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Clinical Cancer Research Vol. 7, 51-57, January 2001
© 2001 American Association for Cancer Research

Clinical Trials

Simultaneous Immunomagnetic CD34+ Cell Selection and B-Cell Depletion in Peripheral Blood Progenitor Cell Samples of Patients Suffering from B-Cell Non-Hodgkin’s Lymphoma1

Michael Mohr, Fikri Dalmis, Eva Hilgenfeld, Elisabeth Oelmann, Michael Zühlsdorf, Karsten Kratz-Albers, Anette Nolte, Christiane Schmitmann, Derya Önaldi-Mohr, Uwe Cassens, Hubert Serve, Walter Sibrowski, Joachim Kienast and Wolfgang E. Berdel2

Departments of Medicine/Hematology and Oncology [M. M., F. D., E. H., E. O., M. Z., K. K., A. N., C. S., H. S., J. K., W. E. B.] and Transfusion Medicine [D. Ö., U. C., W. S.], University of Münster, D-48129 Münster, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The reduction of residual tumor cells is one of the main targets of leukapheresis product (LP) processing. Immunomagnetic enrichment/selection of CD34+ progenitor cells (Baxter Isolex 300i) can achieve a reduction of contaminating B-cells of approximately 2–3 logs in B-cell non-Hodgkin’s lymphoma patients. Specific release of the enriched CD34+ cells (stem cell releasing agent PR34+; Baxter) and the use of antibody-coated immunobeads targeted against B-cell markers (CD10, CD19, CD20, CD22, CD23, and CD37) during this procedure allows the GMP-like simultaneous capture of residual B cells within a closed system. This combination of two purging techniques enhances the B-cell depletion capacity up to 4.5 logs. By performing 10 clinical-scale purging procedures, we could show that the simultaneous immunomagnetic purging method is easy to perform and highly efficient. We evaluated B-cell log depletion by flow cytometry for cases with marker-positive cells detectable before and after the purging procedure. The mean reduction of B-cells in these cases was 3.5 logs; the mean CD34+ cell yield and purity were 47 and 92%. Using three LPs, we tested the procedure on a modified Baxter Isolex 300i device with software adaptations for this procedure (software version 2.0) in direct comparison with CD34+ cell selection only, using the former version (version 1.12). The CD34+ cell yield was 49% (40–54%) for the CD34+ cell selection and 51% (19–72%) for simultaneous double selection. The mean purity was 96% for CD34+ cell selection and 98% for simultaneous double selection. B-cell depletion was 1.9 logs for CD34+ cell selection, and after simultaneous double selection, the B-cell content was decreased by 3.7 log steps (P = 0.0495). Clinical application of double-purged cells has not prolonged the hematopoietic recovery times after high-dose therapy as compared with nonpurged peripheral blood progenitor cell autotransplants. In conclusion, we could show that the simultaneous double selection protocol developed leads to a highly increased B-cell purging efficacy when compared with CD34+ cell selection without any negative effects regarding CD34+ cell yield and engraftment times after high-dose therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the last 5 years, a number of Phase III studies has proved the benefit of HDC3 with stem cell autograft rescue compared with conventional therapy in malignant diseases such as AML (1 , 2) , Hodgkin’s disease (3) , NHL (4 , 5) , and myeloma (6) . For other indications, such as the breast cancer, data are still controversial (7, 8, 9, 10) . Recent publications have shown that the use of PBPCs instead of bone marrow as a source of autologous hematopoietic stem cells used for hematopoietic rescue after HDC leads to a faster hematopoietic recovery (11 , 12) , contributing to feasibility and lower lethality of HD protocols.

Several retrospective studies indicate that tumor cell contamination of stem cell autografts may be an important factor for the prognosis of patients with Hodgkin’s disease, NHL, AML, acute lymphocytic leukemia, and breast cancer after high-dose therapy and retransfusion (13, 14, 15, 16, 17, 18) . Direct evidence for a relapse caused by contaminating tumor cells was presented by gene-marking studies in chronic myelogenous leukemia, AML, and neuroblastoma (19, 20, 21) . Thus far, there exists only one randomized study testing the impact of tumor cell depletion by CD34+ cell selection in myeloma patients after HDC. Although this study with 131 myeloma patients was not powered to assess survival as an end point, there was thus far no significant difference detected in outcome between patients who received selected and nonselected products (22) . Although the negative influence of reinfused TC on the clinical outcome after stem cell retransfusion is not clear, a large number of depletion methods have been developed (23) . The most common technique, the immunomagnetic CD34+ cell enrichment (selection) is a widely applied and effective purging procedure (24, 25, 26) . A sequential combination of CD34+ cell selection and negative B-cell selection can enhance the tumor cell depletion by ~2 log steps (27 , 28) . The use of a specific CD34 release peptide now allows the simultaneous enrichment of CD34+ cells (+ selection) and purging of tumor cells (- selection), which is less expensive and time-consuming than sequential methods (29) .

