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Purging Peripheral Blood Progenitor Cell Grafts from Lymphoma Cells: Quantitative Comparison of Immunomagnetic CD34⁺ Selection Systems

Second Department of Internal Medicine, University of Kiel, Kiel, Germany

Key Words. PBPC • CD34⁺ selection • Lymphoma • Purging • PCR

Correspondence: Dr. Ulrike Paulus, Second Department of Medicine, Chemnitzstr. 33, D-24116 Kiel, Germany.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autologous peripheral blood progenitor cell (PBPC) transplantation is increasingly being used for treatment of indolent lymphomas. Since involvement of bone marrow and peripheral blood is frequent and methods to reduce the lymphoma cell load of PBPC grafts are thus highly desirable, we have studied purging of PBPC comparing two immunomagnetic CD34⁺ selection systems (VarioMACS^TM, Miltenyi Biotech; Bergisch Gladbach, Germany, and Isolex50^TM System, Baxter; Irvine, CA). Samples of freshly collected mobilized PBPCs were contaminated with BALM-3 or KARPAS422 lymphoma cells that had been labeled with the fluorescent DNA stain Hoechst 33342. The mixture was subjected to separation with the two devices and the resulting "CD34⁺" fractions were screened for lymphoma cells by limiting dilution using fluorescence microscopy and by polymerase chain reaction amplification of t(14;18) or CDRIII-rearrangements. Both devices yielded comparable purities (MACS 97% [87%-99%]; Isolex 97% [84%-99%]) and recoveries of CD34⁺ cells (MACS 56% [30%-81%]; Isolex 45% [24%-63%]). The overall depletion of lymphoma cells was 3.9 log (2.6-5.9), however, residual contaminating cells were seen in every single experiment. The purging efficacy was dependent on the type of contaminating lymphoma cell (BALM-3: 4.4 log [3.7-4.8]; KARPAS422: 3.2 log [2.6-4.2]; p = 0.018), whereas the type of selection system used or the percentage of CD34⁺ cells in the starting material had no influence. We conclude that excellent purification of CD34⁺ cells leading to a vigorous depletion of lymphoma cells can be achieved with both CD34⁺ selection systems investigated. However, the efficacy of purging may greatly differ between individual lymphomas, and complete eradication of contaminating cells from PBPC grafts may rarely be achieved with CD34⁺ selection alone.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myeloablative therapy followed by autologous stem cell support (ASCT) is increasingly seen as a new therapeutic option for disseminated low-grade non-Hodgkin's lymphomas (NHL) [1-3] as none of the conventional treatment modalities proved to be curative for this group of diseases. Progression-free survival appears to be significantly prolonged in patients treated with ASCT as compared to historical controls without high-dose intensification [3], although it is still unknown if myeloablative therapy has the potential to eradicate the disease. Due to their favorable engraftment kinetics, peripheral blood progenitor cells (PBPCs) are now the preferred source of stem cells used for ASCT [4-6].

Although lymphoma cell contamination of PBPC grafts from patients with low-grade NHL might be less common compared with bone marrow (BM) grafts [7], PBPCs frequently contain tumor cells as revealed by sensitive techniques, such as polymerase chain reaction (PCR) amplification of t(14;18) or CDRIII rearrangements [4, 8]. Various methods to eliminate lymphoma cells from PBPC grafts are currently under investigation. Besides using B cell-specific monoclonal antibodies for direct depletion of NHL cells [9, 10], effective purging might be achieved by positive selection of CD34⁺ cells [11-13].

CD34⁺ selection is possible with a variety of techniques, such as immunoadsorption or immunomagnetic separation. Although some reports have shown the suitability of CD34⁺ selection for eliminating tumor cells from PBPC grafts [11, 12], data on its efficacy in patients with low-grade NHL are sparse. In the present study, we have compared two immunomagnetic CD34⁺ selection devices ("VarioMACS," Miltenyi Biotech; Bergisch Gladbach, Germany, and "Isolex50," Baxter Immunotherapy Division; Irvine, CA) with regard to their effects on lymphoma cells contaminating PBPC grafts, and to variables influencing CD34⁺ cell enrichment and efficacy of tumor cell depletion. PBPC samples were contaminated with defined amounts of lymphoma cell lines. Employing both a newly developed sensitive limiting dilution analysis (LDA) and a semi-nested PCR, we show that up to a five-log reduction of lymphoma cells can be achieved. Whereas the type of device used or the percentage of CD34⁺ cells present in the starting material had no significant influence on purging efficacy, the latter appeared to be dependent on the cell line used for contamination.

