Purging and Haemopoietic Progenitor Cell Selection by CD34 Cell Separation
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Bone Marrow Transplantation, (1998) 21, 665–671 1998 Stockton Press All rights reserved 0268–3369/98 $12.00 Purging and haemopoietic progenitor cell selection by CD34؉ cell separation ¨ ¨ W Kruger1, M Gruber1, S Hennings1, N Fehse1, B Fehse1, K Gutensohn2, N Kroger1 and AR Zander1 1Bone Marrow Transplantation Unit, 2Blood Transfusion Service, University Hospital Eppendorf, Hamburg, Germany Summary: High-dose chemotherapy supported by autologous stem cell reinfusion for treatment of metastatic and high-risk female breast cancer is currently under investigation in several Tumour cell contamination of autologous peripheral American and European studies.1,2,3 Tumour cell contami- blood stem cell samples (PBSC) and bone marrow (BM) nation of autologous stem cell products has been described is frequent. Enrichment of CD34+ stem cells is a promis- using immunocytochemistry, reverse transcriptase PCR and ing approach to purging tumour cells from autografts cell culture techniques in approximately one third of har- without damaging progenitor cells. Breast cancer cells vests with a range from 0% to 100%.4–7 The clonogenic were seeded (10؊3؊10؊7) into mononuclear cells from and metastatic potential of tumour cells isolated from blood G-CSF-mobilised PBSC and BM harvests from patients and autografts was demonstrated in cell culture assays and without breast cancer. CD34+ cells were enriched from in nude mice.8–10 The metastatic potential of accidentally mixtures either by immunomagnetic separation (Isolex- retransplanted tumour cells could be investigated by gene 50, and MiniMACS) or by biotin-streptavidin immu- marking of autografts prior to infusion followed by the noaffinity columns (Ceprate-LC). CD34؉ cell fractions detection of marked cells after relapse.11 However, due to were determined by FACS, cancer cells were detected the poor transduction efficacy of epithelial cancer cells by immunocytochemically with an anti-pancytokeratin gene transfer, a negative result would not exclude the possi- antibody. The CD34؉ cells were enriched with a median bility of relapse induction by accidentally reinfused tumour Isolex-50), 96.5% cells. Different approaches have been described to purge) (17 ؍ purity of 92.2% (43.5–96.1) (n MiniMACS) and 77.9% (31.4– autografts of tumour cells.12 A major problem of purging) (17 ؍ n) (99.2–66.6) -Ceprate-LC) from PBSC and BM har- procedures using cytotoxic agents is the damage to haemo) (15 ؍ n) (93.6 vests. The percentages of median recovery of CD34+ poietic progenitor cells with subsequent delay of cells were 30.8% (18.6–71.8) (Isolex-50), 69.9% (39.1– engraftment, an increased incidence of severe infections 100) (MiniMACS) and 42.9% (23.7–100) (Ceprate-LC). due to prolonged neutropenia and bleeding complications The median tumour cell reductions in log steps were due to thrombocytopenia.13–15 -Haemopoietic cells necessary for recovery after high (13 ؍ Isolex-50), 3.5 (2.6–4.3) (n) (13 ؍ n) (4.3–2.9) 3.7 -Ceprate-LC). dose therapy and progenitor support express the CD34 anti) (17 ؍ MiniMACS) and 1.5 (0.9–2.9) (n) Results were compared statistically by univariate analy- gen on their surface.16 These cells can easily be enriched sis. Purity was significantly (P Ͻ 0.05) better after Mini- by immunological methods using anti-CD34 antibodies and MACS selection. Recovery rates were significantly dif- separation by biotin-streptavidin immunoaffinity or mag- ferent between all devices tested. Tumour cell purging netic separation with paramagnetic microbeads. Rapid and was superior after immunomagnetic separation (P Ͻ safe engraftment after reinfusion of enriched CD34+ cell 0.001). Tumour cell purging is a main objective of fractions has been described by several investigators after CD34+ selection in the autologous setting. Our in vitro allogeneic and autologous transplantation.17,18 The data clearly indicate that immunomagnetic separation immunologic selection of CD34-positive cells has no toxic is more efficient in the prevention of accidental rein- side-effects on haemopoietic stem cells and progenitors fusion of contaminating tumour cells compared to without prolongation of cytopenia after reinfusion. immunoaffinity. However, it is not yet known if the Furthermore, CD34 positive-cell selection is a promising same results can be obtained with fresh contaminating approach to reducing the incidence or mitigating the sever- tumour cells. ity of graft-versus-host-disease in the allogeneic setting by Keywords: CD34+ cell selection; micrometastases; purg- adjusted or nearly complete T cell depletion. However, the ing; breast cancer major rationale for CD34-positive cell selection in autolog- ous marrow and stem cell transplantation is a reduction