Characterizing Breast Cancer Cells in Blood and Bone Marrow

Institution: Stanford University
Investigator(s): Robert Carlson, M.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10IB-0073 Award: $155,781
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2004)
Small numbers of cancer cells are known to circulate in the blood of many women with newly diagnosed or metastatic breast cancer. In addition, cancer cells can also be found in the blood forming parts of bone in women with newly diagnosed breast cancer. Some studies have found that the presence of these cancer cells increases a woman's risk for recurrent disease, while others have not. The individual breast cancer cells that make up a breast cancer are heterogeneous. However, since only a small proportion of cancer cells are responsible for either recurrence of metastasis, there is a need to sample and analyze breast cancer cells from patients at the individual level. We intend to develop a method that will allow us to tell which patients are most likely to develop a recurrence based upon the characteristics of the cancer cells in the blood or bone.

In our CBCRP-funded project we will acquire samples of blood and bone marrow (during anesthesia for breast surgery) from women with breast cancer. Cancer cells will be isolated and the presence of "biomarkers" analyzed by a special diagnostic instrument called, a high dimensional fluorescence-activated cell sorter (HiD FACS). Using this approach we will be able study the patterns of up to a dozen biomarker characteristics of individual cancer cells from the blood or bone marrow. Among the biomarkers we plan to utilize are Her-2/neu, EGFR, CD44/CD24, osteopontin, COX-2, integrins, chemokine receptors, and others.

Earlier studies of breast cancer cells circulating in the blood or found as isolated cells in the bone marrow have employed only a limited number of biomarkers. This study will simultaneously evaluate up to 12 characteristics of each individual cancer cell. In addition, we will be in a position to compare the "profiles" of blood-borne breast cancer cells with those already having spread to the bone. Then, we will be in a position to assess whether our approach has "predictive power" for recurrent disease. Representatives from the Community Breast Health Project, a community-based educational and advocacy group will be involved in the oversight of the research as full participants in our research meetings.

Final Report (2005)
Many patients with breast cancer are known to have small numbers of cancer cells in their blood and in their bone, called circulating tumor cells (CTCs). Whether CTCs are express biomarkers to predict metastasis to bone and predict disease outcome is controversial. The relationship between CTCs and bone marrow metastases is poorly understood. This project aimed to isolate circulating tumor cells from the blood and isolated tumor cells from bone marrow to study the biological characteristics of the individual cells.

We have identified selected biological characteristics (i.e., cell biomarkers) of interest, including: estrogen receptor, human epidermal growth factor receptor 2 (HER2/neu), epidermal growth factor receptor (EGFR), CD24 or CD44 (potential markers for breast cancer stem cells), or osteopontin (a protein important in establishing cancer spread to the bone). To provide “proof-of-principle” for our method we performed control studies where we “spiked” blood samples collected from normal, disease-free women with a variety of breast cancer tumor cell lines having an established presence of the biomarkers listed above. We studied two commercially available methods of isolating tumor cells, (1) the Milenyi Biotech column-based approach, and (2) the Immunocon CellTracks® AutoPrep System. Both methods use an immunomagnetic protocol where antibodies directed against epithelial antigens are conjugated to magnetic “beads.” Then, use of magnets can achieve a separation of tumor cells from other blood components. However, we have found that both methods either fail to collect enough of the cancer cells or they damage the cancer cells, such that either subsequent biomarker staining (revealed by FACS analysis) is poor or DNA is lost to prevent additional genetic analysis.

Our future efforts will be aimed at identifying methods of isolating CTCs from blood that captures the vast majority of tumor cells and that produces minimal damage, so that their biological characteristics may be accurately studied. Once that is accomplished, the method will be modified to allow the isolation of breast cancer cells from the bone marrow of patients with breast cancer.

Our eventual goal is to provide a better method of predicting the likelihood of breast cancer spread and of identifying whether the breast cancer will recur.