Trial Information

A Feasibility Study of a Novel Technique to Identify Circulating Breast Cancer Cells in Patients 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.