Nipple aspirate fluid—A liquid biopsy for diagnosing breast health

Abstract

PurposeNipple secretions are protein‐rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopathological features. Our aim, in this pilot study, was to investigate the intra‐ and interpatient protein composition of nipple secretions and the implications for their use as liquid biopsies.Experimental designMatched pairs of nipple discharge/nipple aspirate fluid (NAF, n = 15) were characterized for physicochemical properties and SDS‐PAGE. Four pairs were selected for semiquantitative proteomic profiling and trypsin‐digested peptides analyzed using 2D‐LC Orbitrap Fusion MS. The resulting data were subject to bioinformatics analysis and statistical evaluation for functional significance.ResultsA total of 1990 unique proteins were identified many of which are established cancer‐associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals were observed.Conclusions and clinical relevanceThis was the most complete proteomic study of nipple discharge/nipple aspirate fluid to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient‐specific protein levels but changes relating to cancer stage require investigation of a larger cohort.

Evaluation of nipple aspirate fluid as a diagnostic tool for early detection of breast cancer

“…Recently, in a series of experiments to optimise protein separation from a NAF sample, Brunoro et al identified 557 different proteins [ 120 ]. The most complete proteomics study was performed by Shaheed et al [ 121 ], identifying more than 1900 unique gene products including mitogenic growth factors (IGF1, IGF2, EGF, PDGFC, PGGFD, TGFβ1, VEGFA), cell adhesion proteins (CEACAMs, NCAM2, ICAM1), established breast cancer biomarkers (EGFR, mucin-1/CA 15-3, mucin-16/CA-125, MUCL1, cytokeratins 5, 8, 14 and 18) as well as 46 candidate biomarkers under investigation by the National Cancer Institute Early Detection Research Network [ 122 ]. A comparison of matched NAF pairs, from a healthy volunteer and patients with benign, DCIS or invasive carcinoma, detected an average of more than 1200 proteins per sample [ 121 ].…”

“…The most complete proteomics study was performed by Shaheed et al [ 121 ], identifying more than 1900 unique gene products including mitogenic growth factors (IGF1, IGF2, EGF, PDGFC, PGGFD, TGFβ1, VEGFA), cell adhesion proteins (CEACAMs, NCAM2, ICAM1), established breast cancer biomarkers (EGFR, mucin-1/CA 15-3, mucin-16/CA-125, MUCL1, cytokeratins 5, 8, 14 and 18) as well as 46 candidate biomarkers under investigation by the National Cancer Institute Early Detection Research Network [ 122 ]. A comparison of matched NAF pairs, from a healthy volunteer and patients with benign, DCIS or invasive carcinoma, detected an average of more than 1200 proteins per sample [ 121 ]. While matched pairs exhibited strong similarity in profile, individuals showed significant differences, confirming the observations by Brunoro et al, using SDS PAGE and 2D-DIGE analysis of NAF [ 123 ].…”

“…Indeed, the composition of the healthy volunteer samples were disproportionately high in milk proteins, despite the fact that the subject had never breast fed. In this case, milk proteins were diagnostic for galactorrhea caused by prescribed medication inducing a nipple discharge [ 121 ]. The different protein profiles identified in NAF samples, collected by different groups, clearly highlight the potential for identifying biomarkers that could be related to the early development of breast cancer.…”

New horizon for breast cancer biomarker discoveries: What might the liquid biopsy of nipple aspirate fluid hold?

“…showing different genetic and epigenetic profiles, allowing tumor to adapt to the microenvironment changes, surviving to the treatments and proning cells to spread in the body. In this field, the technology of LB (applied to a fluid produced by breast gland, named nipple aspirate fluids, NAF) highlights the fascinating power of combining basic and clinical research, as pointed out in a wide clinical proteomic study published in this issue of our journal .…”

“…The LB technology using NAF may represent the outstanding breakthrough of proteomic cancer research revealing novel diagnostic and prognostic applications. In conjunction to metabolomic and degradome profiling, the use of NAF as liquid biopsy approach will improve the detection of changes in the cellular microenvironment of the breast tumors, understanding molecular and biochemical mechanisms which drive breast tumor initiation, maintenance and progression, enhancing the development of novel drug targets and new treatment strategies.…”