	Materials and Methods

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Cells
PBPC samples of leukapheresis products obtained from 10 individuals for autologous or allogeneic transplantation were investigated. All donors had given informed consent. Nine donors were male, one was female, and their median age was 46 years. Five of them suffered from a high-grade NHL, two from a low-grade NHL, one from Hodgkin's disease and two were healthy donors. PBPCs were mobilized either with the Dexa-BEAM protocol (dexamethasone, BCNU, etoposide, cytarabine, and melphalan) plus G-CSF as previously described [14] or with G-CSF alone (Table 1). The lymphoma cells used for contamination of PBPC were derived from the NHL cell lines BALM-3 (kindly provided by J. Steinmann, Kiel) and KARPAS422 (kindly provided by A. Karpas, Cambridge). KARPAS422 cell lines carry the translocation t(14;18) characteristic of follicular lymphomas and BALM-3 lines have a specific CDRIII rearrangement [15, 16]. According to flow cytometric analyses both cell lines are negative for the CD34^– antigen. The cell lines were kept at 37°C and 5% CO₂ in medium that consisted of RPMI 1640 (BRL Life Technologies; Eggenstein, Germany), 15% fetal calf serum (Seromed; Berlin, Germany) and 1% L-glutamine. It was supplemented alternatingly with kanamycin and penicillin/streptomycin.

CD34⁺ Selection
Immunomagnetic separation of CD34⁺ cells was performed with either the VarioMACS or the Isolex50 system. Both are small-scale separation devices based on antibody-mediated magnetic labeling of CD34⁺ cells. CD34⁺ selection with Isolex was carried out as previously described [18]. Briefly, the PBPC/tumor cell mixture was incubated with 400 µl (= 200 µg) of a monoclonal murine antihuman anti-CD34 antibody (9C5) and subsequently rosetted with 500 µl of Dynabeads (bead/cell ratio = 0.5). For separation of rosettes from unbound cells the suspension was repeatedly exposed to magnetic forces. Each time the supernatant was removed and collected (CD34^– fraction) while the rosettes retained by the magnet were resuspended afterwards (CD34⁺ fraction). The CD34⁺ cells were released from the beads by enzymatic cleavage of the 9C5-binding epitope using chymopapain.

VarioMACS experiments were performed according to the manufacturer's instructions. The unfractionated sample was first incubated with 400 µl of human immunoglobulin (FcR blocking agent) and 400 µl of a monoclonal hapten-conjugated CD34 antibody (QBEND/10) and afterwards with 400 µl of colloidal super-paramagnetic microbeads conjugated to antihapten antibodies. For separation of bead-bound cells from unbound cells, the cell suspension was passed through a column with a ferromagnetic core that had been attached to a powerful permanent magnet. The column was washed with medium and the effluent collected (CD34^– fraction). After removal of the magnet the retained cells were eluted and passed through another newly prepared column. The final product obtained after the second passage was assigned as CD34⁺ fraction.

Flow Cytometry
For immunophenotypical quantification of CD34⁺ cells, samples were suspended with phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugated specific monoclonal antibodies or PE-/FITC-conjugated irrelevant isotype-specific antibodies (DAKO; Hamburg, Germany) in phosphate-buffered saline containing 0.2% sodium azide. After 30 min incubation and fixation with 1% formaldehyde, flow cytometry was performed with a FACScan flow cytometer (Becton Dickinson; Heidelberg, Germany). The antibodies used were: HPCA-2-PE (anti-CD34), and HLe-1-FITC (anti-CD45, both from Becton Dickinson). Analyses were done using FACScan software (Becton Dickinson). Absolute numbers of CD34⁺ cells were calculated from the total percentage of HPCA-2⁺/HLe-1⁺ cells which were side scatter low [19).

