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Comparison between Bone Marrow and G-CSF-Mobilized Peripheral Blood Allografts Undergoing Clinical Scale CD34⁺ Cell Selection

Bone Marrow Transplantation Center, Hamburg University Hospital Eppendorf, Hamburg, Germany

Key Words. Allogeneic transplantation • CD3 • CD34 • CFU-GM • Cryopreservation • Stem cells

Dr. med. H.T. Hassan, Bone Marrow Transplantation Center, Hamburg University Hospital Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany.

	Abstract

Top Abstract Introduction Materials and Methods Clinical-Scale CD34+ Cell... Results Discussion References

 
Allogeneic transplantation of selected CD34⁺ cells, rather than conventional transplantation of bone marrow (BM) harvest or peripheral blood (PB) leukapheresis products, has the advantage of reducing volume, facilitating storage and decreasing the amount of dimethylsulfoxide (DMSO) and cell lysis products, as well as reducing the number of T-lymphocytes responsible for graft-versus-host disease (GVHD). Using biotin-avidin immunoaffinity columns (Ceprate^TM SC system, CellPro; Bothell, WA), CD34⁺ cells were selected from each of 20 allografts (12 G-CSF-mobilized PB and 8 BM ) collected from 14 HLA-identical normal healthy donors for transplantation. After the clinical-scale selection, the median concentration of CD34⁺ cells was 44.6% (range, 13% to 91%) in BM and 50.4% (range, 15% to 77%) in PB. Whereas 75% of the PB allografts had a CD34⁺ cell yield of more than 65%, only 37.5% of the BM allografts achieved such a yield, p < 0.01. The number of T-lymphocytes in the selected CD34⁺ cell allografts was reduced by two to three logs from a median of 4.2 x 10⁹ to 7.8 x 10⁵ CD3⁺ cells. The enrichment in CD34⁺ cells was 240-fold (range, 24- to 382-fold) in PB versus only 34-fold (range, 14- to 108-fold) in BM. Also, the enrichment in clonogenic cells was significantly more in PB (median value of 38.6-fold) than in BM (median value of 19.2-fold) and more in allografts from younger (< 50 years old) rather than older ( 50 years old) adult donors. A correlation was found between the percentage of CD34 or CD3⁺ cells before and after selection (r = 0.58 or r = 0.60, respectively, p < 0.05). Selective enrichment of the colony forming units-granulocyte-macrophage (CFU-GM) was found in all 20 allografts. The progenitor cell recovery after freezing and thawing was similar in BM and PB allografts, with a mean of about 60% for the CFU-GM and BFU-E. In the same six donors, the CD34⁺ cell yield was significantly more in the PB after mobilization (median 78.5%, range 50% to 90%) than in the BM before mobilization (median 41.5%, range 25% to 87%), p < 0.01. Ten patients with hematologic malignancies have been allotransplanted with 14 of the 20 selected CD34⁺ cells either combined BM + PB (n = 4) or single (n = 6) grafts. Seven patients did not develop acute GVHD, and only two patients developed grade II GVHD, one of whom developed only grade II GVHD that resolved after brief treatment with corticosteriods. Only one patient showed chronic GVHD (skin and liver). The low incidence and severity of GVHD seen in the present study (only 30%) could be due to the two- to three-log reduction of T-lymphocytes in the selected CD34⁺ cell allotransplants. All 10 patients had stable hematological recovery, and seven had full donor hematopoiesis. In conclusion, G-CSF-mobilized PB leukapheresis products undergoing selection of CD34⁺ cells have a greater yield and enrichment of progenitor cells than BM harvests collected from HLA-identical normal healthy donors for allogeneic transplantation. The low incidence and severity of both acute and chronic GVHD (30%) seen in the present study are very encouraging.

	Introduction

Top Abstract Introduction Materials and Methods Clinical-Scale CD34+ Cell... Results Discussion References

 
Transplantation of selected CD34⁺ cells offers several clinical advantages compared to conventional transplantation with buffy coated bone marrow (BM) harvests or mobilized peripheral blood (PB) leukapheresis products in both the autologous and allogeneic settings. Transplantation using CD34⁺ cells concentrates might significantly reduce the incidence and severity of infusional toxicity due to a twenty- to forty-fold reduction in the volume of the infused graft and dimethylsulphoxide (DMSO), as well as 200-fold reduction in the number of nonengrafting cells potentially at risk of lysis during the freeze-thaw cycle [1].