In this report, we present our results of a simultaneous immunomagnetic double purging procedure (+/- selection) for LP from NHL patients using new selective removal devices and software in the Isolex 300i system.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Characteristics and Therapy.
We performed 10 runs of the simultaneous +/- purging procedure with LP in a group of eight NHL patients. PBPC collection was performed with a Cobe Spectra cell separator (Cobe, Heimstetten, Germany) after the second or third cycle of DexaBEAM (dexamethasone, 3 x 8 mg, days 1–10; BCNU, 60 mg/m2 BS; melphalan, 20 mg/m2 BS; 1-ß-D-arabinofuranosylcytosine, 800 mg/m2 BS; VP-16, 500 mg/m2 BS) chemotherapy and 10 µg/kg body weight/day of s.c. granulocyte-colony stimulating factor (Filgrastim; Amgen, Thousand Oaks, CA) (30) . Within 18–24 days after DexaBEAM, the CD34+ cell counts, as monitored daily in the peripheral blood, reached numbers higher than 10/µl. In all patients, one single collection was needed to reach CD34+ cell numbers of >=5 x 106 cells/kg prior to cell processing. For one patient, two leukapheresis procedures were required by the treatment protocol (German multicenter CLL3 study), and for one patient the leukapheresis had to be repeated because of cell damage caused by a defect in the freezing process. For all patients, an unpurged LP was collected after the first cycle of DexaBEAM and cryopreserved as a back-up.

Patients were treated on different multicenter or institutional study protocols (after approval of the institutional ethical board), according to their specific disease entity. Thus, HDC consisted of CY/TBI (n = 2; cyclophosphamide, 120 mg/kg body weight + 12 Gy irradiation), BEAM (n = 4; BCNU, 300 mg/m2 BS; melphalan, 140 mg/m2 BS; 1-ß-D-arabinofuranosylcytosine, 1600 mg/m2 BS; VP-16, 800 mg/m2 BS) or CVB (n = 1; cyclophosphamide, 6 g/m2 BS; VP-16, 1200 mg/m2 BS; BCNU, 300 mg/m2 BS). This was followed by stem cell rescue with +/- selected stem cells (>=1 x 106 CD34+ cells/kg) and daily s.c. granulocyte-colony stimulating factor (5 µg/kg/day). Patients were entered on trial only after informed consent was obtained.

Purging Procedure.
After storage of the collected cells of the LP (culture medium:human serum albumin, 1:1; 4°C for a maximum of 24 h), the immunomagnetic CD34+ cell enrichment (Isolex 300i, standard set and software; Baxter, Munich, Germany) was performed (temperature <25°C) as described by the manufacturer with the following modifications. During the procedure, following the transfer of the specific competitive stem cell-releasing agent (PR34+; Baxter) to the separation column, we added monoclonal antibodies against NHL markers (CD10, CD19, CD20, CD22, and CD37; Baxter) bound to immunobeads (Dynabeads M450; Baxter) for additional negative selection (purging) of B cells. To avoid cell clumping during the procedure, the separation column was prefilled with 5000 units of Pulmozyme (Roche, Grenzach, Germany) plus 0.1 mM Mg2+(Cl-)2. The process is visualized in Fig. 1Citation . Using software version 2.0, the procedure takes ~20 min longer than CD34+ cell enrichment only and induces a cost increase of ~20%, if one purging antibody is used and an additional 10% for each additional purging antibody.



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  		Fig. 1. Simultaneous CD34+ cell selection and B-cell depletion.

 
Using LPs of three patients, we tested the +/- purging procedure on a modified Baxter Isolex 300i device with software adaptations for this procedure (software version 2.0; Baxter) in direct comparison with + selection only, using the former version (versus 1.12). In these cases, we used 1:1 split LPs and performed two runs with the same starting time on both devices. All steps were performed according to the manufacturers description. After high-dose therapy, only the +/- selected products were used for retransfusion. For these patients, the CD34+ cell-enriched products were cryopreserved and stored as a back-up.

Evaluation of Immunomagnetic CD34+ Cell Selection.
The efficiency of the immunomagnetic separation procedure to select CD34+ cells from peripheral blood mononuclear cells (+ selection) was evaluated by applying definitions as published (26) :


The BCD was expressed as described below. The cell product was cryopreserved after selection with computer assistance at a rate of -1°C/min and stored in liquid nitrogen until retransfusion. Extra vials were frozen for quality control. The following parameters were tested after thawing: (a) number and purity of CD34+ and propidium iodide-negative (viable) cells; (b) viability and cell count in trypan blue dye exclusion; (c) colony count in the CFU assay (see below); and (d) microbiological sterility control.

