Immunomagnetic enrichment and flow cytometric isolation of breast cancer circulating tumor cells (CTCs) for genomic characterization

We developed a novel approach to isolate tumor cells with high purity from blood via immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) and examined copy number alterations in these cells. Magnetic beads coated with EpCAM mAb were added to blood to enrich for tumor cells. Enriched samples were then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. DNA from isolated tumor cells was subjected to whole genome amplification (WGA) and copy number analysis via array comparative genomic hybridization (CGH). The assay was evaluated using BT474 and MCF7 breast cancer cell lines and in CTCs from 5 metastatic breast cancer (MBC) patients with matched archival primary tumors and later extended to an additional 97 MBC patients. Evaluation of the assay on isolated breast cancer cell lines spiked into blood correctly identified the known genomic alterations with high reproducibility. In clinical studies, comparison of CTCs with matched archival primary tumors confirmed shared lineage with notable divergence. In addition, serial testing of CTCs confirmed reproducibility, and indicated genomic change over time. Analysis of genomic profiles of CTCs from 102 MBC patients revealed common copy number alterations including gains in 1q and 8q and losses in 8p and 11q. Comparison with a published CGH dataset of primary breast tumors revealed similar frequencies of recurrent genomic copy number aberrations.