Ex vivo expansion of CD34+ peripheral blood progenitor cells: implications for the expansion of contaminating epithelial tumor cells

Cytokine-supported ex vivo expansion of peripheral blood progenitor cells (PBPCs) offers new perspectives for autografting after high-dose chemotherapy. One of the potential advantages is the possibility to reduce the volume of blood processed from the patient, thus allowing reduction of the overall tumor cell number in the final autograft. However, ex vivo expansion will only be advantageous if contaminating tumor cells are not expanded concomitantly. This question has not previously been addressed. Therefore, we analyzed unseparated PBPC preparations, CD34(+)-selected cell fractions, and ex vivo-expanded cell preparations from stage IV (n = 16) and high-risk stage II/III (n = 8) breast cancer patients for the presence of human epithelial antigen-(HEA) or cytokeratin (CK)-positive tumor cells. We found that three of 16 (18.8%) of the unseparated PBPC products from stage IV patients were HEA- and/or CK-positive, whereas none of the stage II/III patients were found to be positive after two cycles of induction chemotherapy with etoposide (VP16), ifosfamide, cisplatin, and epirubicin (VIP-E). After CD34+ cell selection (Ceprate SC; CellPro, Bothell, WA) and stem-cell factor (SCF), interleukin (IL)-1, IL-3, IL-6, and erythropoietin (EPO)-mediated ex vivo expansion of the CD34+ cells for 14 to 21 days, no tumor cells could be detected in these primary breast cancer patients at a sensitivity of 1 tumor cell per 4 x 10(5) nucleated cells. Thus, to answer the question of whether tumor cells are expanded concomitantly on ex vivo expansion of normal CD34+ cells, we cocultured defined numbers of primary renal carcinoma cells (RS-85), xenograft-derived breast cancer cells, and small-cell lung cancer cells with CD34+ cells selected from normal donors or cancer patients, either in serum or serum-free culture media. We found that none of the three epithelial tumor cell types increased significantly in number during a 14-day coculture period when compared with normal CD34+ cells alone or tumor cells alone, which increased 110- +/- 77-fold and 45- +/- 26-fold, respectively. However, during coculture, the tumor cells did not undergo cell death and were able to regrow when maintained in serum for longer time periods. We conclude that cytokine-supported expansion cultures of positively selected CD34+ PBPCs from primary high-risk stage II/III or stage IV breast cancer patients do not contain detectable tumor cells, which suggests that there is no increased risk of concomitantly expanding tumor cells. Moreover, cocultures of exogenously mixed tumor cell lines with normal CD34+ cells showed a relative disadvantage of tumor cell growth compared with the growth of hematopoietic cells, again without an apparent risk of concomitantly expanding tumor cells. However, considering the pronounced heterogeneity of tumor cell kinetics, ex vivo-expanded PBPC from cancer patients should be monitored for minimal residual disease.