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Supplemental Figure 1: CONSORT diagram for flow of potential and actual subjects enrolled on study. Supplementary Table 1. Demographic and risk information at baseline. Supplementary Table 2. Change in fatty acid content in all five compartments. Supplementary Table 3. Change in anthropomorphic variables. Supplementary Table 4: Changes in levels of cytokines and adipokines in RPFNA specimens, by Luminex. Supplementary Table 5: Changes in levels of mRNA in RPFNA specimens, by RT-qPCR. Supplementary Table 6: Changes in levels of all peptides and specific phosphopeptides in RPFNA specimens, by Reverse Phase Protein Array.

Supplementary Tables 1-3

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.

Supplemental Table 1: Specimen collection and assay methods details. Supplemental Table 2 Adverse Events Supplemental Table 3. Value and changes in ratio of (DHA+EPA)/AA in erythrocyte phospholipids. Supplemental Table 4. Median relative difference (percent) between baseline and 12 months for 19 serum biomarkers (of a total of 24 assessed) which exhibited a statistically significant change over time* either for the total cohort of 35 women completing the 12-month trial and/or for groups defined by randomization or by dichotomization at 10% weight loss achieved at 6 months. Supplemental Table 5: Biomarker change at 12 months by subgroups defined by randomization arm (placebo vs ω-3 FA) and dichotomization by weight loss (10%). Biomarkers are grouped into categories of adipokines, cytokines, hormones/growth factors, and insulin. A total of 24 biomarkers or ratios were assessed at 12 months. Supplemental Table 6: Levels and changes for adiponectin assessed in serum collected both fasting and non-fasting, and in benign breast tissue acquired non-fasting by RPFNA. Supplemental Table 7: Baseline, 6-month, and 12-month values and change over time for Ki-67, Masood cytomorphology score.

Supplementary Figures and Tables

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.

Interventions that relieve vasomotor symptoms while reducing risk for breast cancer would likely improve uptake of chemoprevention for perimenopausal and postmenopausal women. We conducted a pilot study with 6 months of the tissue selective estrogen complex bazedoxifene (20 mg) and conjugated estrogen (0.45 mg; Duavee) to assess feasibility and effects on risk biomarkers for postmenopausal breast cancer. Risk biomarkers included fully automated mammographic volumetric density (Volpara), benign breast tissue Ki-67 (MIB-1 immunochemistry), and serum levels of progesterone, IGF-1, and IGFBP3, bioavailable estradiol and testosterone. Twenty-eight perimenopausal and postmenopausal women at increased risk for breast cancer were enrolled: 13 in cohort A with baseline Ki-67 < 1% and 15 in cohort B with baseline Ki-67 of 1% to 4%. All completed the study with > 85% drug adherence. Significant changes in biomarkers, uncorrected for multiple comparisons, were a decrease in mammographic fibroglandular volume (P = 0.043); decreases in serum progesterone, bioavailable testosterone, and IGF-1 (P < 0.01), an increase in serum bioavailable estradiol (P < 0.001), and for women from cohort B a reduction in Ki-67 (P = 0.017). An improvement in median hot flash score from 15 at baseline to 0 at 6 months, and menopause-specific quality-of-life total, vasomotor, and sexual domain scores were also observed (P < 0.001). Given the favorable effects on risk biomarkers and patient reported outcomes, a placebo-controlled phase IIB trial is warranted.