Flow Cytometry.
CD34+ hematopoietic cells were quantified by three-color immunofluorescence using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) under assistance of CellQuest software. The following antibodies were used: CD34-PE and CD45 FITC (Becton Dickinson). For viability testing after cryopreservation, propidium iodide was added as a third fluorescence. For B-cell determination CD19, CD20, CD5, and/or cCD79a antibodies in relation to the tumor immune phenotype were used. Detection limit of the method for cCD79a or CD20/CD5 coexpression is 0.01% of the nucleated cells. Aliquots of the cell suspensions were investigated before and after the purging procedure for their B-cell content. The B-cell depletion was expressed in log steps as follows:

Considering that in this calculation a bad CD34+ cell yield would be expressed as additional BCD, we decided to calculate with the "yield corrected" BCD:

CFU Assay.
The hematopoietic progenitors, clusters (<40 cells), CFU-GM (granulocyte-macrophage lineage), BFU-E and CFU-E (erythroid lineage), and CFU-GEMM (mixed early progenitors with granulocytic, monocytic, megakaryocytic, and erythroid potential), were assayed using a standardized culture assay (Methocult H4431; Stemcell Technologies, Vancouver, Canada). We have plated 105 cells in 1 ml of medium for the unselected samples and 103 cells in 1 ml of medium for the selected samples. Each sample was plated in triplicate assays, and the calculated means are given.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In this series of experiments, we have established and tested a new simultaneous immunomagnetic double purging procedure (+/- selection) for LPs from B-NHL patients using new selective removal devices in the Isolex 300i system. Furthermore, we tested the +/- selection procedure on a new device modified especially for this application (version 2.0) in direct comparison with the former version (version 1.12).

Purging Procedure.
By processing LPs from 10 B-NHL patients, simultaneous immunomagnetic +/- selection led to a mean CD34+ cell purity of 92% (range, 68–99%). The mean yield of CD34+ cells was 47% (range, 19–72%; Table 1Citation ).


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  		Table 1 Patients’ graft data (+/- selection)

 
Hematopoietic Recovery.
Up to now, seven of the patients were treated with high-dose chemotherapy, as described above, followed by stem cell rescue with simultaneously +/- selected CD34+ cells (target, >1 x 106 CD34+ cells/kg). One patient developed FUO and died of septicemia resistant to antibiotics 3 days after reinfusion of the stem cells. The hematopoietic recovery times of the six remaining patients were 11.3 days for >2,000 leukocytes (WBCs)/µl and 16 days for >50,000 platelets/µl (Table 2)Citation . Thus, recovery times were well within the range expected after autograft with untreated or chemopurged PBPCs (30 , 31) , and all patients recovered promptly with hematopoiesis with up-to-normal counts.


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  		Table 2 Patients’ recovery data (+/- selection)

 
Purging Efficacy.
The B-cell depletion after the simultaneous double selection, as assessed by flow cytometry, ranged between 0.9 and 4.6 logs with a mean of 3.5 logs. The yield corrected BCD ranged between 0.6 and 4.0 logs with a mean of 3.2 log steps (see Table 1Citation ). Molecular data on minimal residual disease are available only for one patient (CLL3 protocol), with the immunoglobulin gene rearrangement being positive for minimal residual disease in all products before and after selection.

Direct Comparison.
In three direct comparison runs, we used cCD79a for the determination of the purging efficacy to avoid any possible interference of the purging and the fluorescence-activated cell sorter antibodies. Here we demonstrated that the yield corrected BCD reached by +/- selection was ~1.8 log steps higher as compared with the CD34+ cell selection only. The detected difference was statistically significant (P = 0.0495, Mann-Whitney test; Fig. 2Citation and Table 3Citation ).



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  		Fig. 2. Direct comparison of BCD measured by cytoplasmic CD79a staining (patient 6, product 8).

 

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  		Table 3 Direct comparison of CD 34+ cell selection only with simultaneous double selection (n = 3)

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The clinical relevance of tumor cell purging from hematopoietic stem cell autografts prior to reinfusion after high-dose therapy is still an open question. Gribben et al. (14) could show a significantly increased disease-free survival for patients who received bone marrow that was depleted from residual lymphoma cells compared with those who received contaminated marrow. This, however, could also reflect different prognostic subgroups by disease-load prior to high-dose therapy. Recently, Mizuta et al. (15) presented results on high-dose chemotherapy and autologous transplantation with purged PBPCs in acute lymphocytic leukemia patients. In this group of patients, the 3-year disease-free survival was significantly related to the grade of minimal residual disease before and after purging of the autograft but not to the purging efficacy. Thus far, the only randomized study in myeloma patients could show no significant benefit of CD34+ cell selected compared with unselected autografts with regard to time to progression (22) .

