Background: Ductal lavage is a method of minimal epithelial sampling of the breast, with potential utility for repeat sampling and biomarker analysis in chemoprevention studies. We report here the baseline findings from a study designed to assess the utility of ductal lavage in this setting. Methods: Tamoxifen-eligible, high-risk women underwent ductal lavage; epithelial cell number (ECN) and morphology were assessed on Papanicolaou-stained slides. Additional slides were immunostained for estrogen receptor (ER) A, Ki-67, and cyclooxygenase-2, and the labeling index (LI) was established by counting negative and positive cells. The ductal lavage supernatant (DLS) was assayed for estradiol, several of its precursors, progesterone, cathepsin D, interleukin-6, and epidermal growth factor (EGF). Results: One hundred sixty-eight women have entered the study (mean age, 51 years; mean 5-year Gail score, 2.8).

Recent developments in breast epithelial sampling techniques (nipple fluid aspiration, ductal lavage, and random fine needle aspiration) provide new opportunities for the acquisition of hormonal and cellular biomarker data in asymptomatic women, and thereby the possibility of developing a unified vision of how the hormonal environment of the breast may interact with the cellular expression of proteins, and with other evolving candidate markers of breast cancer risk. The purpose of this review is to integrate available information regarding cellular and breast fluid biomarkers of hormone action on the breast, to identify candidate biomarkers for studies of breast cancer risk and prevention. These include the estrogen receptors a and b, markers of proliferative and apoptotic response, and protein markers of estrogen action in breast cells and nipple fluid. Studies of breast hormone levels in nipple aspiration fluid (NAF) show that estrone sulphate is present in large quantities in the normal breast, while the differences in serum ovarian steroids that are seen in preand postmenopausal women are blunted in NAF. The variability of several estradiol precursors in NAF over time is relatively small, a useful attribute of potential biomarkers of breast cancer risk, particularly if they are reversible with intervention in Phase 2 prevention trials. These studies are already providing new insights into the hormonal etiology of breast cancer, and should lead to the identification of robust, reversible biomarkers for use in breast cancer prevention studies.

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To the Editors: Robust and reversible biomarkers are needed for the successful completion of phase II prevention trials, an area where Fabian and colleagues have made enormous contributions. One potential marker is cell proliferation, measured by Ki-67 labeling; Khan et al. present data obtained by random periareolar fine-needle aspiration in 147 women at high-risk, which they interpret as providing preliminary support for use of Ki-67 as a response biomarker in prevention trials (1). They base this on an association between morphologically defined hyperplasia with atypia, and Ki-67 labeling index. These results are based on a single random periareolar fine-needle aspiration procedure, so that a key attribute of a useful biomarker (reproducibility; ref.2) remains untested.Our experience at Northwestern University using ductal lavage for similar analyses has both similarities and differences to these data. In an ongoing phase II chemoprevention study with tamoxifen as the intervention agent, we found a mean Ki-67 labeling index in ductal lavage samples of 0.72% (based on results from 117 women with >100 cells at baseline lavage). Although we observed a good correlation of Ki-67 labeling index with total epithelial cell yield (P = 0.001), no correlation was seen with cytological atypia. In agreement with Khan et al., we see a very strong correlation between cytology and total cell number, and note with interest that when Khan et al. performed a multivariate analysis to predict Ki-67 expression, a significant correlation of Ki-67 with cytomorphology was seen only after cell number was excluded from the analysis. This, together with our own findings in relation to the correlation of cell number with biomarkers such as endoplasmic reticulum, cyclooxygenase-2, and Ki-67 expression, suggests that epithelial cell yield is at least as good a biomarker of response to preventive intervention as Ki-67 labeling.We have compared the mean Ki-67 labeling index in women who did not take tamoxifen with those who accepted tamoxifen therapy, at baseline and 6 months later. In the no-intervention group, Ki-67 labeling index dropped from 0.73 to 0.27 (P = 0.003) while in the tamoxifen group, comparable values were 0.55 and 0.37 (P = 0.35; ref. 3). In phase Ia and Ib studies of women with hormone receptorpositive breast cancer, Fabian et al. found no significant difference in Ki-67 labeling, although proliferating cell nuclear antigen -labeled cells decreased significantly following arzoxifene intervention (4).Considering these results together, we remain skeptical whether parameters such as Ki-67 labeling and morphological atypia are robust and reproducible indicators of response. In Response: Drs. Bhandare and Khan in their letter to the editor question whether Ki-67 labeling and cytomorphologic evidence of atypia in cytology specimens are sufficiently robust biomarkers to be used as response end points in early phase prevention trials. This is based on their experience with a limited number of ductal lavage specimens in which sampl...