The inflammation-resolving and insulin-sensitizing properties of eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids have potential to augment effects of weight loss on breast cancer risk. In a feasibility study, 46 peri/postmenopausal women at increased risk for breast cancer with a body mass index (BMI) of 28 kg/m2 or greater were randomized to 3.25 g/day combined EPA and DHA (ω-3-FA) or placebo concomitantly with initiation of a weight-loss intervention. Forty-five women started the intervention. Study discontinuation for women randomized to ω-3-FA and initiating the weight-loss intervention was 9% at 6 months and thus satisfied our main endpoint, which was feasibility. Between baseline and 6 months significant change (P < 0.05) was observed in 12 of 25 serum metabolic markers associated with breast cancer risk for women randomized to ω-3-FA, but only four for those randomized to placebo. Weight loss (median of 10% for trial initiators and 12% for the 42 completing 6 months) had a significant impact on biomarker modulation. Median loss was similar for placebo (−11%) and ω-3-FA (−13%). No significant change between ω-3-FA and placebo was observed for individual biomarkers, likely due to sample size and effect of weight loss. Women randomized to ω-3-FA exhibiting more than 10% weight loss at 6 months showed greatest biomarker improvement including 6- and 12-month serum adiponectin, insulin, omentin, and C-reactive protein (CRP), and 12-month tissue adiponectin. Given the importance of a favorable adipokine profile in countering the prooncogenic effects of obesity, further evaluation of high-dose ω-3-FA during a weight-loss intervention in obese high-risk women should be considered.Prevention Relevance:This study examines biomarkers of response that may be modulated by omega-3 fatty acids when combined with a weight-loss intervention. While focused on obese, postmenopausal women at high risk for development of breast cancer, the findings are applicable to other cancers studied in clinical prevention trials.

Summary of Assessment of Ki-67 Staining Problems. Supplementary Table 1: Summary of Values, Changes, and Statistical Results for Ki-67 Assessments. Supplementary Table 2: Summary of Analyses of Different Subsets of Subjects.

Background: Nearly 10% of breast cancers (BC) are diagnosed in premenopausal woman under age 45 and of childbearing potential. Women considering future childbearing are typically excluded from BC prevention trials and are ineligible for standard of care chemoprevention. More biomarkers are needed to support BC prevention trials in this young cohort. Women of childbearing potential are encouraged to supplement diet with minimum of 300mg of EPA + DHA omega-3 fatty acids (FA) per day. EPA and DHA are thought to have a favorable effect on the gut microbiome implicated in cancer development. Few studies have characterized the breast microbiome by core biopsy or surgical sample but to our knowledge no studies have explored the feasibility of breast microbiome collection using the less invasive technique Random Periareolar Fine Needle Aspiration (RPFNA). In a pilot study, we demonstrated feasibility of recruiting premenopausal women considering future pregnancy to a BC prevention trial with a 6-month intervention of omega-3 FA supplementation (19-A-1921-SABCS). RPFNA was used to collect breast tissue for biomarker analysis, which is a mildly invasive technique used for repeated sample collection in BC prevention trials. Objectives: 1) To determine the feasibility of characterizing breast microbiome from specimens collected by RPFNA in premenopausal woman at high risk for BC, 2) To identify changes in the breast and stool microbiome with omega-3 FA supplementation in this population. Methods: Ten women between the ages of 21 and 40 who were considering future pregnancy and at high risk for BC were enrolled to a pilot study and took Omega-3-Acid Ethyl Ester (total of 750mg DHA and 930mg EPA) daily for 6 months. Tissue collection with RPFNA of breast as well as blood, urine and stool were completed at baseline and off-study visit. RPFNA samples from the first 2 passes at each site of breast (4 sites total) were collected for microbiome and placed in a 2mL tube with 0.5 – 1cc of PBS and flash frozen and stored at -80C. DNA was isolated from RPFNA samples using QIAamp DNA Mini Kit (51304). Microbiome profiling analysis was performed by Veracet using 16S V4 rRNA gene sequencing on the Illumina MiSeq platform. Wilcoxon signed rank test was used to compare paired samples. Results: Of the 10 women enrolled, median age was 33 years (range 22-37). 90% (9 of 10) returned for off-study visit. Of the 9 women who completed the off-study visit, 2 elected to not undergo off-study RPFNA. There were 16 total stool samples and 17 total RPFNA samples for microbiome evaluation. There were 6 paired (baseline and off-study) stool and 7 paired RPFNA samples. Mean DNA concentration from RPFNA samples was 10.36ng/µl (range 0.62 – 74.10). From all samples, 52.1% of operational taxonomic units (OTUs) were classified at the genus level. Breast samples were sequenced to a depth of mean 27,767 reads (range 5,745 – 125,445) and stool to a depth of mean 119,296 reads (range 72,979 – 188,867). The alpha diversity metric of OTU richness was 1069 (breast) and 438 (stool). Shannon diversity was 4.51 (breast) and 3.89 (stool). Mean OTU richness for baseline and off-study RPFNA samples were 1098 and 1028 respectively (V = 25, p value = 0.076). Mean OTU richness for baseline and off-study stool samples were 440 and 437 respectively (V = 15, p value = 0.40). Conclusion: We demonstrated feasibility of analyzing breast microbiome from an RPFNA specimen. Additional investigation with modifications to technique and/or sample population is. needed to achieve adequate sequencing depth for characterization of breast microbiome. Lower depth of sequencing in breast samples is thought to reflect differences in microbial DNA quantity. We were unable to assess change in microbiome composition in breast or stool samples with omega-3 fatty acid supplementation due to small sample size. Citation Format: Lauren E Nye, Jennifer R Klemp, Kandy R Powers, Anne P O'Dea, Amy L Kreutzjans, Trina Metheny, Teresa A Phillips, Susan E Carlson, Bruce F Kimler, Carol J Fabian. Feasibility of microbiome analysis from random periareolar fine needle aspiration in premenopausal women at increased risk for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-17.

