Early detection of breast cancer by organ-specific and circulatory liquid biopsies

Breast cancer is the most common type of cancer in women, representing approximately one-fourth of all cancers diagnosed in women, both worldwide and in the Netherlands. The most recent numbers from the Netherlands show that in 2019, a total of 17,114 women were diagnosed with primary ductal carcinoma in situ (DCIS) or invasive breast cancer. This high incidence of breast cancer led to the implementation of screening programs to detect breast tumors at an early stage. A disadvantage of current programs that rely on imaging is the fact that imaging only detects breast cancer when a tumor can be visualized. In view of the surge of liquid biopsy research (i.e., research focused on biofluids as sources of tumor-derived material shed by (pre)cancerous cells), it is not unlikely that a combination of imaging and measurement of biomarkers could be an approach used in the near future. Liquid biopsies can be collected minimally invasively or non-invasively and allow an easy and regular (even serial; e.g., every half year) measurement of cancer biomarkers. A liquid biopsy of special interest for early detection of breast cancer is nipple aspirate fluid, on account of its breast-specific source for biomarkers. Nipple aspirate fluid (NAF) refers to a fluid that is present in the ductal tree of the breasts and that can be obtained non-invasively using adapted breast pumps. Given its ductal (and possibly lobular) origin, which is where breast tumors develop, the acquisition of NAF and evaluation of its biomarker content are hypothesized to provide relevant information about early changes inside the breast. Another advantageous characteristic of NAF is the ability to synchronously acquire matched pairs of bilateral NAF samples, which provides intra-patient control samples for unilateral disease. NAF samples are a feasible source for the evaluation of biomarkers. The focus of investigation in this thesis was the biomarker class of microRNAs (miRNAs). MiRNAs are short, non-coding RNAs which regulate gene expression at the post-transcriptional level by binding to messenger RNA (mRNA). Clinically, miRNAs are biomarkers of interest because, in combination, they can form a signature or fingerprint for oncological diseases. Furthermore, miRNAs can be of value as biomarkers to understand the biology of disease or disease-related risk factors, such as high breast density for breast cancer. To investigate the potential of miRNAs in NAF and blood as biomarkers for early breast cancer detection, NAF, serum, and plasma samples were collected from three cohorts of women: a longitudinal high-risk cohort with repeated sample collection, a healthy cohort, and a cohort of women with established invasive breast cancer. Blood was also collected to compare the diagnostic value of biomarkers found in each fluid. All samples were immediately biobanked after collection. The first part of this thesis (Chapters 2-4) provides background information about NAF research, summarizes the practical aspects of creating the biobank, and describes the NAF collection process from the perspective of participants. The second part (Chapters 5-6) reports the first results of miRNAs analyses in NAF. In Chapter 7, we summarize the data obtained