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    Awarded Grants
    Identification and Validation of Biomarkers for Breast Cancer in Nipple Aspiration Fluid

    Scientific Abstract:
    Background: Nipple aspiration fluid (NAF) potentially offers a superior source of biomarkers for breast cancer since the proteins present are specifically from breast tissue. In a pilot study which consisted of 5 cases of primary invasive breast cancer and 5 normal controls, we have established a comprehensive proteomic approach that is applicable for biomarker discovery and validation in NAF. Preliminary findings and illustration of the approach are as follows: 1) Proteomic profiling using protein chip arrays and mass spectrometry. This technology is highly sensitive, we were able to obtain reproducible protein profiles using 1µg of NAF protein. 2) Biomarker discovery using a combination of bioinformatics tools. We have used unsupervised cluster analysis to recognize patient subgroups and supervised analysis for biomarker discovery. Two subgroups were observed in this cohort, and a panel of three proteins were needed to correctly identify all cancer cases. 3) Protein identification and 4) Independent validation using quantitative immunoassay. One marker has been identified as neutrophil alpha-defensin (HNP). HNP is peptide antibiotics made principally by neutrophils even though some tumors might also produce HNP with the same capabilities. Recent studies have implicated diverse functional activities of HNP, including on tumor cell proliferation in renal cell carcinoma. Objective / Hypothesis: The objective of this study is to further evaluate and validate our preliminary findings in a large patient cohort. Specific Aims and Study Design: We will obtain NAF samples from a currently on going multi-center clinical trial. Approximately 200 samples from both breasts of five categories (unaffected, benign, atypical, DCIS, and invasive) will be available. 1) We will use immunoassay to evaluate the prevalence of elevated HNP in breast cancer, and its specificity. 2) We will validate and optimize the current panel of biomarkers on protein chip arrays; biomarkers may be added (if new patient subgroups appear) or removed if not specific to cancer. 3) We will separate and purify each protein that constitutes the optimized biomarker panel. 4) We will develop immunoassays that can be performed easily in a clinical laboratory. Potential outcomes and Benefits of the Research: We anticipate identifying a protein pattern in NAF that can capture breast cancer with high sensitivity and specificity. The result of this study will potentially lead to the development of a non-invasive and facile test for breast cancer early detection, and the discovery of new therapeutic targets.

    Lay Abstract:
    Breast cancer is the most commonly diagnosed cancer among women and is highly heterogeneous. Single tumor markers that are clinically applicable for breast cancer early detection do not exist at the present time, due to inadequate sensitivity and specificity. Rather than targeting a specific abnormality that may only be present in a small subgroup of patients, researchers look for a common “patterns of changes” by globally compare protein expressions in biological samples of the disease and non-disease group, an approach called proteomics. Compared to serum, nipple aspiration fluids offer a superior source of biomarkers since the proteins present are specifically from breast tissue. In a pilot study which consisted of 5 cases of primary invasive breast cancer and 5 normal controls, we have established a comprehensive proteomic approach that is applicable for biomarker discovery and validation in breast fluid. We were able to obtain reproducible protein profiles using just 1µg of protein (The sensitivity of the detection technology is critical since breast fluid has limited protein yield). Using a combination of bioinformatic tools, we have recognized two patient subgroups and identified a panel of three proteins needed to correctly classify all cancer cases. One of the marker has been identified as alpha-defensin (HNP). HNP is peptide antibiotics made principally by human neutrophils even though some tumors might also produce HNP with the same capabilities. Recent studies have implicated diverse functional activities of HNP, including on tumor cell proliferation in renal cell carcinoma. Based on this pilot study, we propose to evaluate and validate our preliminary findings in a large patient cohort. Approximately 200 samples from both breasts of five categories of women (unaffected, benign, atypical, DCIS and invasive) will be available. First, we will use immunoassay to evaluate the prevalence of elevated HNP in breast cancer, and its specificity. Second, we will validate and optimize the current panel of biomarkers by protein profiling. Finally, we will separate and purify each protein that constitutes the optimized biomarker panel and develop immunoassays that can be performed easily in a clinical laboratory. We anticipate identifying a protein pattern in breast fluid that can capture breast cancer with high sensitivity and specificity. The result of this study will potentially lead to the development of a non-invasive and facile test for breast cancer detection, and the discovery of new therapeutic targets.