High-Throughput Proteomic Profiling of Nipple Aspirate Fluid from Breast Cancer Patients Compared with Non-Cancer Controls: A Step Closer to Clinical Feasibility

Abstract

Background: Early detection of breast cancer (BC) is critical for increasing survival rates. However, current imaging approaches can provide ambiguous results, requiring invasive tissue biopsy for a definitive diagnosis. Multi-dimensional mass spectrometric analysis has highlighted the invaluable potential of nipple aspirate fluid (NAF) as a non-invasive source of early detection biomarkers, by identifying a multitude of proteins representative of the changing breast microenvironment. However, technical challenges with biomarker validation in large cohorts remain due to low sample throughput, impeding progress towards clinical utility. Rather, by employing a high-throughput method, that is more practicable for clinical utility, perturbations of the most abundant NAF proteins in BC patients compared with non-cancer (NC) controls could be monitored and validated in larger groups. Method: We characterized matched NAF pairs from BC (n = 9) and NC (n = 4) volunteers, using a rapid one dimensional liquid chromatography-mass spectrometry (1D LC-MS/MS) approach. Results: Overall, 198 proteins were relatively quantified, of which 40 were significantly differentiated in BC samples, compared with NC (p ≤ 0.05), with 26 upregulated and 14 downregulated. An imbalance in immune response and proteins regulating cell growth, maintenance and communication were identified. Conclusions: Our findings show 1D LC-MS/MS can quantify changes reflected in the NAF proteome associated with breast cancer development.

Understanding Breast Cancer Aggressiveness and Its Implications in Diagnosis and Treatment

“…Proteomic profiling of plasma samples revealed the significant relationship between the upregulated proteins in breast cancer and their association with the complement system and protease inhibitor activities. Mapping the myriad of proteins and their role in activating or inhibiting the immune system may help address the disease and early detection [ 13 ]. Several pathways in the cancer cell or tumor environment are altered in BC [ 8 ].…”

“…Integrated bioinformatics and systems biology can play a role in understanding and testing key expressed genes related to BC recurrence. Therefore, using omics technologies to identify BC specific biomarkers is an indispensable approach for BC early diagnosis that can prevent metastasis, improve treatment, and reduce relapse [ 13 , 16 ].…”

The application of high-throughput proteomics in cytopathology

“…Regarding breast cancer, most published cytology-based proteomics studies utilized nipple aspirate fluid (NAF), whereas a smaller number FNA samples (Table 1). A few reported significant proteomic profile differences between the NAF of patients with breast cancer compared to non-malignant controls [39,[42][43][44]. In a breast FNA-based study performed by Franzen et al [45], expression levels of several immune-related proteins differed between cancer and controls, while a few were associated with estrogen receptor, Ki-67 status, and tumor grading.…”

Nipple Aspirate Fluid at a Glance

“…The latter three publications comprise the most recent publications about NAF, together with publications from George et al [ 126 ] and Jiwa et al [ 48 , 71 , 127 ]. The first described a proteomic evaluation of NAF [ 126 ] whereas the latter described in a systematic review that the diagnostic accuracy of nipple smear cytology is limited by poor sensitivity [ 48 , 127 ] and, in a questionnaire-based study, that there is a great readiness of women to undergo NFA [ 71 ]. Based on ongoing clinical studies using NAF samples (see Table 3 ), future data on NAF’s content and clinical potential will be generated and reported.…”