In the autologous setting, the main advantage for using selected CD34⁺ cells has been shown to be a reduction of the tumor cells contaminating the autografts in particular solid tumors up to four logs, without pharmacologic agents such as 4-hydroperoxycyclophosphamide or immunotoxins which damage the engrafting cells as well as tumor cells [2, 3].

In the allogeneic setting, the main advantage for using selected CD34⁺ cells has been suggested to be a reduction of the high T-lymphocyte content that contributes to graft-versus-host disease (GVHD), while retaining enough cells for immune reconstitution and a potential graft-versus-leukemia (GVL) effect, without T cell depletion methods such as complement-mediated lysis or counter flow centrifugal elutriation which have been complicated by graft failure and/or increased rate of relapse [4, 5].

Recently, several pilot clinical studies of autologous transplantation with selected CD34⁺ cells from BM or G-CSF-mobilized PB using the Ceprate^TMSC system (CellPro; Bothell, WA), have been reported showing a sustained trilineage engraftment equivalent to that of conventional PB stem cell autotransplantation after high-dose chemotherapy in breast cancer [2, 6-9], lymphoma [3, 10-15], myeloma [10, 11, 16] and sarcoma [17] patients.

Also, preliminary results of several pilot clinical studies of allogeneic transplantation with selected CD34⁺ cells from G-CSF-mobilized PB ± BM using the Ceprate^TMSC system have shown a sustained trilineage engraftment equivalent to that of conventional peripheral blood progenitor cell (PBPC) allotransplantation after myeloablative therapy in leukemia and lymphoma patients [18-28].

The present study reports our experience of clinical-scale CD34⁺ cell selection using the Ceprate^TM SC system in 20 allografts (12 G-CSF-mobilized PB and 8 BM) collected from 14 HLA-identical normal healthy donors for transplantation at our center with the focus on: 1) the progenitor cell and T-lymphocyte composition of the 20 allografts processed; 2) their progenitor cell recovery after freezing and thawing; 3) comparison between the composition of PB and BM allografts processed, and 4) the incidence and severity of GVHD and hematological recovery after allotransplantation with selected CD34⁺ cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Clinical-Scale CD34+ Cell... Results Discussion References

 
Normal Healthy Donors
From 14 normal healthy HLA-identical related donors, either the BM harvests from the posterior iliac crests were collected, under general anesthesia, by 50-70 aspirations of 20 ml yielding a total volume of 1 to 1.2 l, or the PB leukapheresis products were collected after G-CSF mobilization. The median age of the donors was 47 years (range, 19 to 63 years). The BM samples were collected 12 weeks prior to mobilization while the PB samples were collected after mobilization from six of the donors. The potential risks and benefits were explained in detail and, according to our institutional guidelines, written informed consent obtained from all donors and patients. Mobilization of PBPCs was performed in 12 donors by s.c. injections of 12 µg/kg G-CSF twice daily (Filgrastim; Amgen; Rockville, MD) for six consecutive days. Daily leukapheresis was started on day 5 of filgrastim administration and continued until 3 x 10¹⁰ mononuclear cells/kg were collected using a COBE Spectra continuous flow blood cell separator. A total of 8 to 10 liters of blood per leukapheresis was processed at flow rates of 50 to 70 ml/min. In all donors, 2 x 10⁸ mononuclear cells/kg from BM and/or PB were stored unprocessed to serve as a back-up.

	Clinical-Scale CD34+ Cell Selection from Allografts Using the CeprateTMSC System and Cryopreservation

Top Abstract Introduction Materials and Methods Clinical-Scale CD34+ Cell... Results Discussion References

 
The second part of mononuclear cells was processed for CD34⁺ cell selection as described before [29]. Briefly, mononuclear cells were washed twice with one liter phosphate buffered saline ([PBS], calcium and magnesium free) and concentrated in a 150 ml final volume of PBS with 0.1% human serum albumin (HSA). The cell suspension containing a median of 2 x 10¹⁰ cells (1 to 5 x 10¹⁰) was then incubated with 20 µg/ml of the biotinylated 12.8 monoclonal antibody for 25 min at room temperature. After washing with PBS to remove any antibody excess, this antibody-sensitized mononuclear cell fraction was filtered through the Ceprate^TM avidin column. In this system, the CD34⁺ cells link to the avidin-coated polyarcylamide beads through an "avidin-biotin-anti-CD34 antibody-CD34 cell" complex. After washing the column with 300 ml of PBS-HSA 1% to remove nonspecifically bound cells, the CD34⁺ cells were released from the avidin beads by mechanical agitation and recovered with 90 ml of PBS-HSA 1% supplemented with 10 IU/ml heparin. Aliquots were taken from the mononuclear, CD34⁺ and CD34^– fractions for both flow cytometric analysis and progenitor cell culture assays. The selected CD34⁺ suspension was centrifuged for 10 min at 500 g to a final volume of 5 ml and mixed with the same volume of minimal essential medium (MEM) containing 20% DMSO. The final 10 ml product was transferred into freezing bags and frozen to –100°C with a computer-controlled cryopreservation device. The frozen cells were transferred into the liquid phase of nitrogen and stored at –196°C for later infusion into patients.

