In a prospective pilot study, we performed breast fine needle aspirations (FNAs) on 21 3 high-risk and 30 low-risk women and analyzed these aspirates for cytologic changes and biomarker abnormalities of aneuploidy and overexpressed estrogen receptor (ER), epidermal growth factor receptor (ECFR), p53 and HER-2lneu. High-risk women were those with a first degree relative with breast cancer (73%), prior biopsy indicating premalignant breast disease (26%), a history of breast cancer (1 3%), or some multiple of these risk factors (1 1 %). Median ages of the high-risk and low-risk groups were 44 and 42, respectively. Sixty-three percent of the high-risk and 73% of the low-risk group were premenopausal, Sixty-eight percent of the high-risk and 1 7% of low-risk women had cytologic evidence of hyperplasia with or without atypia ( P < ,0001). Aneuploidy and overexpression of EGFR and p53 occurred in 25%, 36%, and 28% of high-risk subjects but in less than 4% of low-risk subjects ( P < .0002). Overexpression of ER and HER-2lneuoccurred in 8% and 1 respectively of high-risk women; no low-risk women had these abnormalities. Sixty-eight percent of high-risk women and 7% of low-risk women had abnormalities of one or more of these biomarkers exclusive of cytcilogy. Thirty-one percent of high-risk women, but no low-risk women had abnormalities of two or more biomarkers IP = ,0004). Biomarker abnormalities were more frequent with increasing cytologic abnormality. Eighteen percent of women with normal cytology, 29% of women with epithelial hyperplasia and 60% of women with hyperplasia with atypi,] had abnormalities of two or more biomarkers ( P = ,048 and <.0001, respectively). Restricting the analysis to those three biomarkers most frequently overexpressed in the high-risk group (ploidy, ECFR, p53), 13% of high-risk women with normal cytology, 20% of high-risk women with epithelial hyperplasia and 51 YO of high-risk women with atypical hyperplasia had abnormalities of 2 or more of these 3 biomarkers. At a median follow up of two years, 8 of 21 3 women have been diagnosed with in situ (n = 5) or invasive (n = 3) cancer. Later detection of neoplasia was associated with prior FNA evidence of atypical hyperplasia ( P < .0001) and multiple biornarker abnormalities in the 5 test battery ( P = ,006) by univariate analysis. By multivariate analysis, development andlor detection of cancer was primarily predicted by atypical hyperplasia ( P = ,0047) and secondarily by multiple biomarker abnormalities ( P = 0.021). Atypical hyperplasia, EGFR, and p53 in breast FNAs have promise as risk markers and as surrogate endpoint biomarkers for kireast cancer chemoprevention trials.