The Physiological MicroRNA Landscape in Nipple Aspirate Fluid: Differences and Similarities with Breast Tissue, Breast Milk, Plasma and Serum

Abstract

Background: MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum. Methods: MiRNA RT-qPCR profiling of NAF (n = 41) and serum (n = 23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared. Results: NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. Conclusion: This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics.

Nipple Aspirate Fluid at a Glance

“…Many biomarker classes have been investigated in NAF, such as hormones (e.g., estrogen, testosterone), tumor markers (e.g., carcinoembryonic antigen and prostate-specific antigen (PSA)) and biochemical components (e.g., aluminum). Moreover, NAF contains molecular markers, such as DNA [ 43 , 44 ], RNA, microRNA [ 45 ] and proteins [ 46 , 47 ]. These findings have been summarized in two reviews from Edward Sauter and Ferdinando Mannello et al [ 46 , 47 ].…”

“…Since pipetting is also hampered by viscous samples, a requirement would be to have automatic positive displacement pipettes to ensure accuracy and reproducibility. Still, current relevant biomarker techniques like real time quantitative reverse transcription PCR (RT-qPCR) [ 45 , 55 ] and mass spectrometry [ 15 , 47 ] have been shown to be feasible for NAF samples.…”

“…We further focused on NAF microRNA analysis, based on the fact that these biomarkers can be detected in several liquid biopsies and can make up a signature for oncological disease. We have proven that microRNAs (miRNAs)are measurable in NAF by RT-qPCR [ 45 ] and are currently investigating the feasibility of small RNA sequencing for miRNA detection in NAF samples. Now that our group has piled up a large sample biobank with blood and NAF from healthy women, women with breast cancer and serial samples from women at increased risk of developing breast cancer, more publications will soon follow [ 122 , 123 , 124 ].…”

Elevated miR-29c-5p Expression in Nipple Aspirate Fluid Is Associated with Extremely High Mammographic Breast Density

“…In the context of MD, the assessment of miRNAs in nipple aspirate fluid (NAF) may be particularly relevant [ 13 ]. NAF contains breast-specific miRNAs, actively or passively secreted from the breast microenvironment into the breast ducts.…”

The changing microRNA landscape by color and cloudiness: a cautionary tale for nipple aspirate fluid biomarker analysis

“…Among the various body biofluids in which miRNAs can be measured, nipple aspirate fluid (NAF) has attracted attention for breast cancer early detection and management because it may reflect pathological changes in the breast microenvironment [14][15][16]. NAF is secreted in small amounts by the breast ducts of adult non-lactating women and can be collected by non-invasive vacuum aspiration preceded by oxytocin nasal spray administration [17][18][19]. The use of oxytocin promotes the release of already existing fluid in the milk ducts, thereby yielding sufficient material (on average 10-20 µl) for molecular analysis in the majority of healthy volunteers and patients [14,15,17,[20][21][22][23].…”