Trina Metheny

Ki-67 Expression in Benign Breast Ductal Cells Obtained by Random Periareolar Fine Needle Aspiration

Ki-67 expression in ductal cells obtained by random periareolar fine needle aspiration (RPFNA) is currently being used as a response biomarker in phase II breast cancer chemoprevention trials; however, Ki-67 in RPFNA has not been well studied as a risk predictor for cancer, which would support its use as a response indicator. We examined the expression of Ki-67 in RPFNA specimens with hyperplasia ± atypia obtained from 147 women at high risk for development of breast cancer. Median Ki-67 was 1.4% (range 0-24%). Ki-67 was higher in specimens from women <50 versus those ≥50 (median 2% versus 0.6%; P = 0.006) and from premenopausal women versus postmenopausal women (P = 0.037); however, hormone replacement therapy (predominately low-dose estrogen without progestins) had no effect. By univariate analysis, Ki-67 was positively correlated with ductal cell number (P = 0.001) and hyperplasia with atypia (P = 0.007). By multivariable analysis, the proportion of ductal cells expressing Ki-67 was again predicted by cell number, which, in turn, was predicted by cytologic atypia. The association of Ki-67 expression with cytologic atypia, a known risk factor for development of breast cancer, provides preliminary justification for its use as a response biomarker in phase II chemoprevention trials.

Reduction in Ki-67 in Benign Breast Tissue of High-Risk Women with the Lignan Secoisolariciresinol Diglycoside

Preclinical and correlative studies suggest reduced breast cancer with higher lignan intake or blood levels. We conducted a pilot study of modulation of risk biomarkers for breast cancer in premenopausal women after administration of the plant lignan secoisolariciresinol given as the diglycoside (SDG). Eligibility criteria included regular menstrual cycles, no oral contraceptives, a >3-fold increase in 5-year risk, and baseline Ki-67 of ≥2% in areas of hyperplasia in breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) during the follicular phase of the menstrual cycle. SDG (50 mg/d) was given for 12 months, followed by repeat RPFNA. The primary end point was change in Ki-67. Secondary end points included change in cytomorphology, mammographic breast density, serum bioavailable estradiol and testosterone insulin-like growth factor-I and IGF-binding protein-3, and plasma lignan levels. Forty-five of 49 eligible women completed the study with excellent compliance (median = 96%) and few serious side effects (4% grade 3). Median plasma enterolactone increased ∼9-fold, and total lignans increased 16-fold. Thirty-six (80%) of the 45 evaluable subjects showed a decrease in Ki-67, from a median of 4% (range, 2-16.8%) to 2% (range, 0-15.2%; P < 0.001, Wilcoxon signed rank test). A decrease from baseline in the proportion of women with atypical cytology (P = 0.035) was also observed. Based on favorable risk biomarker modulation and lack of adverse events, we are initiating a randomized trial of SDG versus placebo in premenopausal women. Cancer Prev Res; 3(10); 1342–50. ©2010 AACR.

Comparison of cytomorphology in specimens obtained by random periareolar fine needle aspiration and ductal lavage from women at high risk for development of breast cancer.

SummaryDuctal lavage (DL) and random periareolar fine needle aspiration (RPFNA) are both being used to harvest epithelial cells for risk assessment as well as response evaluation in chemoprevention trials. The magnitude of increase in relative risk has been defined in a prospective study for RPFNA but not for DL atypia. We attempted both procedures in 26 women at high risk for development of breast cancer. Median age was 43 (range 32–57); 15 women were premenopausal, with 6 of the postmenopausal women on HRT. Collection of nipple aspirate fluid (NAF) was attempted and, if successful, was followed by DL; RPFNA was then performed on all women. Both procedures were attempted the same day (follicular phase of menstrual cycle if premenopausal) in 24 subjects and within three months for two subjects. Twenty-three subjects produced NAF, 17 of the 23 (74%) had a successful duct cannulation as part of the DL procedure, with 16 yielding sufficient ((10) ductal cells for morphologic assessment. Twenty-five of 26 (96%) subjects had a successful RPFNA procedure with adequate cellularity for morphology. There was concordance between DL and RPFNA specimens for traditional cytologic category assessment in 10/16 (63%), Masood index score in 13/16 (82%), and Consensus Panel assessment in 12/16 (75%) of specimens. We conclude that same day DL and RPFNA is feasible, with 62% and 96% of high-risk women having a successful procedure with evaluable cytomorphology. RPFNA was more likely to yield an evaluable specimen, but if a cellular DL specimen was obtained, morphology was generally similar.