Flow Cytometry Analysis
For determination of CD34 and CD3⁺ cells, 1 x 10⁶ cells from each of the mononuclear, CD34⁺ and CD34^– fractions were incubated for 30 min at 4°C in darkness with the phycoerythrin (PE)-conjugated monoclonal antibody anti-CD34 (HPCA-2) and anti-CD3 (Leu-4), respectively, obtained from Becton Dickinson then washed twice with PBS/1% bovine serum albumin (BSA). The cells were analyzed on a fluorescence-activated cell sorter (FACS)can flow cytometry equipped with LYSIS II software (Becton Dickinson; Heidelberg, Germany). A minimum of 20,000 events was acquired for both control and positive samples in order to determine a resolvable CD34⁺ population.

The total number of CD34 or CD3⁺ cells was calculated by multiplying the number of cells per fraction by the purity percentage of either CD34 or CD3⁺ cells in the fraction tested.

The yield of CD34⁺ cells was calculated by dividing the total number of CD34⁺ cells in the selected fraction by the initial total number of CD34⁺ cells in the input mononuclear fraction.

Hematopoietic Progenitor Cell Culture Assay
The colony-forming units-granulocyte-macrophage (CFU-GM) and BFU-E assays were performed from the mononuclear, CD34⁺ and CD34^– fractions of each BM or PB allograft as described before [30]. Briefly, 1 x 10⁵ mononuclear or CD34^– cells and 5 x 10³ CD34⁺ cells were plated in 35 mm petri dishes in 1 ml aliquots of Iscove's modified Dulbecco'smedium containing 30% fetal bovine serum, 1% BSA, 2 mM L-glutamine, 10^–4 M 2-mercaptoethanol, 10% agar leucocyte conditioned medium and 3 U/ml erythropoietin (Stem Cell Technologies; Vancouver, Canada, catalog no. H4431). Dishes were incubated in quadruplicate at 37°C in humidified 5% CO₂ for 14 days. On day 14, both CFU-GM and BFU-E were scored using an inverted microscope. The values for CFU enrichment (fold) were obtained by dividing the cloning efficacy in the final selected CD34⁺ cell fraction by the cloning efficacy in the mononuclear cell fraction before selection. The cloning efficacy was calculated as the percentage of clonogenic cells, i.e., the number of clonogenic cells in 100 mononuclear or CD34⁺ cells. The colony forming cell (CFC) recovery was calculated by dividing the total number of colonies detected in the selected CD34⁺ cells fraction by the total number of colonies in the input mononuclear fraction.

Allogeneic Transplant Recipients (Patients) and Their Myeloablative Treatment
Ten patients, four with acute myeloid leukemia (AML) in remission, three with high-risk acute lymphocytic leukemia (ALL) Philadelphia positive (Ph⁺) in remission, two with plasmacytoma III in relapse, and one with polycythemia vera in remission, were transplanted with four combined allografts (BM + PB from the same donor) or six single allografts (two BM and four PB). Two patients (one with AML[M5] in remission and one with high-risk ALL Ph⁺ in remission) received PB CD34⁺ cells as a boost for weak BM engraftment, 107 and 146 days after previous allogeneic bone marrow transplantation. The median age was 42 years (range, 15 to 52 years).

As a myeloablative regimen, the seven leukemia patients and the polycythemia vera patient received total body irradiation, high-dose cyclophosphamide and etoposide, and the two patients with plasmacytoma received high-dose busulphan and cyclophosphamide.

