Ex Vivo Expansion of Bone Marrow from Breast Cancer Patients: Reduction in Tumor Cell Content Through Passive Purging

Bone Marrow Transplantation, (1998) 22, 153–159 1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt Ex vivo expansion of bone marrow from breast cancer patients: reduction in tumor cell content through passive purging BI Lundell1, JJ Vredenburgh2, C Tyer2, K DeSombre2 and AK Smith1 1Aastrom Biosciences, Ann Arbor, MI; and 2Duke University Bone Marrow Transplant Program, Duke University Medical Center, NC, USA Summary: a trend of higher relapse rates in breast cancer patients receiving tumor cell positive PBPC collections has been High-dose chemotherapy (HDC) with hematopoietic reported.7 support appears promising in the treatment of breast High-dose chemotherapy with hematopoietic rescue cancer, although reinfusion of contaminating tumor appears promising in the treatment of breast cancer,10,11 but cells may contribute to disease relapse. Ex vivo expan- the presence of contaminating tumor cells in the reinfused sion may reduce tumor cell content through use of a hematopoietic cell product remains a concern. Retrovirally small inoculum volume and by passive purging during mediated gene marking techniques and retrospective clini- culture. We assessed the ex vivo expansion potential of cal trials have demonstrated in patients with acute myelo- tumor cell positive bone marrow (BM) from breast can- genous leukemia, neuroblastoma, B cell lymphoma, chronic cer patients and the effect of ex vivo expansion on tumor lymphocytic leukemia, or chronic myelogenous leukemia cell content. Cryopreserved/thawed mononuclear cell that reinfusion of contaminating tumor cells present in the (C/T MNC) BM harvests with known tumor cell con- BM harvest can contribute to disease relapse in the BM tamination (n = 7) were assessed for tumor cells pre- itself or in extramedullary sites.12–16 The contribution of and post-expansion using immunocytochemical (ICC) reinfused tumor cells to the relapse of breast cancer is cur- staining. Pre-expansion inoculum samples contained a rently unclear. Studies monitoring the reinfusion of tumor × 6 range of 6–2128 tumor cells per 5.0 10 nucleated cells. positive hematopoietic cell products in breast cancer Ex vivo expansion resulted in fold expansions of 6.67 patients were unable to detect a correlation between tumor and 11.37 for total cells and CFU-GM, respectively. cell contamination and site of relapse.6,8 Tumor cells were undetectable in four of the seven post- In vitro clonogenic assays suggest, however, that tumor expansion samples and were reduced in the remaining cells remaining in BM harvests or PBPC collections can three samples. The data demonstrate passive purging of be viable and clonogenic12,13,17,18 potentially contributing to breast cancer cells during ex vivo expansion, with hema- disease relapse. The homing of reinfused tumor cells to topoietic progenitor cell expansion comparable to that sites of previous disease may prevent the distinction of normal BM. Reduction in tumor cell number con- between relapse due to residual tumor cells or reinfused tained in the small volume culture inoculum combined tumor cells. Altered adhesion receptor expression or func- with passive purging during the ex vivo expansion pro- tion, similar to that previously demonstrated in breast can- cess suggest a potential 2–4+ log reduction in tumor cell cer tumor cells,19 may contribute to the homing of reinfused content in the reinfused cell product. tumor cells from the circulation to a microenvironment cap- Keywords: bone marrow transplantation; ex vivo expan- able of supporting tumor cell growth.20,21 Many HDC trials sion; tumor cell contamination are currently conducted without some form of tumor cell purging. Removal of tumor cells from the reinfused hematopoietic stem cell product may result in improved disease- Immunologic techniques have revealed tumor cell infil- free and overall patient survival. tration of the BM in as many as 48% of early stage breast Previous ex vivo expansion studies using CD34+ PBPC cancer patients at the time of diagnosis.1–4 The presence of derived from breast cancer patients have reported median tumor cells in the BM has been shown to be predictive of progenitor expansions of 10- to 46-fold,22 with a relative relapse in early stage5–7 and advanced stage8,9 breast cancer disadvantage of tumor cell growth compared to hematopo- patients. In addition, the number of tumor cells identified ietic cell growth in cultures seeded with tumor cell lines.23 in the BM has been correlated with shorter disease-free and The ex vivo expansion potential of primary tumor cells and overall survival.6 Although the prognostic significance of their impact on hematopoietic progenitor cell expansion in tumor cells in PBPC collections requires further evaluation, a stromal-based culture system has not been evaluated. The intent of this study was (1) to evaluate the ex vivo expansion potential