Breast ductal lavage for assessment of breast cancer biomarkers.

Lavage of the ductal systems of the breast provides fluid (DLF) containing hormones and products of hormone actions that may represent more accurately the composition of the breast than samples collected from blood or urine. The present study was undertaken to assess the presence of potential cancer biomarkers, their variation among individuals at high risk for breast cancer, and differences associated with menopause and tamoxifen treatment. Seventy seven tamoxifen-eligible subjects with a 5-year breast cancer risk estimate (Gail > 1.6%)(N = 53) or recently diagnosed breast cancer (N = 24) were offered tamoxifen therapy; those not accepting tamoxifen were under observation only. After six months, all subjects underwent ductal lavage (DL) in an unaffected breast. Estradiol (E2), estrone sulfate, androstenedione, dehydroepiandrosterone (DHEA), DHEA sulfate, progesterone, cathepsin D and epidermal growth factor (EGF) were measured in DLF by immunoassays. Data were expressed as the mass of analyte per mg of protein in DLF and normalized by natural log transformation. With the exception of DHEA, none of the analytes measured were significantly lower in postmenopausal women than in premenopausal women. The mean log(e) concentration difference in estradiol was 10.9%. Tamoxifen treatment for 6 months did not result in a significantly greater concentration of E2 or in any of the other analytes in DLF of pre- or postmenopausal women. The between-duct variance of the concentration of free steroids within the same breast averaged 51% less than that between subjects, and was similar to that of non-diffusible proteins. The maintenance of estradiol concentrations in the breast after menopause demonstrates the importance of local biosynthesis. The fact that DLF E2 does not reflect the high serum concentrations of E2 during tamoxifen treatment indicates that breast concentrations of estradiol may be under feedback control. Unlike studies of low risk populations, progesterone concentrations were not significantly less in postmenopausal than in premenopausal women. The similarity in variance of free steroids and protein analytes between ducts of a breast indicates little transfer of steroids between lobules.

Ductal lavage is an inefficient method of biomarker measurement in high-risk women.

Effective methods of serial epithelial sampling to measure breast-specific biomarkers will aid the rapid evaluation of new preventive interventions. We report here a proof-of-principle phase 2 study to assess the utility of ductal lavage (DL) to measure biomarkers of tamoxifen action. We enrolled women with a 5-year breast cancer risk estimate >1.6% or the unaffected breast of women with T1a or T1b breast cancer. After entry DL, participants chose tamoxifen or observation and underwent repeat DL 6 months later. Samples were processed for cytology and immunohistochemistry for estrogen receptor alpha, Ki-67, and cyclooxygenase-2. Of 182 women recruited, 115 (63%) underwent entry and repeat DL; 85 (47%) had sufficient cells for analysis from > or =1 duct at both time points; in 78 (43%), cells were sufficient from > or =1 matched ducts. Forty-six women chose observation and 39 chose tamoxifen. We observed greater reductions in the tamoxifen group than in the observation group for Ki-67 (adjusted P = 0.03) and estrogen receptor alpha (adjusted P = 0.07), but not in cyclooxygenase-2 (adjusted P = 0.4) labeling. Cytologic findings showed a trend toward improvement in the tamoxifen group compared with the observation group. Interobserver variability for cytologic diagnosis between two observers showed good agreement (kappa = 0.44). Using DL, we observed the expected changes in tamoxifen-related biomarkers; however, poor reproducibility of biomarkers in the observation group, the 53% attrition rate of subjects from recruitment to biomarker analyses, and the expense of DL are significant barriers to the use of this procedure for biomarker assessment over time.

Endocrine biomarkers in ductal lavage samples from women at high risk for breast cancer.

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) alpha, 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). Ductal lavage was accomplished in 145 (86.3%) women. Data were analyzed by duct and by woman (averaging data across all ducts). Mild atypia was seen in 43 of 145 (29.6%), whereas severe atypia was seen in 2 (1.4%) of women. We observed significant positive correlations between ECN and cytologic atypia, ER LI, cyclooxygenase-2 LI, and Ki-67 LI. EGF levels in supernatant were significantly associated with estrogenic precursors, ER LI and ECN. A factor representing the DLS hormone and protein variables explained 36% of the variance; total ECN was highest when factor score and ER LI were high and was lowest when both were low (P for interaction = 0.001). CONCLUSIONS: Biomarker analyses in epithelial cells and DLS are feasible. The significant associations of EGF with other markers suggest a possible role in increasing epithelial cell mass.