The inflammation-resolving and insulin-sensitizing properties of eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids have potential to augment effects of weight loss on breast cancer risk. In a feasibility study, 46 peri/postmenopausal women at increased risk for breast cancer with a body mass index (BMI) of 28 kg/m2 or greater were randomized to 3.25 g/day combined EPA and DHA (ω-3-FA) or placebo concomitantly with initiation of a weight-loss intervention. Forty-five women started the intervention. Study discontinuation for women randomized to ω-3-FA and initiating the weight-loss intervention was 9% at 6 months and thus satisfied our main endpoint, which was feasibility. Between baseline and 6 months significant change (P < 0.05) was observed in 12 of 25 serum metabolic markers associated with breast cancer risk for women randomized to ω-3-FA, but only four for those randomized to placebo. Weight loss (median of 10% for trial initiators and 12% for the 42 completing 6 months) had a significant impact on biomarker modulation. Median loss was similar for placebo (−11%) and ω-3-FA (−13%). No significant change between ω-3-FA and placebo was observed for individual biomarkers, likely due to sample size and effect of weight loss. Women randomized to ω-3-FA exhibiting more than 10% weight loss at 6 months showed greatest biomarker improvement including 6- and 12-month serum adiponectin, insulin, omentin, and C-reactive protein (CRP), and 12-month tissue adiponectin. Given the importance of a favorable adipokine profile in countering the prooncogenic effects of obesity, further evaluation of high-dose ω-3-FA during a weight-loss intervention in obese high-risk women should be considered. Prevention Relevance: This study examines biomarkers of response that may be modulated by omega-3 fatty acids when combined with a weight-loss intervention. While focused on obese, postmenopausal women at high risk for development of breast cancer, the findings are applicable to other cancers studied in clinical prevention trials.

We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was −1.8% in the SDG arm (P = 0.001) and −1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = −2.2%; P = 0.002) but not placebo (median = −1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.

