5 x 10(9)/L and a median of 9 days (range, 4 to 53+ days) to achieve a platelet count of > or = 20 x 10(9)/L. A clear dose-response relationship was evident between the number of CD34+ cells per kilogram infused between the number of CD34+ cells per kilogram infused and neutrophil and platelet engraftment kinetics. Factors potentially influencing the engraftment kinetics of neutrophil and platelet recovery were examined using a Cox regression model. The single most powerful mediator of both platelet (P = .0001) and neutrophil (P = .0001) recovery was the CD34 content of the PBPC product. Administration of a post-PBPC infusion myeloid growth factor was also highly correlated with neutrophil recovery (P = .0001). Patients receiving high-dose cyclophosphamide, thiotepa, and carboplatin had more rapid platelet recovery than patients receiving other regimens (P = .006), and patients requiring 2 mobilization procedures versus 1 mobilization procedure to achieve > or = 2.5 x 10(6) CD34+ cells/kg experienced slower platelet recovery (P = .005). Although a minimal threshold CD34 dose could not be defined, > or = 5.0 x 10(6) CD34+ cells/kg appears to be optimal for ensuring rapid neutrophil and platelet recovery.     

Gene therapy improves immune function in preadolescents with X-linked severe combined immunodeficiency

Retroviral gene therapy can restore immunity to infants with X-linked severe combined immunodeficiency (XSCID) caused by mutations in the IL2RG gene encoding the common gamma chain (gammac) of receptors for interleukins 2 (IL-2), -4, -7, -9, -15, and -21. We investigated the safety and efficacy of gene therapy as salvage treatment for older XSCID children with inadequate immune reconstitution despite prior bone marrow transplant from a parent. Subjects received retrovirus-transduced autologous peripherally mobilized CD34(+) hematopoietic cells. T-cell function significantly improved in the youngest subject (age 10 years), and multilineage retroviral marking occurred in all 3 children.     

Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte-macrophage colony-stimulating factor for hematologic support after high-dose intensification for breast cancer.

High-dose therapy with autologous marrow support results in durable complete remissions in selected patients with relapsed lymphoma and leukemia who cannot be cured with conventional dose therapy. However, substantial morbidity and mortality result from the 3- to 6-week period of marrow aplasia until the reinfused marrow recovers adequate hematopoietic function. Hematopoietic growth factors, particularly used after chemotherapy, can increase the number of peripheral blood progenitor cells (PBPCs) present in systemic circulation. The reinfusion of PBPCs with marrow has recently been reported to reduce the time to recovery of adequate marrow function. This study was designed to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized PBPCs alone (without marrow) would result in rapid and reliable hematopoietic reconstitution. Sixteen patients with metastatic breast cancer were treated with four cycles of doxorubicin, 5-fluorouracil, and methotrexate (AFM induction). Patients responding after the first two cycles were administered GM-CSF after the third and fourth cycles to recruit PBPCs for collection by two leukapheresis per cycle. These PBPCs were reinfused as the sole source of hematopoietic support after high doses of cyclophosphamide, thiotepa, and carboplatin. No marrow or hematopoietic cytokines were used after progenitor cell reinfusion. Granulocytes greater than or equal to 500/microL was observed on a median of day 14 (range, 8 to 57). Transfusion independence of platelets greater than or equal to 20,000/microL occurred on a median day of 12 (range, 8 to 134). However, three patients required the use of a reserve marrow for slow platelet engraftment. In retrospect, these patients were characterized by poor baseline bone marrow cellularity and poor platelet recovery after AFM induction therapy. When compared with 29 historical control patients who had received the same high-dose intensification chemotherapy using autologous marrow support, time to engraftment, antibiotic days, transfusion requirements, and lengths of hospital stay were all significantly improved for the patients receiving PBPCs. Thus, autologous PBPCs can be efficiently collected during mobilization by chemotherapy and GM-CSF and are an attractive alternative to marrow for hematopoietic support after high-dose therapy. The enhanced speed of recovery may reduce the morbidity, mortality, and cost of high-dose treatment. Furthermore, PBPC support may enhance the effectiveness of high-dose therapy by facilitating multiple courses of therapy.     

A randomized multicenter comparison of CD34(+)-selected progenitor cells from blood vs from bone marrow in recipients of HLA-identical allogeneic transplants for hematological malignancies

OBJECTIVE: Peripheral blood progenitor cells (PBPC) have been established as an alternative source of hematopoietic stem cells for allogeneic transplantation, but an increased incidence of both acute and chronic graft-vs-host disease (GVHD) has become apparent. We performed a prospective randomized trial comparing bone marrow transplantation (BMT) vs PBPC transplantation (PBPCT) using CD34(+) selection for T-cell depletion (TCD) in both study arms. PATIENTS AND METHODS: Between January 1996 and October 2000, 120 patients with a diagnosis of acute leukemia, myelodysplasia, multiple myeloma, or lymphoma were randomized to receive either filgrastim-mobilized PBPC or BM from HLA-identical sibling donors after standard high-dose chemoradiotherapy. Patient characteristics did not differ between study arms. RESULTS: Recipients of PBPC received more CD3(+) T cells (median: 3.0 vs 2.0 x 10(5)/kg, p<0.0001) and more CD34(+) cells (median: 3.6 vs 0.9 x 10(6)/kg, p<0.0001). Neutrophil and platelet recoveries occurred significantly faster after PBPCT. The cumulative incidence of acute GVHD grades II-IV was 37% after BMT vs 52% after PBPCT and was most significantly (p=0.007) affected by the number of CD3(+) T cells in the graft. Acute GVHD appeared strongly associated with increased treatment-related mortality (TRM) in a time-dependent analysis. Higher numbers of CD34(+) cells were associated with less TRM. With a median follow-up of 37 months (range: 12-75), overall survival at 4 years from transplantation was 60% after BMT and 34% for recipients of PBPCT (p=0.04), which difference was largely due to increased GVHD and TRM in PBPC recipients receiving T-cell dosages greater than 2 x 10(5)/kg. CONCLUSION: Outcome following T cell-depleted PBPCT critically depends on the number of CD3(+) T cells, whereby high T-cell numbers may blunt a favorable effect of higher CD34(+) cell numbers.     

Selection and use of chemotherapy with hematopoietic growth factors for mobilization of peripheral blood progenitor cells

Peripheral blood progenitor cells (PBPCs) have become the preferred means of stem cell support for high-dose chemotherapy in recent years. The biology of PBPC mobilization is complex and may be influenced by several variables. Signals from both stromal and hemopoietic cells may induce downregulation of adhesion molecules and upregulate the expression of metalloproteinases. Cytokines alone can mobilize PBPCs but a synergistic effect has been shown when they are used in conjunction with chemotherapy. Disease-specific mobilization strategies appear to have the advantage of less toxicity, greater stem cell yield, and enhanced antitumor activity. Studies have demonstrated that the number of peripheral blood CD34+ cells can be used as a predictor for the timing of apheresis and for estimating PBPC yield. Similarly the CD34+ cell dose is the strongest predictor of hematologic recovery after PBPC transplant. Age, prior radiotherapy, marrow involvement, and prior chemotherapy (especially with alkylating agents) are important factors influencing the yield of stem cells.