However, gene-marking studies, case reports, and the well-known fact that even in remission occult tumor cells can be detected in bone marrow and peripheral blood argue in favor of the application of purging procedures (19, 20, 21 , 23 , 24 , 32, 33, 34) . Thus, different purging methods have been developed (23 , 26 , 29 , 35, 36, 37) .

In this study, we have examined the safety and efficacy of purging PBPCs from B-NHL patients by developing and using a new simultaneous immunomagnetic +/- selection protocol. Using LPs from B-NHL patients, simultaneous immunomagnetic +/- selection led to a mean CD34+ cell purity of 92% and a mean yield of CD34+ cells of 47%. Hematopoietic recovery of NHL patients after high-dose therapy and stem cell rescue was prompt, and no major delay was observed.

Purging efficacy was evaluated by flow cytometric B-cell determination, and the yield corrected BCD after double selection ranged between 0.6 and 4.0 log steps. In direct comparison with CD34+ cell selection only, the double selection protocol reported here demonstrated an 1.8 log steps increased BCD, the detected difference in B-cell log depletion was statistically significant. The method is easy, fast, and safe to perform under GMP-like conditions within a closed system. However, the BCD in our tests did not reach the BCD of 4.6–6 log steps as achieved by sequential +/- selection as reported by Paulus et al. (28) .

Today, mainly three different purging methods are used clinically, which are still intensively tested for methodological improvement and clinical relevance. Apart from positive stem cell selection (e.g., immunomagnetically with anti-CD34 antibodies), there are negative selection techniques in which tumor cells are eliminated either immunologically by antitumor antibodies or chemically/pharmacologically by toxin or drug exposure (14 , 16 , 38, 39, 40, 41, 42) .

Choosing a positive stem cell selection system offers multiple advantages. CD34+ hematopoietic cells can be enriched up to >90% (our data) before reinfusion into the patient. This means less volume, less freezing medium, and therefore less potential toxicity for the patient. CD34+ cells of LP show a full capability of restoring hematopoiesis when administered after high-dose therapy (43) and identical reconstitution of hematopoiesis as with unseparated LPs (44) . However, immunomagnetic CD34+ cell selection can decrease tumor cell numbers by 1.9–3.1 log steps as reported (24, 25, 26) .

On the other hand, negative selection techniques bear the risk of purging damages for hematopoietic stem cells (39 , 45) . Some studies show depletion of normal hematopoietic cells, delay in engraftment, and an increasing risk of myelosuppressive complications. This can be attributable to simultaneous toxic effects by chemotherapeutics and immunotoxins on hematopoietic precursor cells. Removal of 1–4 logs of tumor cells with one purging cycle was seen when using a mixture of three different antitumor antibodies (41) . Anderson et al. (36) combined immunomagnetic purging with 4-HC and eliminated up to 5 logs of clonogenic breast cancer cells but with a reduced recovery (<50%) of GM-CFU.

The B-cell purging method described here combines positive CD34+ cell enrichment with negative B-cell purging by a simultaneous immunomagnetic procedure. Looking at safety and efficacy, it compares favorably with the other techniques published before. It is as easy to perform as the positive selection of CD34+ cells with the same system, and cost increase is limited. We recommend simultaneous immunomagnetic double purging for clinical testing within a wider range of high-dose therapy trials.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a research grant from Baxter Germany. Back

2 To whom requests for reprints should be addressed, at Medizinische Klinik A, Universitätsklinikum Münster Albert-Schweitzer Strasse 33, D-48129 Münster, Germany. Phone: 49-251-834-7586; Fax: 49-251-834-7588; E-mail: berdel{at}uni-muenster.de Back

3 The abbreviations used are: HDC, high-dose chemotherapy; AML, acute myelogenous leukemia; NHL, non-Hodgkin’s lymphoma; B-NHL, B-cell NHL; BCD, B-cell depletion; PBPC, peripheral blood progenitor cell; LP, leukapheresis product; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BS, body surface; VP-16, etoposide; CFU, colony-forming unit. Back

Received 4/26/00; revised 10/30/00; accepted 11/ 2/00.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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M. Witzens-Harig, C. Heilmann, M. Hensel, M. Kornacker, A. Benner, R. Haas, S. Fruehauf, and A. D. Ho
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