In this study, we assessed the functional and kinetic characteristics of highly purified hematopoietic CD34+ cells from the apheresis products of 16 normal donors undergoing glycosylated granulocyte colony-stimulating factor (G-CSF ) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation in allogeneic recipients. Mobilized CD34+ cells were evaluated for their colony-forming capacity and trilineage proliferative response to selected recombinant human (rh) CSF in vitro and the content of very primitive long-term culture initiating cells (LTC-IC). In addition, the cycling status of circulating CD34+ cells, including committed clonogenic progenitor cells and the more immature LTC-IC, was determined by the cytosine arabinoside (Ara-C) suicide test and the acridine orange flow cytometric technique. By comparison, bone marrow (BM) CD34+ cells from the same individuals were studied under steady-state conditions and during G-CSF administration. Clonogenic assays in methylcellulose showed the same frequency of colony-forming unit cells (CFU-C) when PB-primed CD34+ cells and BM cells were stimulated with phytohemagglutinin–lymphocyte-conditioned medium (PHA-LCM). However, mobilized CD34+ cells were significantly more responsive than their steady-state BM counterparts to interleukin-3 (IL-3) and stem cell factor (SCF ) combined with G-CSF or IL-3 in presence of erythropoietin (Epo). In cultures added with SCF, IL-3, and Epo, we found a mean increase of 1.5- ± 1-fold (standard error of the mean [SEM]) of PB CFU–granulocyte-macrophage and erythroid progenitors (burst-forming units-erythroid) as compared with BM CD34+ cells (P < .05). Conversely, circulating and BM megakaryocyte precursors (CFU-megakaryocyte) showed the same clonogenic efficiency in response to IL-3, granulocyte-macrophage–CSF and IL-3, IL-6, and Epo. After 5 weeks of liquid culture supported by the engineered murine stromal cell line M2-10B4 to produce G-CSF and IL-3, we reported 48.2 ± 35 (SEM) and 62.5 ± 54 (SEM) LTC-IC per 104 CD34+ cells in PB and steady-state BM, respectively (P = not significant). The Ara-C suicide assay showed that 4% ± 5% (standard deviation [SD]) of committed precursors and 1% ± 3% (SEM) of LTC-IC in PB are in S-phase as compared with 25.5% ± 12% (SD) and 21% ± 8% (SEM) of baseline BM, respectively (P < .001). However, longer incubation with Ara-C (16 to 18 hours), in the presence of SCF, IL-3 and G-CSF, or IL-6, showed that more than 60% of LTC-IC are actually cycling, with no difference being found with BM cells. Furthermore, studies of cell-cycle distribution on PB and BM CD34+ cells confirmed the low number of circulating progenitor cells in S- and G2M-phase, whereas simultaneous DNA/RNA analysis showed that the majority of PB CD34+ cells are not quiescent (ie, in G0-phase), being in G1-phase with a significant difference with baseline and G-CSF–treated BM (80% ± 5% [SEM] v 61.9% ± 6% [SEM] and 48% ± 4% [SEM], respectively; P < .05). Moreover, G-CSF administration prevented apoptosis in a small but significant proportion of mobilized CD34+ cells. Thus, our results indicate that mobilized and BM CD34+ cells can be considered equivalent for the frequency of both committed and more immature hematopoietic progenitor cells, although they show different kinetic and functional profiles. In contrast with previous reports, we found that PB CD34+ cells, including very primitive LTC-IC, are cycling and ready to progress into S-phase under CSF stimulation. This finding should be taken into account for a better understanding of PBSC transplantation.

Summary.We studied the effects of an intensified induction/ consolidation treatment containing fludarabine (ICE/FLAN/ FLAN) on the mobilization and collection of peripheral blood stem cells (PBSC) in 31 consecutive untreated acute myeloid leukaemia (AML) patients. The complete remission (CR) rate was comparable to classic inductions (68% after ICE; 84% after ICE-FLAN I). To mobilize PBSC, 19 patients received 10 mg/kg/d of granulocyte-colony stimulating factor (G-CSF) starting at day 13 after FLAN, 13 (69%) of whom were found to be nonmobilizers. When a second G-CSF administration was performed in 10/13 patients mobilization was either not achieved (8/10) or was considered insufficient (<1 × 10 6 CD34 þ cells/kg) (2/10) and all 13 were subsequently submitted to bone marrow harvest. The harvest was considered adequate in 12/13 (92%) patients and autologous BMT (ABMT) has so far been performed in 10/12 cases with a mean of 8·6 × 10 8 /kg nucleated reinfused cells. The median times to neutrophil and platelet recovery after ABMT did not significantly differ from those of two previous series of patients treated with ABMT without fludarabinecontaining regimens. Adequate amounts of PBSC were obtained in 6/19 (31%) patients, who were then reinfused. Median times for platelet recovery were significantly longer than in a previous series of 26 AML cases reinfused with PBSC after treatment with the ICE-NOVIA induction/ consolidation regimen (125 v 20 d to 20 × 10 9 plt/l, P < 0·02; 218 v 37 d to 50 × 10 9 plt/l, P < 0·02). In addition, times for platelet recovery after ICE/FLAN/FLAN were not significantly different from those in a previous group treated with ABMT performed after ICE/NOVIA,without fludarabine. We conclude that fludarabine-containing regimens severely impair mobilization and collection of PBSC in AML patients and seem unsuitable when PBSC autotransplantation is programmed.

