Breast cancer screening by methylation analysis of tumor suppressor genes in breast fluid

Breast cancer is the major cause of cancer death in the Western world. Despite the fact that women undergo imaging-based screening at regular intervals, many cancers are still discovered in between screening visits. This indicates an urgent need for novel screening and risk assessment modalities that could be of additive value to imaging-based screening. Breast cancer is initiated and progresses by genetic and epigenetic events resulting in aberrant gene function. Epigenetic alterations occur through mechanisms other than changes in the primary nucleotide sequence of a gene. One of the most important epigenetic mechanisms is DNA methylation. The term DNA methylation describes the addition of a methyl group to a cytosine base in the DNA. In the normal situation DNA methylation is involved in the regulation of many cellular maintenance processes. However, during tumorigenesis, hypermethylation of promoters of tumor suppressor genes is associated with silencing of transcription. In this way methylation contributes to cancer initiation and progression. Methylation alterations frequently occur in early stages of tumor development and can also be detected in non-cancerous cells adjacent to the tumor, which indicates a methylation "field defect". Moreover, epigenetic changes are reversible and therefore potential therapeutic targets. Analyzing DNA promoter methylation of tumor suppressor genes in nipple fluid could be a new screening modality. Nipple fluid is normally produced in small amounts in the ducts of non-lactating women and can be obtained by non-invasive vacuum aspiration. This fluid contains intact ductal epithelial cells together with free DNA derived from such cells, which can be analyzed for methylation aberrations. Using oxytocin nasal spray, nipple fluid can be aspirated from more than 90% of women, including high-risk women that previously underwent local or systemic treatment. We and others previously showed that methylation of a panel of tumor suppressor genes such as CCND2, SCGB3A1, APC, RASSF1 and RARB is associated with the presence of breast cancer. Using Quantitative Multiplex Methylation-Specific PCR, methylation alterations can reliably be assessed in the limited amounts of DNA present in nipple fluid. In conclusion, analyzing patterns of promoter methylation of tumor suppressor genes in nipple fluid can potentially serve as a valuable breast cancer screening method. A large prospective study is currently ongoing in our institute to determine the added value of this modality to existing screening methods.