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    Research Grants Awarded

    Molecular Characterization Of Disseminated Tumor Cells

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    Background: The development of specific targeted therapies directed to functional cancer biomarkers has lead to improvements in breast cancer survival. To continue this progress, it is essential to identify new functional biomarkers on specific subpopulations of breast cancer cells which are prognostic and can be therapeutically targeted. An important area for exploration is the molecular characterization of disseminated breast tumor cells (DTCs). These cancer cells reside in bone marrow and appear to be intermediaries in the metastatic process. We have developed a methodology to enrich and study DTCs using immunoselection and gene microarray analysis. Using this strategy, we have identified 67 genes which are highly expressed in bone marrow of breast cancer patients and are likely associated with DTCs. In our initial studies analyzing the association of disease recurrence with expression of these genes in bone marrow from breast cancer patients, we found the expression of one of these genes, TWIST1, in the bone marrow of breast cancer patients, was significantly associated with the development of early recurrent disease. Our overall goals are two-fold: 1. to develop a multi-gene assay which predicts the presence of metastatically competent DTC and 2. To understand the function of these genes in the metastatic process for future therapeutic targeting. Hypothesis: We hypothesize that TWIST1 as well as a subset of genes from the 67 gene signature will be excellent functional biomarkers indicative of the presence of disseminated tumor cells which are associated with early distant relapse and studying the function of these genes will lead to new insights into the metastatic process. Specific Aims: 1. To evaluate TWIST1 expression in a larger cohort of patient bone marrow specimens to establish its role as a biomarker for metastatic, chemopersistent disease. 2. Determine the ability of the remaining genes in the 67 gene signature to predict disease recurrence. 3. To test whether the genes identified in Specific Aim #2 can alter the metastatic behavior of breast cancer cells in models of metastases. Methods: To accomplish these goals we will validate TWIST1 and candidate genes from the 67 gene signature using 700 banked bone specimens collected from 120 patients with clinical stage II/III breast cancer who received identical chemotherapy regimens and have a median 2 year follow-up. Genes which are identified as predictive of recurrent disease will be ectopically expressed or silenced in human breast cancer cell lines. The effect of these gene expression alterations on metastatic disease formation will be determined using mouse models of metastases. To ensure the success of this project, we have assembled an expert team of clinician scientists, molecular biologists, and a statistician representing experience in all areas of the proposed experimentation. Anticipated Results: In preliminary experiments, we have identified 7 genes from the 67 gene signature which appear to be excellent candidates for further evaluation in human samples and in animal models. Validation of these genes will allow clinicians to identify patients at high risk of early recurrent disease development, lead to new targets for intervention, and provide new insights into the metastatic process.

    Lay Abstract:
    Background: Breast cancer is the most frequently diagnosed cancer in women with an estimated 608,380 new cases in 2007. Despite a recent decrease in incidence, breast cancer remains the second leading cause of cancer death in American women. Mortality from breast cancer is a consequence of metastatic foci formed by cells released from the primary tumor. Breast tumors shed a large number of cells into the circulation, however only a very small fraction of these cells may be able to complete all the necessary steps for formation of a metastatic focus. Sub-populations of disseminated breast cancer cells (disseminated tumor cells, DTCs) detected in the bone marrow of breast cancer patients appear to be intermediaries in the metastatic process. Targeting the metastatically competent sub-population of DTCs should result in a decrease in metastatic disease development and improved survival. To study these cells, we have developed a methodology for enriching for and performing gene array analysis of DTCs from breast cancer patients. We have identified 67 genes which are over expressed in bone marrow from breast cancer patients compared to bone marrow from normal volunteers. We have found the expression of one of genes in the 67-gene signature, TWIST1, a known metastases regulator, is over expressed in the bone marrow of patients who have rapid onset of metastatic disease. Hypothesis: We hypothesize that TWIST1 as well as a subset of genes from the 67 gene signature will be excellent functional biomarkers indicative of the presence of disseminated tumor cells which are associated with early distant relapse and studying the function of these genes will lead to new insights into the metastatic process and provide new therapeutic targets. Specific Aims: 1. To evaluate TWIST1 expression in a larger cohort of patient bone marrow specimens to establish its role as a biomarker for metastatic, chemopersistent disease. 2. Determine the ability of the remaining genes in the 67 gene signature to predict disease recurrence. 3. To test whether the genes identified in Specific Aim #2 can alter the metastatic behavior of breast cancer cells in models of metastases. Methods: To accomplish these goals we will validate TWIST1 and candidate genes from the 67 gene signature using 700 banked bone specimens collected from 120 patients with clinical stage II/III breast cancer who received identical chemotherapy regimens and have a median 2 year follow-up. Genes which are identified as predictive of recurrent disease will be turned-off or silenced in human breast cancer cell lines. The effect of the silencing these genes on metastatic disease formation will be determined using mouse models of metastases and in vitro assays. To ensure the success of this project, we have assembled an expert team of clinician scientists, molecular biologists, and a statistician representing experience in all areas of the proposed experimentation. Goals and Anticipated Results: Our overall goals are two-fold: 1. to develop a multi-gene assay which predicts the presence of metastatically competent DTC and 2. To understand the function of these genes in the metastatic process for future therapeutic targeting. In preliminary experiments, we have identified 7 genes from the 67 gene signature which appear to be excellent candidates for further evaluation in human samples and in animal models. Validation of these genes will allow clinicians to identify patients at high risk of early recurrent disease development, lead to new targets for intervention, and provide new insights into the metastatic process.