Modulation of Breast Cancer Risk Biomarkers by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Postmenopausal Women.

Associational studies suggest higher intakes/blood levels of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) are associated with reduced breast cancer risk. We performed a pilot study of high-dose EPA + DHA in postmenopausal women to assess feasibility before initiating a phase IIB prevention trial. Postmenopausal women with cytologic evidence of hyperplasia in their baseline random periareolar fine needle aspiration (RPFNA) took 1,860 mg EPA +1500 mg DHA ethyl esters daily for 6 months. Blood and breast tissue were sampled at baseline and study conclusion for exploratory biomarker assessment, with P values uncorrected for multiple comparisons. Feasibility was predefined as 50% uptake, 80% completion, and 70% compliance. Trial uptake by 35 study entrants from 54 eligible women was 65%, with 97% completion and 97% compliance. Favorable modulation was suggested for serum adiponectin (P = 0.0027), TNFα (P = 0.016), HOMA 2B measure of pancreatic β cell function (P = 0.0048), and bioavailable estradiol (P = 0.039). Benign breast tissue Ki-67 (P = 0.036), macrophage chemoattractant protein-1 (P = 0.033), cytomorphology index score (P = 0.014), and percent mammographic density (P = 0.036) were decreased with favorable effects in a proteomics array for several proteins associated with mitogen signaling and cell-cycle arrest; but no obvious overall effect on proteins downstream of mTOR. Although favorable risk biomarker modulation will need to be confirmed in a placebo-controlled trial, we have demonstrated feasibility for development of high-dose EPA and DHA ethyl esters for primary prevention of breast cancer. Cancer Prev Res; 8(10); 922–31. ©2015 AACR. See related article, p. 912.

Optimization of estrogen receptor analysis by immunocytochemistry in random periareolar fine-needle aspiration samples of breast tissue processed as thin-layer preparations

Immunostaining of estrogen receptor alpha (ER) in samples of benign breast tissue obtained by random periareolar fine-needle aspiration (RPFNA) is a practical tool for breast cancer chemoprevention trials. The authors report an optimized method of ER immunostaining for use with thin-layer preparations of modified Cytolyt-fixed benign breast tissue acquired by RPFNA. Samples of benign breast tissue and MCF-7 controls processed as thin-layer preparations were tested for the effects of antibody titer, antigen retrieval temperature (90° or 115°C), buffer (20% nuclear decloaker, pH 9.3; 10 mM citrate buffer, pH 6), and blocking solution (0.01% glucose oxidase or 0.3% H2O2) on ER immunostaining. The prevalence of positively stained breast epithelial cells, mean intensity of ER staining, and composite immunostaining score were evaluated for effect of immunostaining protocol. RPFNA samples and MCF-7 cells processed using nuclear decloaker and low-temperature antigen retrieval had more ER-positive cells (P<0.0001) and increased mean staining intensity and weighted staining indices (P<0.05) compared with samples prepared with citrate buffer and high-temperature antigen retrieval. Glucose oxidase increased ER-positive cells in comparison to hydrogen peroxide (P<0.04) when combined with low-temperature antigen retrieval and the use of nuclear decloaker. Staining was negative for all non-immune controls regardless of protocol. The combination of low-temperature antigen retrieval, diluted commercial nuclear decloaker solution, and a glucose oxidase blocking step yielded optimal ER immunostaining for thin-layer preparations of benign breast tissue harvested by RPFNA.

