Thirty percent of all women newly diagnosed with breast cancer and no clinical evidence of metastatic disease will have disseminated tumor cells (DTC) detectable in their bone marrow at the time of diagnosis. The presence of DTC is associated with an increased risk of metastatic disease development as well as cancer death. Chemotherapy can eliminate DTC in some breast cancer patients. However, those women with persistent DTC after chemotherapy are five times more likely to die from their breast cancer. This data suggests that chemotherapy-resistant DTC are likely to be precursor cells capable of forming metastatic foci. Biologically, DTC are a heterogeneous population of cells and clinical studies suggest that only a subset have the ability to form metastases. Identification of specific DTC that correlate with poor clinical outcome would result in a new prognostic marker and identify women at high risk for developing metastatic disease and breast cancer death. We propose that persistent disseminated tumor cells present after chemotherapy represent a unique subpopulation of all DTC, are predictors of a poor response to chemotherapy, and correlate with poor clinical outcome. We hypothesize that chemotherapy-resistant DTC can be identified by their expression of a unique constellation of tumor marker proteins which may be similar to those expressed by breast cancer stem cells. In this proposal, our specific aims are: 1) characterize tumor markers expressed by DTC which are present after chemotherapy, 2) compare the expression of these markers to that on DTC detected prior to chemotherapy, 3) correlate expression of the defined tumor markers on DTC with clinical outcome of breast cancer patients to identify those markers that are predictive of disease recurrence, 4) Utilize biomarkers identified in Specific Aims 1 and 2 to isolate purified DTC for further molecular analysis. ? ? Specimens to be analyzed are from the Siteman Cancer Center Tissue Procurement Center where processed and cryopreserved bone marrow cells and cytological slides have been prepared from women with locally advanced breast cancer before and after treatment with neoadjuvant chemotherapy. Tumor marker expression by DTC will be determined by double immunocytochemistry using anti-cytokeratin antibodies and a panel of antibodies directed against cell surface proteins known to be associated with metastatic potential, chemotherapy resistance, and breast cancer stem cell markers. Information on biomarker expression from Specific Aims 1 and 2 will be utilized for immunomagnetic isolation of DTC and further molecular analysis of these cells. We have successfully used both double immunostaining and immunomagnetic separation to detect breast cancer cells in model systems of bone marrow containing DTC. ? ? The results of the proposed experiments will identify a unique constellation of tumor makers expressed by DTC persistent after chemotherapy and associated with the metastatic potential of the cells. Identification of biologically important DTC will allow us to stratify women newly diagnosed with breast cancer into high and low risk groups for the development of metastatic disease. Furthermore, defining tumor markers expressed by DTC and performing molecular analysis of these cells will lead to new insights into the biology of the disseminated tumor cells and to the development of new targeted therapies. ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Cancer Biomarkers Study Section (CBSS)
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Lively, Tracy (LUGO)
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Washington University
Schools of Medicine
Saint Louis
United States
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