Ductal lavage findings in women with mammographic microcalcifications undergoing biopsy.

BACKGROUND: We designed a prospective study to assess the likelihood that early lesions presenting as mammographic calcifications could be accessed for cytological diagnosis by ductal lavage (DL). METHODS: Consenting women with calcifications (Breast Imaging Reporting and Data System 4 or 5) underwent DL of fluid-yielding ducts (FYDs) before stereotactic core or excisional biopsy. The DL catheter was used to inject .2 to 1 mL of Isovue 300 into the duct to determine whether the FYD corresponded to the duct containing calcifications (designated overlap). Additional FYDs were injected, if possible, until overlap was identified. DL cytology was compared with histology. RESULTS: Twenty women were enrolled (mean age, 54.2 years); the mean size of the calcification-bearing area was 190 mm(2). The histological findings were as follows: 1 invasive cancer, 9 ductal carcinomas-in-situ (DCIS), 5 atypical hyperplasias, and 5 usual hyperplasias or fibrocystic changes. Four women had no FYD. In 15 women who underwent DL and ductography, overlap of dye and calcifications was seen in 4 (27%): 1 fibrocystic change, 1 hyperplasia, 1 atypical hyperplasia (cytological diagnosis mildly atypical), and 1 DCIS (cytological diagnosis benign). Of the remaining 8 DCIS lesions, 4 had no nipple aspiration fluid, 1 showed extravasation, and 3 were lavaged but the duct did not overlap. CONCLUSIONS: These results are consistent with data from women undergoing mastectomy for larger invasive cancer and DCIS and show that cancer-containing ducts do not yield nipple fluid in most cases.

Ductal lavage findings in women with known breast cancer undergoing mastectomy.

BACKGROUND: Ductal lavage has the potential to detect cancer by sampling breast epithelium in asymptomatic high-risk women. To assess the utility of ductal lavage as a cancer diagnostic test, we investigated the association between ductal lavage cytologic findings and histologic findings in women with known breast cancer undergoing mastectomy. METHODS: Ductal lavage was performed in the operating room before mastectomy on 44 breasts from 32 women with known cancer and on eight breasts from seven women undergoing prophylactic mastectomy, two with occult malignancy. If the ductal lavage sample from one or more ducts contained enough epithelial cells for a cytologic diagnosis, lavaged ducts were injected with a mixture of colored dye, gelatin, and a radiographic contrast compound after mastectomy, and breast tissue was radiographed and sectioned. Histologic findings in ducts with and without dye were recorded. Associations between cytologic results and histologic results were examined by univariate and multivariable analyses. RESULTS: At least one duct was lavaged in 36 breasts (mean = 1.4 ducts per breast); all histologic and cytologic procedures were completed in 28 breasts and in 39 ducts. Markedly atypical or malignant cytology was found in five cancer-containing breasts. In 39 ducts with complete cytologic and histologic data and when marked atypia or malignant cells defined a positive cytologic test, sensitivity was 43% (95% confidence interval [CI] = 23% to 72%), specificity was 96% (95% CI = 86% to 100%), and accuracy was 77% (95% CI = 63% to 89%). When mild or marked atypia or malignant cells defined a positive cytologic test, sensitivity was 79% (95% CI = 57% to 96%), specificity was 64% (95% CI = 46% to 83%), and accuracy was 69% (95% CI = 55% to 83%). When all 31 cytologically evaluable breasts were analyzed, sensitivity was 17% (95% CI = 7% to 35%), specificity was 100% (95% CI = 5% to 100%), and accuracy was 19% (95% CI = 9% to 38%). CONCLUSION: In breasts with cancer, ductal lavage appears to have low sensitivity and high specificity for cancer detection, possibly because cancer-containing ducts fail to yield fluid or have benign or mildly atypical cytology.