Clinical Trial of Acolbifene in Premenopausal Women at High Risk for Breast Cancer

The purpose of this study was to assess the feasibility of using the selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women. To do so, we assessed change in proliferation in benign breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) as a primary endpoint, along with changes in other risk biomarkers and objective and subjective side effects as secondary endpoints. Twenty-five women with cytologic hyperplasia ± atypia and ≥2% of breast epithelial cells staining positive for Ki-67, received 20 mg acolbifene daily for 6–8 months, and then had benign breast tissue and blood risk biomarkers reassessed. Ki-67 decreased from a median of 4.6% [interquartile range (IQR), 3.1%–8.5%] at baseline to 1.4% (IQR, 0.6%–3.5%) after acolbifene (P < 0.001; Wilcoxon signed-rank test), despite increases in bioavailable estradiol. There were also significant decreases in expression (RT-qPCR) of estrogen-inducible genes that code for pS2, ERα, and progesterone receptor (P ≤ 0.026). There was no significant change in serum IGF1, IGFBP3, IGF1:IGFBP3 ratio, or mammographic breast density. Subjective side effects were minimal with no significant increase in hot flashes, muscle cramps, arthralgias, or fatigue. Objective measures showed a clinically insignificant decrease in lumbar spine bone density (DEXA) and an increase in ovarian cysts but no change in endometrial thickness (sonography). In summary, acolbifene was associated with favorable changes in benign breast epithelial cell proliferation and estrogen-inducible gene expression but minimal side effects, suggesting a phase IIB placebo-controlled trial evaluating it further for breast cancer prevention. Cancer Prev Res; 8(12); 1146–55. ©2015 AACR.

Abstract P5-12-03: Initial report of a randomized trial of letrozole in high risk women taking hormone replacement therapy

Background: On the basis of positive results in a pilot study, we initiated a randomized, double-blind, placebo-controlled trial of the aromatase inhibitor letrozole in post-menopausal women at high risk for development of breast cancer who were taking hormone replacement therapy (HRT). The objective was to determine if risk biomarkers for breast cancer in benign breast tissue sampled by random peri-areolar aspiration (RPFNA) could be favorably modulated. Methods Women who exhibited cytologic hyperplasia +/- atypia and Ki-67 immunocytochemistry staining ≥1.5% on screening RPFNA were eligible to be randomized 1:1 between placebo and letrozole (2.5. mg daily) for six months, followed by repeat RPFNA. Women were then given the option to receive open-label letrozole for a second six months, and a third RPFNA. The primary analysis was a difference between the two groups for the change in Ki-67 between baseline and 6 months. The initial accrual goal was 108 subjects, with the expectation of 96 subjects evaluable for the baseline∼6 months comparison. Results 55 subjects were enrolled between March 2007 and March 2014, when accrual was closed. From the time of our successful pilot study to present, there had been a steady decline in the use of HRT by women in our high risk cohort, both in frequency of women using as well as the type and strength of HRT. The result was fewer potential subjects for screening and fewer still that satisfied the 1.5% Ki-67 criterion. Thus, the trial was closed early. Of 55 enrolled subjects, two dropped out prior to 6 months; 52 completed 6 months and provided evaluable RPFNA specimens for analysis, with one subject scheduled for repeat aspiration in September. Six subjects went off study between 6 and 12 months; 42 have completed the entire 12 month schedule, and 5 are still on trial. At baseline, 18 women displayed hyperplasia (Masood score 13-15) and 37 had hyperplasia with atypia (Masood score 14-17). Median Ki-67 was 3.0%, with a range from 1.6 – 15.4%. For 52 comparisons between baseline and 6 months, 8 women had no change by Masood score, 8 had an increase and 36 exhibited a decrease, i.e., less abnormality. Two women had no change in Ki-67 staining, 13 exhibited increased Ki-67 and 37 showed a decrease; median at 6 months 1.7%, median change -1.4%. For 42 comparisons between baseline and 12 months, 11 women had no change by Massod score, 9 increased, and 22 decreased. One woman had no change in Ki-67 staining, 10 exhibited increased Ki-67 and 31 showed a decrease. The decreases in Masood score and Ki-67 between baseline and 12 months (when all subjects had received letrozole, either for 6 or 12 months) were statistically significant (p Conclusion While pending final analysis of the primary (blinded) endpoint, preliminary analysis indicates favorable modulation of cytomorphology and proliferation by the aromatase inhibitor letrozole in high risk post-menopausal women taking hormone replacement therapy. Funding: NIH RO1 CA122577; Novartis Pharmaceuticals Corp. Citation Format: Carol J Fabian, Bruce F Kimler, Jennifer L Nydegger, Trina Metheny, Carola M Zalles, Brian K Petroff, Hung-wen Yeh, Michael D Alvarado. Initial report of a randomized trial of letrozole in high risk women taking hormone replacement therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-12-03.