Summary:We compared the feasibility and efficacy of autologous bone marrow (ABMT) and peripheral blood progenitor cell transplantation (PBSCT) performed after an identical induction/consolidation in adults with acute myeloid leukemia (AML). From January 1993 to June 1996 91 consecutive AML patients were enrolled in a program consisting of anthracycline-based induction and highdose cytarabine consolidation (NOVIA). Until May 1994 ABMT was performed; from June 1994, if PBSC collection was adequate, PBSCT was performed. Out of 88 evaluable patients, 73 obtained a complete remission (CR) and 15 were resistant. Allogeneic bone marrow transplantation was performed in 16 patients. Fortyfour (50%) were given autologous stem cell transplantation. ABMT was performed in 21 cases; twenty-nine patients were given G-CSF mobilization after NOVIA administration. An adequate number of PBSC was obtained in 23/29 (79%) cases, which were then reinfused. Median times to both neutrophil and platelet recovery from transplant were significantly shorter for the PBSC group (17 vs 36 days to 500 PMN/l, P Ͻ Ͻ Ͻ 0.01; 20 vs 150 days to 20 000 platelets/l, P Ͻ Ͻ Ͻ 0.02; 37 vs 279 days to 50 000 platelets/l, P Ͻ Ͻ Ͻ 0.03), as were days of hospitalization after the reinfusion (18 vs 33, P Ͻ Ͻ Ͻ 0.03) and median days to transfusion independence. Toxicity was not significant in either group. After a minimum follow-up for live patients of 24 months (longer than the mean time for relapse observed for the PBSC series -14 months) the percentage of relapses was similar: 11 of 21 (52.4%) and 12 of 23 (52.1%) in the ABMT and PBSC groups, respectively. Our results indicate that autologous PBSC transplantation, performed after an intensive chemotherapy regimen, is not inferior to ABMT in terms of disease-free survival and allows faster recovery times and reduced need for tranfusion support.

Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

The feasibility of sequential positive and negative selection of mobilized CD34+ B-lineage negative cells to achieve tumour-free autografts in multiple myeloma (MM) patients was evaluated. Peripheral blood stem cells (PBSC) of 14 patients with advanced disease were mobilized. CD34+ cells were enriched in 12 of the patients by the avidin-biotin immunoabsorption technique. Subsequently, CD10+, CD19+, CD20+ and CD56+ cells (B-lin cells) were removed by immunomagnetic depletion. Minimal residual disease (MRD) was detected by flow cytometry and PCR-based molecular analysis of the patient specific IgH complementary-determining region III (CDRIII). Positive selection of stem cells produced a median recovery of 54.7% of the initial content of CD34+ cells (median purity 71.9%). Negative depletion of B-lineage cells reduced the number of CD34+ cells to 33.3% of the baseline value (median purity 72.7%). However, long-term culture assays showed the recovery of >60% of primitive haemopoietic progenitor cells after depletion of the B-lineage-positive cells. All evaluable patients had detectable disease in PBSC collections. The first step of positive selection of CD34+ cells resulted in >2 logs of tumour cell purging. However, molecular assessment showed the persistence of the disease in 6/7 cases. Immunofluorescence analysis demonstrated 1 additional log of B-cell purging by negative depletion. More importantly, molecular evaluation of IgH CDRIII region showed the disappearance of myeloma cells in 6/7 patients. 12 patients received a median of 3.9 x 106 CD34+ B-lin- cells/kg after conditioning with high-dose melphalan and showed a rapid reconstitution of haemopoiesis. These results were similar to two similar cohorts of patients who received either unmanipulated PBSC or positively selected CD34+ cells after the same conditioning regimen. Severe extrahaematological toxicity was limited to mucositis; no late infections were observed. We concluded that autotransplantation of purified CD34+ B-lin- cells was associated with a rapid and sustained recovery of haemopoiesis and low peritransplant morbidity. Sequential positive and negative enrichment of stem cells reduced tumour cell contamination in B-cell malignancies below the lower limit of detection of molecular analysis.