Identifying Circulating Breast Cancer Cells in Women With Metastatic Breast Cancer

OBJECTIVES:

Primary

To compare identification of circulating breast cancer cells (CBCCs) in blood or pleural or peritoneal fluid by a novel technique using stem cell marker retinaldehyde dehydrogenase (ALDH) and surface antigen expression (CD44+, CD24-) to the standard technique using the CellSearch® system in women with metastatic breast cancer.

Secondary

To determine whether CBCCs have the potential to grow into metastatic lesions.

OUTLINE: Patients undergo sample collection to help develop a new technique using stem cell marker retinaldehyde dehydrogenase (ALDH) and surface antigen expression (CD44+, CD24-) in isolating circulating breast cancer cells (CBCCs) from blood and pleural or peritoneal fluid. Blood may also be drawn to measure the number of circulating tumor cells using the standard CellSearch® system.

Mononuclear cells are isolated by density centrifugation. Cells are stained against surface antigens that provide specific expression patterns for CBCCs (CD44, CD24). Cells are analyzed on a fluorescence activated cell sorting (FACS) Calibur flow cytometer and sequentially gated (ALDHhigh→CD44+ vs CD24-/low or CD44+ vs CD24-/low→ALDHhigh) for detection of CBCCs. For further confirmation of epithelial origin, ALDHhighCD44+CD24-/low cells are isolated using a FACSAria flow sorter, cytocentrifuged onto glass slides then stained for the expression of epithelial-specific cytokeratins 5, 8, 14, 18 and 19 by standard immunohistochemical techniques. Using the phenotype that is found to most highly enrich for epithelial cells, cells are isolated by FACS and assayed for clonogenic growth.