Evaluation of a Device for Negative Selection of CD34+ Cells Alone or in Combination with Breast Cancer Tumor Cell Purging

Abstract

Background: Positive selection of CD34+ cells may be associated with an alteration of the target cells by antigen-antibody interactions and/or a selection of distinct CD34-cell subgroups within the purified cell fraction. Thus, a negative CD34 cell selection procedure (StemSep^™), carried out on leukapheresis samples (LA) of patients with breast cancer (n = 6) or other malignancies (n = 4) was evaluated. Material and Methods: 3 samples per patient (each 1 ml) containing 1 ×10⁸ leukocytes (median 3.3% (range 0.9–9.4%) CD34+ cells), were processed as follows: set A CD34+ cell selection alone; set B CD34+ cell selection with concomitant breast cancer cell purging; or set C CD34+ cell selection followed by breast cancer cell purging. The samples of sets B and C were spiked with 1% MCF-7 breast cancer cells. Furthermore, the antibodies included in the breast cancer purging cocktail were evaluated regarding sensitivity and specificity by means of flow cytometry and immunocytochemistry. Results: In the first 2 procedures (sets A/B) the median CD34 cell yield was 49% (range 24–90%), resulting in a CD34 cell purity of 72% (range 32–95%) and in a 3 log reduction of the other cells. The sequential procedure (set C) caused significantly (p < 0.005) higher CD34+ cell losses (16% yield, 81% purity) with a nonsignificantly higher depletion of the other cell types. The selection caused no changes in CD34+ cell clonogenicity for CFU-GM/BFU-E and no loss of viability (>90%). Both simultaneous and sequential selection achieved a tumor cell reduction of more than 3 orders of magnitude (>3 log depletion) whereby APAAP and quantitative RT-PCR for CK19 showed corresponding results in 80% of all samples. Only 2 (4A6 and 5E11) of the 5 tested epithelial breast cancer antibodies showed a binding to MCF-7 and BT-474 cells comparable to the anti-cytokeratin antibody Cam5.2. Conclusions: The negative CD34 cell selection approach investigated in combination with simultaneous specific tumor cell depletion is comparable to other small-scale devices while the sequential procedure had unacceptable CD34 cell losses.