Primary prevention of Breast Cancer by Monitoring Gene Promoter Methylation in Nipple Aspirates

Collaboration: departments of Pathology (Prof. dr. PJ van Diest, drs. K Suijkerbuijk), Oncology (prof. dr. E van der Wall), and Clinical Genetics (dr. M Ausems, dr. R van der Luijt)

Focus: oncology

Previous funding: KWF Resident Research Year Award for Karijn Suijkerbuijk, Pink Ribbon/American Women’s Club, IKMN start up grant, Ate Visser Foundation

Background
Breast cancer is the leading cause of cancer death in women in the Western world. In the Netherlands, the incidence is about 12,000 per year, which means that eventually every ninth woman will get breast cancer. Several hormonal and lifestyle factors have been identified as risk factors for the development of breast cancer, but the most well-established risk factor is the presence of a germline mutation in the BRCA1 or BRCA2 genes, which indicates a life time risk of 45-80% to get breast cancer 1-3. Regular screening by clinical breast examination, mammography and/or Magnetic Resonance Imaging (MRI) is offered to these high-risk women, but one out of four breast tumors are missed by these screening modalities 4. The most effective form of primary prevention for high-risk women is bilateral mastectomy 5, which gives a considerable breast cancer risk reduction 6. As this procedure is highly mutilating, many women opt out and those who decide to undergo prophylactic surgery prefer to postpone it as much as possible, with the subsequent risk of developing invasive breast cancer in the mean time. In contrast, the procedure has to be seen as over treatment in the 15%-55% of BRCA carriers that would never have developed breast cancer, since up to now no procedures are available that accurately predict who of these high-risk women will and who will not develop breast cancer.

A second group of women with a similar high-risk of developing invasive breast cancer comprises women with ductal carcinoma in situ (DCIS) of the breast. They have a relative increased risk of 10-11x to develop invasive breast cancer. Today, local treatment is as intensive as in invasive breast cancer whereas no method can accurately predict if a woman with DCIS will indeed develop invasive cancer.

A new primary prevention modality for these high-risk women could very well be found in the analysis of nipple fluid 7. Nipple fluid, that contains breast epithelial cells, free DNA and proteins secreted by them, is produced in small amounts in the breast ducts of non-lactating women and can be collected in a non-invasive way by vacuum-aspiration.

Aim
This project aims to set up a program to monitor development of epigenetic changes in nipple aspirates to optimally time preventive breast surgery in high-risk women. Epigenetic changes are non- gene specific changes to the DNA, e.g. methylation, that lately have been recognized as important contributors in carcinogenesis.We previously showed that methylation in breast tissue is a good predictor of sporadic as well as hereditary breast malignancy. Furthermore, we proved that intranasal oxytocin enables harvesting nipple fluid in almost all women and allows to assess methylation patterns in the DNA isolated from this fluid.

Approach
This project aims to implement genetic monitoring of nipple aspirates as a primary prevention tool for breast cancer. On short notice, we will do more validation studies by preoperatively taking aspirate nipple fluid from:

1) All women just having been diagnosed with DCIS or invasive breast cancer by core biopsy in our hospital. Comparing methylation patterns in the nipple fluid to the methylation in the excised tumor will allow us to find out how well methylation aberrations in nipple fluid correspond with methylation aberrations in a breast tumor present in the same breast.

2) High-risk women undergoing therapeutic or preventive surgery. High-risk women are defined as having a life time breast cancer risk over 20%. This population includes carriers of a BRCA1 or BRCA mutation, women without a known BRCA mutation that have a pedigree-based breast cancer risk > 20% and women that have a history of DCIS. Comparing methylation patterns in nipple fluid with various stages of carcinogenetic changes (hyperplasia, DCIS, invasive cancer) allows to establish how well aberrant nipple aspiration methylation patterns point to the development of cancer in this target population.

Clinical significance
Positive results of this validation study will permit setting up a prospective trial to establish clinical decision making based on thresholds of specific methylation patterns. Next to methylation analysis, we will also work on detection of oncogene amplification and proteomics. Eventually, we want to establish a specific pattern of epigenetic changes that points to a progression in breast cell behavior from benign to malignant and therefore indicate the time point for prophylactic surgery. On the other hand, should this pattern never occur, prophylactic surgery could be completely avoided. If proven successful, monitoring the development of epigenetic changes in nipple fluid could become a true valuable screening method in high risk women allowing primary prevention of breast cancer.

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