LDAs
LDAs were performed for exact quantification of very small amounts of contaminating tumor cells. Briefly, a 1:2 dilution series of the cell sample was prepared. Eighteen replicates of each concentration were pipetted into wells of a 72-well Terasaki plate. The fluorescence of labeled tumor cells is usually bright enough to detect a single fluorescent cell per visual field when assessed microscopically with UV light. For each concentration the number of "positive" (one or more fluorescent cells) and "negative" (no fluorescent cells) replicates was determined. With the knowledge of the fraction of negative wells for each concentration step of the 1:2 dilution series of the given cell sample, its tumor cell frequency could be calculated according to Strijbosch et al. [20]. For validation of LDA, preliminary experiments were carried out to prove that the results obtained are exact and reproducible; in three independent experiments defined amounts of fluorescence-labeled tumor cells were seeded into samples of PBPC (0.1%, 0.01%, 0.001% BALM 3 cells). For an actual tumor cell content of 0.1% contamination was calculated to be 0.064%-0.072%, an actual tumor cell content of 0.01% was determined to be 0.01%-0.013% and actual 0.001% corresponded to calculated 0.0016%. Ninety-five percent confidence intervals did not exceed 0.4 log. Although LDA appears to consistently underestimate in particular larger actual proportions of contaminating cells (0.1%-10%, see also Results section), the difference was always less than one log. A representative experiment is shown in Figure 1. Thus, the frequencies determined by this newly developed LDA proved to be a reliable approximation of the actual tumor cell content allowing a sufficiently exact comparative quantification of even very small amounts of tumor cell contamination.

View larger version (16K): [in this window] [in a new window]   Figure 1. Representative experiment for validation of LDAs. Samples of a PBPC graft were contaminated with defined amounts of H33342-stained BALM-3 cells (* 10%, 0.1%, • 0.01%, 0.001%). For each of these four contaminations LDAs were performed as described under Material and Methods and resulted in the following tumor cell frequencies as calculated according to Strijbosch et al. (expected/ calculated): * 10%/6.98%, 0.1%/0.064%, • 0.01%/0.011%, 0.001%/0.001%. Abscissa: dilution of samples (number of cells per well). Ordinate: fraction of "negative" wells (without detectable fluorescent cells) of all replicates at a given dilution.

Statistical Analysis
Nonparametric Mann-Whitney tests were used to compare quantitative parameters. Correlations were calculated with Spearman's rank correlation test.

	Results

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CD34⁺ Selection
As determined by flow cytometry, the median CD34⁺ cell content in the 17 unfractionated samples was 1.17% (0.2%-5.2%). After immunomagnetic separation, this proportion increased to a purity of 96.6% (84.2%-99.5%) in the CD34⁺ cell fractions, which means CD34⁺ cells were enriched 114-fold (19-450). A median of 46% (24%-81%) of the CD34⁺ cells present in the starting fractions was recovered in the CD34⁺ cell fractions. No significant differences with regard to purity, enrichment, recovery, or residual CD34⁺ cells in the CD34^– fraction were found for the two different selection procedures investigated (MACS versus Isolex; Table 2). However, the purities of the CD34⁺ fractions were positively correlated with the percentage of CD34⁺ cells in the starting material (rs = 0.77; Spearman's rank correlation).

View larger version (12K): [in this window] [in a new window]   Figure 2. Results of LDAs (experiments four and five). The results of LDAs are presented for unfractionated samples (contaminated with 5% KARPAS422) and CD34+ fractions obtained after either the MACS procedure A) or the Isolex procedure B). Again, cell dilutions are given on the abscissa and the respective proportion of negative wells on the ordinate. A) unfractionated sample, 1:78 tumor cells; * CD34+ fraction, 1:6,452 tumor cells; B) unfractionated sample, 1:69 tumor cells; * CD34+ fraction, 1:6,944 tumor cells.