CD34⁺ Cell Infusion, Supportive Care, GVHD and Engraftment
The frozen CD34⁺ fraction was rapidly thawed, diluted in 30 ml of heparinized (10 IU/ml) MEM and infused via a central venous line on day 0. Supportive care for all patients in our center has been carried out as described in detail before [31]. All patients received G-CSF 5 µg/kg/day s.c. daily after allotransplantation until the absolute neutrophil count reached 1000/µl. The diagnosis and grading of GVHD were done on a clinical evaluation with histologic confirmation as described before [32]. Both cyclosporine and methotrexate were given to all patients as GVHD prophylaxis. Engraftment was monitored by daily differential blood counts. The day of granulocyte or platelet engraftment was defined as the first of three consecutive days with neutrophil count 500/µl, or the day the platelet count 20,000/µl without platelet transfusion for at least five days thereafter.

	Results

Top Abstract Introduction Materials and Methods Clinical-Scale CD34+ Cell... Results Discussion References

 
Composition of Allografts Before CD34⁺ Cell Selection
Filgrastim administration was generally well-tolerated, with bone pain and headache relieved with analgesics as the only side effects, and no donor had to discontinue G-CSF because of symptoms. In the eight BM harvests, the concentration of CD34⁺ cells varied from 0.4 % to 3.1%, with a median value of 0.93%, and the cloning efficacy varied from 0.07% to 0.6%, with a median of 0.17% (Fig. 1). In the 12 G-CSF-mobilized PB leukapheresis products, the concentration of CD34⁺ cells varied from 0.04 % to 3.3%, with a median value of 0.22%, and the cloning efficacy varied from 0.04% to 0.5%, with a median value of 0.15% (Fig. 1). Similar results have been reported in small-scale samples of five unprimed BM and six G-CSF-mobilized PB from healthy adult donors [33]. The median concentration of CD3⁺ cells was 19.8% (range, 16% to 22%) in the eight BM harvests and 27.6% (range 20% to 46%) in the 12 PB leukapheresis products (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Enrichment of CD34+ and reduction of CD3+ cells. BM: bone marrow harvest; PB: peripheral blood leukapheresis product; Pre: before positive selection; Post: after positive selection.

View larger version (26K): [in this window] [in a new window]   Figure 2. The correlation between CD34+ or CD3+ cells before and after positive selection was evaluated using the Pearson correlation test. Pre: before positive selection; Post: after positive selection.

The enrichment in clonogenic cells was significantly more in selected CD34⁺ cell allografts from young (< 50 years old) rather than from older ( 50 years old) adult donors (Table 2). Also, the CD34⁺ cells yield and purity were significantly better in allografts from young (< 50 years old) than from older ( 50 years old) adult donors (Table 2). Selective enrichment of the CFU-GMs was found in all 20 allografts after using the Ceprate^TMSC system which was significant in 13 allografts, p < 0.01 (Fig. 3, A and B).

View larger version (83K): [in this window] [in a new window]   Figure 3. The CFU-GM enrichment was calculated as the percentage of CFU-GM from the total number of day 14 colonies of both the mononuclear and CD34+ cell fractions. BM: bone marrow harvest; PB: peripheral blood leukapheresis product.

	Discussion

Top Abstract Introduction Materials and Methods Clinical-Scale CD34+ Cell... Results Discussion References

 
A variety of techniques including FACS, positive antibody panning selection (CELLector flasks, AIS; Santa Clara, CA), immunomagnetic bead adsorption (MiniMACS, Miltenyi; Bergisch Gladbach, Germany) and column immunoadsorption (Ceprate^TM) have been compared for small-scale selection of CD34⁺ cells from BM, G-CSF-mobilized PB and cord blood [34, 35]. Both studies have shown the Ceprate^TM and MACS methods to result in a significantly greater yield and purity than the other systems [34, 35]. In the present study, the Ceprate^TMSC system was used for clinical-scale selection of CD34⁺ cells from BM and G-CSF-mobilized PB allografts collected from 14 healthy normal donors for transplantation at our center.

Using the Ceprate^TMSC system, the median yield and purity of CD34⁺ cells from the 20 allografts were 72.5% (range, 25% to 90%) and 51.0% (range, 13% to 91%), respectively. These figures are in good agreement with those of other investigators using the small-scale selection [34, 35]. About 70% of the 20 selected CD34⁺ cell allografts had 2 x 10⁶ CD34⁺ cells/kg body weight of recipient, the recommended threshold for rapid hematologic recovery after PB transplantation [2-28]. The CD34⁺ cell yield and enrichment were significantly greater in PB than BM allografts (Table 1).