of BM samples obtained from stage IV breast Correspondence: Dr BI Lundell, Aastrom Biosciences, Inc., 24 Frank cancer patients which were known to be tumor cell positive Lloyd Wright Drive, Domino’s Farms, Lobby L, Ann Arbor, MI 48105, USA using a small scale culture system, and (2) to determine the Received 21 January 1998; accepted 18 March 1998 effect of ex vivo expansion on tumor cell content. Passive tumor cell purging during ex vivo expansion BI Lundell et al 154 Materials and methods Two BM aspirates from each normal donor were pooled and the nucleated cell concentration was determined. MNC Bone marrow samples were separated by density gradient centrifugation (Ficoll– Paque Plus; Pharmacia Biotech, Uppsala, Sweden; The Duke University Bone Marrow Transplant Program s.g.1.077) at 300 g for 20 min at 25°C. Fresh BM MNC has performed a series of clinical protocols for patients util- were washed twice with LTBMC medium prior to culture. izing a variety of purging techniques. Seven back-up BM samples collected from stage IV breast cancer patients having a median age of 46 years (range 34–51 years) were Small scale cell culture expansion system available for ex vivo expansion. All seven patients exhibited Fresh and C/T MNC expansion cultures were initiated in tumor cell infiltration of the BM by routine histologic 24-well tissue culture plates (Costar, Cambridge, MA, examination of BM biopsies. The BM samples used USA) at a viable nucleated cell density of 0.5 × 106 cells for these studies were obtained using standard BM per well in a final volume of 0.6 ml. Ex vivo expansion harvesting techniques. cultures were performed using LTBMC medium sup- For a controlled comparison, small volume BM aspirates plemented with recombinant human (rh) erythropoietin = (n 7) were collected after obtaining informed consent (Epoetin Alfa; Amgen, Thousand Oaks, CA, USA) at 0.1 from normal volunteers having a median age of 37 years U/ml, rh PIXY321 (Immunex Corporation, Seattle, WA, (range 21–45 years). Briefly, two aspirates were collected USA) at 5 ng/ml, and rh flt-3 ligand at 25 ng/ml (Immunex from adjacent sites on the posterior iliac crest. For each Corporation). Ex vivo expansion cultures were incubated at aspiration, 3 ml of BM was collected from each of four 37°C in a humidified atmosphere with 5% CO and fed ° 2 quadrants, rotating the BM needle bevel 90 between quad- with a 50% medium exchange on days 4, 7, 9, 10 and 11. rants, for a total volume of 12 ml. Cells were anticoagulated To prevent cell loss during feeding, medium removed from with 20 U/ml preservative-free heparin (Apothecon, Prince- each well was centrifuged at 300 g for 5 min. The resulting ton, NJ, USA) and processed within 5 h of collection. cell pellet was resuspended in fresh expansion medium and returned to culture. Expansion cultures were harvested on Sample processing day 12 using trypsin/EDTA (Gibco BRL) and Dulbecco’s PBS (Gibco BRL). The expanded cell product was enumer- An unpurged aliquot of patient BM MNC was control rate ated on a cell counter as described above. cryopreserved and stored for a median period of 33 months (range 12–42 months). Cryopreserved patient samples were Hematopoietic cell assays shipped on dry ice to Aastrom Biosciences, where they were stored in the vapor phase of liquid nitrogen for up CFU-GM: Cells were inoculated in methylcellulose (final to 1 month prior to thawing for initial tumor cell content concentration 1.0%; Sigma) prepared with IMDM sup- assessment and ex vivo expansion. Cryopreserved samples plemented with 2 mm glutamine (Gibco BRL), 30% FBS, were thawed rapidly in a 37°C circulating waterbath, 1.0% BSA, 1% monothioglycerol (Sigma), 0.1% penicillin- diluted with approximately two volumes of long-term bone streptomycin (Gibco BRL), 5 ng/ml rh PIXY321, 5 ng/ml marrow culture medium ((LTBMC; Iscove’s modified Dul- rh G-CSF (Amgen) and 10 U/ml rh erythropoietin. Tripli- becco’s medium (IMDM) with 4 mml-glutamine (Gibco cate 1 ml aliquots containing 1.0–2.0 × 104 cells per ml BRL, Grand Island, NY, USA), 10% fetal bovine serum (fresh and C/T MNC expansion culture inoculums) or 3.0– (FBS; BioWhittaker, Walkersville, MD, USA), 10% horse 6.0 × 103 cells per ml (expanded cell products) were plated serum (Gibco BRL), 20 ␮g/ml vancomycin (Vancocin HCl, in gridded 35-mm dishes (Nunc, Naperville, IL, USA) and Lilly, Indianapolis, IN, USA), 5 ␮g/ml gentamicin incubated at 37°C in a humidified atmosphere with 5% ␮ (Fujisawa USA, Deerfield, IL, USA), and 5 m hydrocorti- CO2. At day 14 of culture, CFU-GM (colony forming units sone (Solu-Cortef, Upjohn, Kalamazoo, MI, USA) plus 200 granulocyte/macrophage) were enumerated as previously ␮g/ml DNAse (Sigma, St Louis, MO, USA) and incubated described.24 at room temperature for 20 min. C/T MNC were pelleted by centrifugation at 300 g for 7 min and resuspended in LTC-IC: To determine the frequency of long-term culture- fresh LTBMC medium.

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