View larger version (27K): [in this window] [in a new window]   Figure 3. Results of PCR amplification of t(14;18) (experiments four and five). Strong tumor-specific signals (arrows) were found in the unfractionated samples (contaminated with 5% KARPAS422, lane eight) and in the CD34– fractions (lane six) of both experiments. Although the intensity of the specific signal was reduced after CD34+ selection with MACS A) or Isolex B), it was still present in both CD34+ fractions (lane seven). Lane one: Phi X 174 RF standard; lanes two and three: aqua; lane four: negative control; lane five: positive control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Various methods aimed at isolation and enrichment of hematopoietic progenitors are available. The most widely used CD34⁺ selection system is the immunoadsorption technique introduced by Berenson et al. [11, 24]. Since in our hands immunomagnetic CD34⁺ selection has been superior to immunoadsorption in terms of purity of CD34⁺ cells and target cell depletion [18], we studied the MACS and Isolex devices (both relying on immunomagnetic separation) with regard to their quantitative efficacy for purging lymphoma cells from PBPC grafts. Our data indicate that both systems result in a very high purity of CD34⁺ cells in the final product (in the vast majority of experiments greater than 90%), matching the results we previously obtained with the Isolex system [18] and exceeding the purities reported for the immunoadsorption technique [11, 18, 23, 25, 26]. A good purity of selected cells appears to be an essential prerequisite not only for tumor cell depletion but also for other purposes, such as progenitor cell analysis or genetic engineering of progenitor cells [21, 27, 28].

There are only a few other studies comparing different CD34⁺ selection devices in head-to-head experiments. Using MACS, de Wynter et al. observed similar recoveries but lower purities (61%-96%) which appeared not to be different from the results found with immunoadsorption [26]. However, the number of experiments was small in their study, and the MACS separations were done without repetition of the final step. (The Isolex system was not included in this comparison.) With two passages, purities higher than 90% have been reported for the MACS system [25]. Whereas, in the present study, yield and purity were not significantly influenced by the kind of device used, the final purity after CD34⁺ selection was positively correlated with the percentage of CD34⁺ cells in the starting fraction, emphasizing that effective PBPC mobilization is an important factor for achieving optimum CD34⁺ purification.

Although both MACS and Isolex gave comparably high tumor cell depletion (up to 5.9 log), residual lymphoma cells were detected by PCR and/or LDA in every single experiment, implying that complete elimination of lymphoma cells may rarely be achieved with CD34⁺ selection alone. Additional steps, such as subsequent negative B cell depletion, may be necessary to fully eradicate NHL cells from PBPC grafts. Nevertheless, our results seem at least equivalent to those achieved by Lemoli et al. who reported a tumor cell depletion of about three log from BM preparations using a similar approach (10% H33342-stained lymphoma cell line contamination) with the immunoadsorption device [29]. However, since LDA was not used in their analysis, the quantitative assessment of purging efficacy in Lemoli's study might be affected by a low sensitivity of tumor cell detection. In spite of better purities achieved with higher CD34⁺ cell numbers in the untreated fractions, a correlation between the percentage of CD34⁺ cells in the starting material and the efficacy of tumor cell depletion was not seen. This may be explained by the fact that lower target cell numbers prior to selection translate into larger depletion of total MNC during the separation procedure which compensates for the possibly higher tumor cell frequencies in the final products.

On the other hand, purging efficacy was significantly influenced by the type of cell line used for contamination, indicating that the performance of the selection systems may greatly differ between individual lymphomas. Among the factors which account for these differences could be the unspecific adhesion of lymphoma cells to CD34⁺ target cells, beads, or other components of the separation devices. Thus, at the moment considerable uncertainty regarding the extent of tumor cell depletion attainable with CD34⁺ selection in individual patients should be taken into account.

Preliminary reports from us and others suggest that similar results in terms of purity and recovery may be achieved with both selection systems also on a clinical scale [30-32]. With the Isolex300 system we were able to achieve a median purity of 89% CD34⁺ cells and 2.4-5.1 log lymphoma cell depletion [30].

Taken together, our data show that excellent purification of PBPC and vigorous depletion of lymphoma cells can be achieved with both CD34⁺ selection systems investigated. However, the efficacy of purging may greatly differ between individual lymphomas. Complete eradication of contaminating cells from PBPC grafts appears to be rarely achieved with CD34⁺ selection alone, and studies aiming at further improvement of cell selection procedures are required.

Recent reports suggest that with a lower initial tumor contamination of the stem cell graft chances are higher to obtain CD34⁺-selected products without detectable tumor cells [33]. Thus efficient in vivo purging prior to stem cell collection appears to be highly important.

	Acknowledgments

 
The excellent technical assistance of Susanne Kell is gratefully acknowledged.

Supported by the Deutsche Krebshilfe (Grant W/9/94/Schm1).

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