The yield and purity of BM-selected CD34⁺ cells were significantly greater in allografts from younger (< 50 years old) rather than from older ( 50 years old) adult donors (Table 2). Also, the cloning efficacy of selected CD34⁺ cells was significantly more in allografts from younger (< 50 years old) rather than from older ( 50 years old) adult donors (Table 2).

More significantly, in the same six normal donors, a comparison between the CFC recovery after CD34⁺ cell selection in the BM before mobilization versus the PB after mobilization revealed greater clonogenic recovery in PB than in BM (Table 3). Therefore, G-CSF-mobilized PB seems to be a better candidate for clinical scale CD34⁺ cell selection than BM harvests, especially from young adult donors.

Using the clinical-scale Ceprate^TMSC system, the number of T-lymphocytes in the CD34⁺ cell concentrates of the allografts was reduced by two to three log to a median of 7.8 x 10⁵ CD3⁺ cells. The selected CD34⁺ cell allografts from PB always had more CD3⁺ cells than those from BM (Table 1). Seven of the ten allotransplanted patients did not develop acute GVHD, and only one patient developed grade II disease that resolved after brief treatment with corticosteriods (Table 5). Also nine patients did not develop chronic GVHD in the 150 or more days after engraftment. The relatively low incidence (30%) and severity of both acute and chronic GVHD seen in the present study are very encouraging. These preliminary data indicate that the likelihood of developing severe acute or chronic GVHD after HLA-identical allogeneic transplantation can probably be reduced using selected CD34⁺ cells instead of the whole buffy coats. However, all 10 patients received both methotrexate and cyclosporine as GVHD prophylaxis, which could also contribute to the relatively low incidence and severity of GVHD seen in the present study. In the two patients who developed severe acute or chronic GVHD (patients 5 and 8), the number of CD3⁺ cells was 1.9 and 2.0 x 10⁶/kg in the allografts (Table 5). Whereas in the seven patients who did not develop acute GVHD, the number of CD3⁺ cells was 0.1-1.1 x 10⁶/kg in the allografts (Table 5).

Therefore, the positive selection of CD34⁺ cells with a concomitant reduction of T cells to below 1 x 10⁶/kg, but still above 1 x 10⁵/kg which was achieved in most BM and PB allografts, could reduce the GVHD and prevent graft failure or rejection. Also, given the clinical characteristics of the 10 patients and their donors including sex match, disease status and other variables known to influence the occurrence of GVHD, it seems unlikely that the absence of these risk factors was responsible for the low incidence of GVHD observed in the present study. The reason(s) for the low incidence of GVHD in the present study could be the two to three log reduction of T-lymphocytes in the enriched CD34⁺ cell allografts and/or the administration of the combination of cyclosporine and methotrexate as GVHD prophylaxis.

However, a larger series of patients will have to be observed to determine the probability of developing acute or chronic GVHD after allogeneic transplantation with selected CD34⁺ cells versus conventional PBPC transplantation. The positive selection of CD34⁺ cells with a concomitant reduction of T cells to below 1 x 10⁶/kg, but still above 1 x 10⁵/kg that has been achieved in the 20 allografts, could decrease the incidence of GVHD without the disadvantages of T cell depletion, including the high risks of graft failure or rejection. Also, the higher number of remaining T-lymphocytes in the PB-selected CD34⁺ cell allografts would be more suitable for immune reconstitution and a potential GVL effect after transplantation.

The advantages of allogeneic transplantation using G-CSF-mobilized PB rather than BM include ease of access, less discomfort for the donor, avoiding the risks of anesthesia and surgical procedure, and faster hematological recovery after myeloablative therapy [18-28]. In addition, a higher yield and clonogenicity of hematopoietic progenitor cells of G-CSF-mobilized PB have been reported compared to BM [33]. In the present study, after clinical-scale CD34⁺ cell selection, G-CSF-mobilized PB had a greater yield, enrichment and clonogenic recovery of hematopoietic progenitor cells than BM harvests collected from normal healthy donors for allogeneic transplantation. In conclusion, G-CSF-mobilized PB seems to be a better candidate for clinical-scale CD34⁺ cell selection than BM harvests collected from healthy donors for allogeneic transplantation.

	Acknowledgments

 
The authors wish to thank Mrs. B. Schleimer, M. Altenöder and B. Biermann for their excellent technical assistance.

	Footnotes

 
Provisionally accepted March 8, 1996.

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