Abstract
Abstract Aims: For early detection of breast cancer the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory and thus a growing field of research. The most common alterations in human cancers including breast cancer are changes in the status of DNA methylation, which are therefore quickly emerging as a new pool of potential biomarkers. Thus, we investigated the feasibility of detecting aberrant tumor suppressor gene methylation in cancer cell-derived free circulating DNA in the bloodstream of breast cancer patients. Methods: Using qualitative MSP, we examined the methylation status of seven biologically significant putative tumor suppressor genes, i.e. ITIH5, DKK3, WIF1, RASSF1A, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum. Free circulating DNA was isolated with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Clinical performance was determined in a training study on 240 serum samples (120 breast cancers, 120 age-matched healthy controls). 20 benign gynaecological disease and 30 colon cancer serum samples were included for additional specificity testing. Results: Based on the training study we could evaluate the top candidate biomarkers with the best values for sensitivity and specificity. A marker panel with DKK3 and ITIH5 detected breast cancer with a sensitivity of 42% (50/120). Specificity of the panel was sufficient with 83%, 100% and 93% in colon cancer samples, benign and healthy control samples, respectively. Control samples revealed unacceptable high methylation rates of SFRP1 and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2 and WIF1 showed a considerable methylation frequency in sera from healthy controls. The well-established potential biomarker RASSF1A was included as a reference gene. RASSF1A was highly specific in the analysed benign and healthy control samples (100%) why we integrated RASSF1A in our independent test study to improve sensitivity and specificity of the marker panel with DKK3 and ITIH5. Conclusions: The current study suggests that cancer-specific methylation of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be valuable biomarkers for early detection of breast cancer. In the second phase of this project we are currently validating with quantitative MSP ITIH5, DKK3 and RASSF1A as reliable methylation biomarkers in an independent test set consisting of 140 breast cancer serum samples, 140 age-matched healthy controls, 40 benign gynaecological disease and 30 colon cancer serum samples for additional specificity testing. To date, this is the first study investigating the potential of ITIH5 and DKK3 in combination with the well-established biomarker RASSF1A as reliable blood-based methylation biomarkers in a large-scale serum collective of breast cancer and matched control samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-382. doi:1538-7445.AM2012-LB-382
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