43 Review www.expert-reviews.com ISSN 1478-9450 © 2009 Expert Reviews Ltd 10.1586/14789450.6.1.43 Breast cancer (BC) is the most frequently diag- nosed cancer in Western countries [1] . The abil- ity to assess a woman’s risk of developing this malignancy remains inadequate and the real key to increasing survival is early diagnosis. In fact, early detection (through screening mammography) decreases the mortality of this multifaceted disease [2] . However, mammo- graphy misses as much as 40% of BC, and breast biopsies after abnormal mammograms confirm cancer in only 20% of cases. Although tools are available to help predict risk for the development of BC, validated biomarkers that are able to more accurately define who is at greatest risk have not yet been identified (e.g., current serum tumor markers are not useful for the diagnosis of BC) [3] . It is well known that BC arises from the epithelial cells lining the ductal–lobular sys- tem and, therefore, the analysis of the fluid contained therein may reveal early signs of precancerous and cancerous transformation [4] . On this basis, nipple aspirate fluid (NAF) has been suggested to be the mirror of the meta- bolic pathways and cellular modifications occurring in the breast microenvironment, both in physiological and pathological condi- tions [5,6] . Since the first evidence, dating back to 1958, of the clinical utility of NAF ana- lysis [7] , ever-growing numbers of studies have demonstrated and/or confirmed that NAF is a potential source of biomarkers for the early diagnosis or risk assessment of BC [8,9] . It has been recognized that the adult nonpregnant and nonlactating breast secretes fluid into the breast ductal system; except during lacta- tion and in the galactorrhea associated with endocrinopathies, the nipple ducts are tightly occluded by keratin plugs, and secretions from the nipple only rarely escape [10] . Initial stud- ies with NAF focused mainly on correlating epithelial cell cytology with BC risk since these are the cells most at risk for transformation to BC [11,12] . More recent studies have begun Ferdinando Mannello † , Virginia Medda and Gaetana A Tonti † Author for correspondence Department of Biomolecular Sciences, Section of Clinical Biochemistry, Faculty of Sciences and Technologies, University Carlo Bo, Via O. Ubaldini 7, 61029 Urbino (PU), Italy Tel.: +39 072 235 1479 Fax: +39 072 232 2370 ferdinando.mannello@uniurb.it Protein and proteomic high-throughput technologies provide the polypeptide signatures of nipple aspirate fluid (NAF), a breast secretion collected noninvasively from healthy individuals and cancer patients. As breast cancer develops from ductal–lobular epithelium, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a useful tool for the analysis of metabolic pathways within the mammary gland, deepening our knowledge of the biomolecular mechanisms of breast cancer initiation and progression. The different protein expression of major NAF proteins, separated using 1D polyacrylamide gels, has proven valuable for the early detection of women with increased risk of cancer. The failure to recognize a single marker with sufficient clinical sensitivity and/or specificity has driven the identification of breast cancer multiple proteins by 2D electrophoresis. Mass spectrometry-based proteomic approaches (SELDI- and MALDI-TOF technologies) have allowed the characterization of differential NAF proteomic fingerprints between healthy individuals and breast cancer patients. The intraductal approach of protein and proteomic analyses may provide a panel of biomarkers to strengthen the armory against breast cancer. KEYWORDS:฀breast฀cancer฀•฀diagnosis฀•฀nipple฀aspirate฀luid฀•฀prognosis฀•฀protein฀•฀proteinase฀•฀proteomics฀ •฀tumor฀marker Protein profile analysis of the breast microenvironment to differentiate healthy women from breast cancer patients Expert Rev. Proteomics 6(1), 43–60 (2009) For reprint orders, please contact reprints@expert-reviews.com