Abstract 158: Modulation of breast tissue biomarkers by high dose omega-3 fatty acid supplementation in women at high risk for development of breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. Previously, we had observed that women at high risk for development of breast cancer were more likely to exhibit cytologic hyperplasia with atypia in specimens acquired by random periareolar fine needle aspiration (RPFNA) if they had low intake and/or low plasma, red blood cell, or breast tissue levels of omega-3 relative to omega-6 fatty acids. We evaluated the effect of high dose omega-3 supplementation on breast tissue markers in two parallel pilot studies, one of pre-menopausal and one of post-menopausal women. Methods. 36 pre-menopausal and 35 post-menopausal women at high risk for breast cancer had breast tissue harvested by RPFNA before and after a 6-month intervention with 4 g daily of omega-3-acid ehyl esters [1.86 g eicosapentaenoic acid (EPA), 1.5 g docosahexaenoic acid (DHA)]. Premenopausal women were aspirated in the follicular phase of the menstrual cycle. Specimens were evaluated for tissue risk biomarkers including cytomorphology, proliferation (Ki-67). Fatty acid composition was determined in plasma, red blood cells, and breast RPFNA specimens. Additional blood and frozen breast tissue was reserved for assessment of hormones, adipokines, cytokines and gene expression. Results. To date, only two of the 71 subjects have discontinued the study early, while 50 subjects have completed study and are evaluable for modulation of tissue biomarkers. Grade 2 or greater gastrointestinal side effects have been reported by only seven subjects. Favorable modulation was observed for cytologic evidence of atypia (70% to 44%; p=0.012), Masood score (medians of 15 to 14; p=0.001), number of epithelial cells recovered (p=0.002), and Ki-67 expression (p=0.059 if all subjects are included even if they did not exhibit Ki-67 staining at baseline, medians of 1.7% to 0.8%; or p=0.001 for 27 women with baseline Ki-67 >1.5%, medians of 3.2% to 1.4%). Modulation was more prevalent (and was statistically significant for all variables) in pre-menopausal women than in post-menopausal women. Fatty acid assessment, adipokine and cytokine assays are batched to minimize variability and all results are not yet available. Preliminary results indicate that the ratio of omega-3:omega-6 fatty acids increased in erythrocytes and plasma by two-fold after 6 months of the high dose omega-3 fatty acid intervention. Conclusion. High dose supplementation with omega-3 fatty acids is well-tolerated in healthy women at high risk for development of breast cancer and was associated with favorable modulation of the tissue risk biomarkers of cytologic atypia and proliferation. This strategy will be explored further as a promising intervention that may reduce risk for development of breast cancer. Supported in part by funding from the Breast Cancer Research Foundation and the Kansas Bioscience Authority. Study agent was provided by GlaxoSmithKline. Citation Format: Carol J. Fabian, Bruce F. Kimler, Carola M. Zalles, Trina Metheny, Jessica A. Box, Jennifer L. Nydegger, Teresa A. Phillips, Brandon H. Hidaka, Susan E. Carlson. Modulation of breast tissue biomarkers by high dose omega-3 fatty acid supplementation in women at high risk for development of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2013-158