Methylation markers for early breast cancer detection: -

Women known with a familial predisposition or a personal history of breast cancer bear an up to 85% lifetime risk of developing breast cancer. Despite regular screening, up to 50% of these women develop “interval malignancies” that are discovered in between screening visits. Therefore, novel ways of screening that could improve the accuracy of imaging-based screening methods are needed. This thesis describes the studies that we have performed in order to establish the potential role of methylation alterations in nipple fluid as a way of early breast cancer detection in women at increased risk. DNA methylation describes the addition of a methyl group to the cytosine base of the DNA. In tumorigenesis, hypermethylation of gene promoters is associated with silencing of transcription and in this way contributes to cancer initiation and progression. Methylation alterations frequently occur in early stages of tumor development and may therefore serve as valuable biomarkers for breast carcinogenesis. We showed that nipple aspiration is feasible in the majority of healthy volunteers and high-risk women. Previously reported success rates were only 27-66%, which prohibited the clinical application of this method. Using oxytocin nasal spray, we were the first to show success rates of 90% in high-risk women, including women that previously underwent chemotherapy, radiotherapy or surgery of the breast. We demonstrated that, using Quantitative Multiplex Methylation-Specific PCR (QM-MSP), a quantitative sodium-bisulfite (NaBi) based methylation assay, we were able to analyze methylation of multiple genes in >99% of nipple aspirates. In a side-by-side comparison, QM-MSP proved to be much more sensitive than other commonly used methylation assays and enabled reproducible methylation analysis in