Background: 11% of women developing breast cancer are pre-menopausal women of childbearing potential under the age of 45. Pregnancy and breast feeding provide long term protection for breast cancer when they occur at an early age. The reasons for protection are poorly understood but likely involve both changes in the immune microenvironment and ductal and lobular epithelial differentiation. This pilot study is addressing a potential prevention strategy in a population otherwise excluded from breast cancer prevention trials and not eligible for standard of care chemoprevention. Methods: Eligible individuals included pre-menopausal women ages 21-40 that were considering future pregnancy and are at high risk for breast cancer based on family history, prior precancerous biopsy, or 5-year Gail model risk estimate of ≥ 1.7% or 10-year Tyrer-Cuzick risk of 2x population risk as listed in the model. Participants were enrolled and baseline tissue collection included random periareolar fine-needle aspiration (RPFNA) of breast, as well as collection of blood, urine and stool. Women were asked to take two capsules of Omega-3-Acid Ethyl Esters daily (a total of 750 mg DHA and 930 mg EPA) for six months. Post-intervention visit included repeat tissue collection. If women were pregnant at time of post-intervention visit, RPFNA was not done. Baseline and post-intervention DHA Food Frequency Questionnaire (FFQ) and the BCPT Questionnaire were administered. The intervention length was shortened for some participants due to the study ending. Objectives: 1) To determine feasibility of a breast cancer prevention intervention study in this cohort of pre-menopausal women at high risk for breast cancer and considering future pregnancy, 2) measure compliance with the omega-3 fatty acid supplement in this population, 3) identify novel biomarkers modulated by moderate dose omega-3 fatty acids in this population. Results: Ten women were successfully enrolled at an average rate of 1.5/month from a single center high risk breast clinic. Of the ten women enrolled, median age was 33 years (range 22-37, ±5.04 stdev), 70% married, 80% Non-Hispanic White, 10% of Ashkenazi Jewish descent and 40% reported having a genetic mutation. Feasibility was achieved with 80% (8 out of 10) of participants returning for post-intervention visit. Reasons given for discontinuation of study were (n=1) side effect from supplement (bloating) and (n=1) scheduling conflicts. Of the eight women who completed the off study visit, two chose not to undergo the off study RPFNA due to discomfort with initial procedure or time commitment. Self-reported pill count showed an average of three missed pills/month. Grade 1 related adverse events reported included odor, nausea and flatulence. Post-intervention, more participants reported diarrhea, vaginal discharge and bleeding, weight gain, general aches and dizziness on BCPT items compared to baseline. Change in benign breast tissue biomarkers will be reported including breast tissue cytomorphology, Ki67, fatty acid analysis and selected gene expression. Conclusion: It is feasible to recruit premenopausal women considering future pregnancy to a breast cancer prevention trial with a minimally invasive sampling procedure. Results from this trial will inform a larger randomized prevention trial. Citation Format: Lauren E Nye, Jennifer R Klemp, Kandy R Powers, Anne P O'Dea, Kendra A Cruz, Amy L Kreutzjans, Trina Metheny, Teresa A Phillips, Susan E Carlson, Bruce F Kimler, Carol J Fabian. Feasibility study of moderate dose omega 3 fatty acid supplementation in premenopausal women at high risk for breast cancer considering future pregnancy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-14-01.