or removal of contaminating tumour cells accidentally cohar- vested during leukapheresis to avoid their reinfusion after high-dose therapy.19,20 + + ¨ Correspondence: W Kruger, Bone Marrow Transplantation Unit, Univer- In this report we compare CD34 cell enrichment, CD34 sity Hospital Eppendorf, Martinistraße 52, 20246 Hamburg, Germany cell recovery, and the efficacy of purging of experimentally Received 22 September 1997; accepted 11 November 1997 seeded breast cancer cells from bone marrow and G-CSF- Purging by CD34+ cell selection ¨ W Kruger et al 666 mobilised peripheral blood stem cell collections between Only gravity was used for cell flow through selection three devices using immunoaffinity and magnetic columns. microbeads for separation of anti-CD34 antibody-labelled + + CD34 cells. We show that CD34 enrichment leads to a FACS analysis decrease in the total number of contaminating tumour cells up to 4.3 log steps with reasonably high recovery of CD34- The flow cytometric analyses were performed before and positive progenitor cells. after CD34+ cell selection on a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany). The instrument settings were established for linear amplification of light Material and methods scatter and logarithmic amplification of fluorescence chan- nels. The cells were stained following standard protocols with a phycoerythrin (PE)-coupled antibody (HPCA-2; Bone marrow and leukapheresis samples Becton Dickinson) not interfering with the antibodies used + Aliquots of 21 bone marrow samples and 37 leukaphereses for CD34 enrichment and recommended by the suppliers were obtained after informed consent. Nineteen volunteer of selection devices. Controls were performed with an anti- donors and two patients with acute myeloid leukaemia isotype IgG1-PE antibody (Becton Dickinson). Signals were underwent bone marrow harvests from the iliac crest under presented graphically as a dot plot and data were analysed general or spinal anaesthesia without prior growth factor with Lysis-II software (Becton Dickinson).21 mobilisation. Thirty-seven leukapheresis aliquots were obtained from 18 patients after preceding 5-day G-CSF Immunocytochemistry mobilisation with a daily dose of 10–24 g/kg body weight injected subcutanously. The underlying diagnoses of the Cytospin slides were prepared with a Shandon cytospin stem cell donors were non-Hodgkin’s lymphoma (n = 8), centrifuge (Shandon, Runcorn, UK). Two hundred thou- multiple myeloma (n = 3), sarcoma (n = 2) and Hodgkin’s sand cells were spun onto each slide and one to five slides were prepared per sample. The number of slides available disease. Four patients were volunteer donors for allogeneic + transplantation. Mononuclear cells were isolated from mar- depended on the amount of recovered cells after CD34 cell + row and stem cell samples by Ficoll centrifugation follow- separation. Due to the limited CD34 cell count available for immunocytochemistry, we could test for samples spiked ing standard protocols. Most marrow and stem cell aliquots Ϫ Ϫ were too small to split them into three aliquots to run the with 10 6 or 10 7 cells only for relative tumour cell three devices. enrichment. Slides were air-dried, fixed, and stored at Ϫ20°C. Tumour cells on cytospin slides were detected by the anti- Cell lines and tumour cell spiking cytokeratin antibody KL1 (Boehringer Mannheim, Mannheim, Germany) and subsequent APAAP (alkaline The breast cancer cell lines MCF-7 and MDA-MB453 were phosphatase anti-alkaline phosphatase) stain. Stained cyto- used for tumour cell spiking experiments. The cell lines spin slides were evaluated by light microscopy, and the were purchased from the American Type Culture Collec- KL1-positive cells were counted.22 tion (ATCC), Rockville, MD, USA, and cultured and main- tained under the conditions recommended by ATCC. For spiking experiments, the cells were trypsinised to obtain Data analysis single cell suspensions. Suspensions were checked by light Data were analysed using the computer software Excel microscopy. Density of tumour cells was calculated using (Microsoft, Munich, Germany) and WinSTAT (Kalmia Co, a Neubauer chamber and mononuclear cell fractions of Cambridge, MA, USA). For the comparison of the two marrow, and leukapheresis samples were counted after Fic- groups, the independent t-test or the Mann–Whitney U test oll separation using a Coulter counter (Coulter, Krefeld, were used, and processing data from all devices were com- Germany). Leukapheresis and bone marrow samples were pared by univariate analysis. spiked with tumour cells MCF-7 and MDA-MB453 in log- arithmic dilutions from 10Ϫ3 (0.1% tumour cells) to 10Ϫ7 (0.00001% tumour cells). For purging experiments tumour Results cells from 10Ϫ3 to 10Ϫ7 were performed for each device at least twice – once with each cell line – with higher