Simultaneous quantitative assessment of tumor cells in blood and bone marrow in primary breast cancer patients.

Abstract

#302 Background: Both circulating tumor cells (CTCs) in peripheral blood and disseminated tumor cells (DTCs) in bone marrow have been detected in primary breast cancer, but typically not quantitatively, simultaneously and using the same methodology. Here, we simultaneously assessed CTC and DTC levels in 390 primary breast cancer patients (Stages I-III), including treatment-naïve and post-neoadjuvant patients.
 Materials and Methods: CTCs/DTCs were enumerated via a novel two-step assay (IE/FC) consisting of immunomagnetic enrichment (IE) and flow cytometry. First, samples were enriched for epithelial cells using magnetic beads coated with anti-EpCAM monoclonal antibody (mAb). Next, fluorescently labeled mAb specific for an alternate EpCAM epitope was combined with a mAb against CD45 and a nucleic acid stain in multiparameter flow cytometry. Tumor cells were also isolated by IE followed by FACS sorting.
 Results: The assay quantitatively detected tumor cells in either blood or bone marrow obtained at time of breast surgery. Tumor cells were detected with greater frequency and at generally higher levels in bone marrow than in blood. In blood, CTCs ranged from 0 to 33.7 cells/mL (mean = 0.9 ± 2.4 cells/mL) and in bone marrow DTCs ranged from 0 to 501.8 cells/mL (mean = 16.8 ± 38.7 cells/mL). Direct comparisons in each patient revealed complex but significant correlation (Spearman's rank correlation = 0.12, p = 0.02) between CTC and DTC levels. In the subgroup of patients receiving neoadjuvant therapy (N=84), 51/79 (64%) were considered positive for DTC (>5.5 cells/mL, with range 0.3-262.2, mean 34±17.1) while 15/79 (19%) were considered positive for CTC (>0.8 cells/mL, with range 0-38.7, mean 1.4±5.7.) Treatment-naïve patients were more likely to be considered positive in the blood (29% vs 19%, p=0.03.) than neoadjuvant patients, possibly reflecting treatment effect. CTCs or DTCs were isolated from selected patients with high cell burden and subjected to expression or genomic analyses, which confirmed the malignant nature of the cells and indicated the feasibility of molecular profiling of isolated CTCs/DTCs.
 Discussion: This approach provides quantitative detection and isolation of CTCs/DTCs in blood and bone marrow, and may be useful for analysis of early events in cancer progression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 302.