Background: FOXA1 is a pioneer transcription factor necessary for full expression of estrogen receptor activity. In collaboration with Grainyhead-like 2 (GRHL2), increased FOXA1 activity can result in endocrine resistance through increase of the endoplasmic reticulum protein AGR2 and its receptor LYPD3. Hyperglycemia and hyperinsulinemia increase FOXA1. Since eicosapentaenoic and docosahexaenoic omega-3 fatty acids (EPA and DHA) improve insulin sensitivity and reduce IGFR-related signaling, we explored the effect of EPA + DHA supplementation on FOXA1 and AGR2 expression in benign breast epithelium. Methods: Reserved excess cells from fine needle aspirations performed pre and post 6 months of supplementation given to postmenopausal women at increased risk for breast cancer were utilized. Cells had been stored at -20 C in a methanol fixative for ~10 y (Fabian et al, Ca Prev Res 2015). Of the 35 women entered, 28 had reserved excess cells available for blocks and after obtaining 4-5 sections per block, 12 had sufficient epithelial cells on both pre and post intervention slides as determined by H and E staining to proceed with immunohistochemistry assessment. Of the 12, median age was 53 and 7/12 were on low dose hormone replacement during the study. Slides were stained with FOXA1 (2F83 Mouse Monoclonal Antibody) at a 1:50 dilution and AGR2 rabbit polyclonal (Proteintech,12275-1-AP) at a 1:800 dilution. F0XA1 exhibits a nuclear staining pattern whereas AGR2 is predominately cytoplasmic. IHC staining was assessed individually by two readers (TM and TP). The most abnormal cell clusters were preferentially assessed. Up to 500 cells are evaluated for percent positive stain and staining intensity. An Allred score was computed as the sum of a categorical score for percent positive cells and a categorical score for the predominant staining intensity. Results: For FOXA1 there was a statistically significant decrease in percent positive cells (11/12, p=0.019) and Allred score (9/12, p=0.022) by Wilcoxon signed rank test. Of the 11 which showed a decrease in FOXA1, seven were on HRT throughout the study (usually estrogen alone). There was no statistically significant change in AGR2 IHC. However, there was a robust linear relationship between stain positivity for FOXA1 and AGR2 (p Conclusions: If this finding is confirmed in other studies, FOXA1 protein expression may be a useful tissue response biomarker in breast cancer prevention trials of omega-3 fatty acids. Citation Format: Carol J. Fabian, Trina Metheny, Teresa A. Phillips, Marsha Danley, Bruce F. Kimler. High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2207.

Objectives: The primary objective was to determine tolerability of ω-3 fatty acids (2150 mg of eicosapentaenoic acid (EPA) and 1050 docosahexaenoic acid (DHA) ethyl esters) vs placebo in women in undergoing a behavioral weight loss intervention (6 months loss and 6 months maintenance). Secondary objectives were to explore potential differences in modulation of blood and benign breast tissue risk biomarkers, satiety and quality of life indices, and weight loss. Results: 46 peri and postmenopausal women were randomized and 42 completed the 6 months of the weight loss intervention and were biomarker evaluable (22 placebo and 20 ω-3 FA). Median baseline BMI in the 42 evaluable women was 31 kg/m2 with a median 6-month relative weight loss of 11% and relative fat mass loss of 20% (DXA). Median 12-month relative mass loss was 10% in the 35 women completing 12 months of the intervention. ω-3 fatty acids increased the ratio of (EPA+DHA): arachidonic acid 2.6-fold (median, range 1.8 - 3.8) vs no change for placebo. There was no difference by randomization group in relative weight or fat mass loss at 6 or 12 months, grade 2 and 3 adverse events, early discontinuation, satiety or other quality of life measures. More serum biomarkers exhibited significant within-group improvement at 6 and 12 months for evaluable women randomized to ω-3 FA than to placebo. At 6 months, significant change (P10% weight (median 15%) showed significant within-group improvement in adiponectin, leptin, adiponectin:leptin ratio, insulin, lipocalin-2, resistin, PAI-1, HGF, CRP, SHBG, bioavailable estradiol and bioavailable testosterone. For women with 10% weight loss. Given the dramatic effect of weight loss on biomarkers, we examined within-group and between-group change from baseline to 6 and 12 months for the four subgroups (10-11 women in each) defined by ω-3 FA or placebo and < or > 10% weight loss at 6 months. The subgroup of >10% loss + ω-3 FA had the greatest within-group change in the proportion of significantly modulated biomarkers at 6 months. >10% loss + ω-3 FA was the only subgroup with a significant within-group increase in adiponectin at both 6 and 12 months and achievement of a beneficial ratio of adiponectin (ug/ml) to leptin (ng/ml) of > 1.0 in 100% of participants. There was a significant between-group effect for adiponectin for >10% loss + ω-3 FA vs each of the other groups. Biomarkers were assessed in tissue acquired by random periareolar fine needle aspiration (RPFNA). There were no significant differences in change in cytomorphology or Ki-67 between women randomized to ω-3 FA or placebo but there were significant within-group increases in benign breast adiponectin (pg/ug protein) at 12 months (p=0.014) for women randomized to ω-3 FA. Conclusions: EPA + DHA ethyl esters (3150 mg/day), added to a behavioral weight loss program in overweight women at increased risk for breast cancer, is well-tolerated and may further improve risk biomarker modulation. The increase in adiponectin when ω-3 FA is added to weight loss is of particular interest given that adiponectin opposes the oncogenic effect of leptin and is associated with improved insulin sensitivity and reduced mTOR signaling. Further study is warranted with enough subjects to detect between-group differences. Citation Format: Carol J Fabian, Christie A Befort, Debra K Sullivan, Susan E Carlson, Jennifer L Nydegger, Amy L Kreutzjans, Kandy R Powers, Teresa A. Phillips, Trina Metheny, Carola M Zalles, Erin D Giles, Stephen D Hursting, Bruce F Kimler. Randomized trial of 12 months of omega-3 fatty acids vs placebo during a weight loss intervention in post-menopausal women at increased risk for breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-02.

