Detection of breast cancer in nipple aspirate fluid by CpG island hypermethylation

Purpose: New approaches to the early detection of breast cancer are urgently needed as there is more benefit to be realized from screening. Nipple aspiration is a noninvasive technique that yields fluid known to contain breast epithelial cells. Silencing of tumor suppressor genes such as p(16INk4a), BRCA1, and hMLH1 have established hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection. Experimental Design: Using sensitive methylation-specific PCR, we searched for aberrant promoter hypermethylation in a panel of six normally unmethylated genes: glutathione S-transferase pi 1 (GSTP1); retinoic acid receptor-beta2 (RARbeta2); p16(INK4a); p14(ARF); RAS association domain family protein 1A (RASSF1A); and death-associated protein kinase (DAP-kinase) in 22 matched specimens of tumor, normal tissue, and nipple aspirate fluid collected from breast cancer patients. Results: Hypermethylation of one or more genes was found in all 22 tumor DNAs (100% diagnostic coverage) and identical gene hypermethylation detected in 18 of 22 (82%) matched aspirate fluid DNAs. In contrast, hypermethylation was absent in benign and normal breast tissue and nipple aspirate DNA from healthy women. Conclusions: Promoter hypermethylation of important cancer genes is common in breast cancer and could be detected in matched aspirate DNAs from patients with ductal carcinoma in situ or stage I cancer. Promoter hypermethylation represents a promising marker, and larger studies may lead to its useful application in breast cancer diagnosis and management.