Interventions that relieve vasomotor symptoms while reducing risk for breast cancer would likely improve uptake of chemoprevention for perimenopausal and postmenopausal women. We conducted a pilot study with 6 months of the tissue selective estrogen complex bazedoxifene (20 mg) and conjugated estrogen (0.45 mg; Duavee) to assess feasibility and effects on risk biomarkers for postmenopausal breast cancer. Risk biomarkers included fully automated mammographic volumetric density (Volpara), benign breast tissue Ki-67 (MIB-1 immunochemistry), and serum levels of progesterone, IGF-1, and IGFBP3, bioavailable estradiol and testosterone. Twenty-eight perimenopausal and postmenopausal women at increased risk for breast cancer were enrolled: 13 in cohort A with baseline Ki-67 < 1% and 15 in cohort B with baseline Ki-67 of 1% to 4%. All completed the study with > 85% drug adherence. Significant changes in biomarkers, uncorrected for multiple comparisons, were a decrease in mammographic fibroglandular volume (P = 0.043); decreases in serum progesterone, bioavailable testosterone, and IGF-1 (P < 0.01), an increase in serum bioavailable estradiol (P < 0.001), and for women from cohort B a reduction in Ki-67 (P = 0.017). An improvement in median hot flash score from 15 at baseline to 0 at 6 months, and menopause-specific quality-of-life total, vasomotor, and sexual domain scores were also observed (P < 0.001). Given the favorable effects on risk biomarkers and patient reported outcomes, a placebo-controlled phase IIB trial is warranted.

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.

Breast cancer prevention strategies for young pre-menopausal women must have minimal side effects and accommodate potential future child-bearing. Based on epidemiologic evidence and informed by a single-arm pilot study, we conducted a multi-institutional, placebo-controlled Phase IIB trial of the lignan secoisolariciresinol diglycoside (SDG) found in high concentrations in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from pre-menopausal women at increased risk for breast cancer during the follicular phase as estimated by dates. Those with cytologic hyperplasia and ≥2% positive cells by Ki-67 immunocytochemistry were eligible for randomization 2:1 to daily Brevail® (50 mg SDG) or placebo. After 12 months, RPFNA and blood for hormone assays were repeated. The primary endpoint was difference in change in Ki-67 between the randomization groups. A planned accrual of 230 was to provide an 80% power of detecting a 2.5% reduction in Ki-67 for the SDG group vs no reduction in the placebo group. Accrual was slower than anticipated and the study was closed with 180 enrollees at five sites. 152 (51 placebo, 101 SDG) sets of paired specimens were evaluable for the primary endpoint. Baseline Ki-67 was a median of 4.1% (range, 2.0 - 26.8%), with no difference between arms (Mann-Whitney nonparametric test, p=0.34). Both arms showed a decrease in percent Ki-67 over time (Wilcoxon signed rank test; p=0.034 for placebo, p=0.001 for SDG). Although Ki-67 reduction was greater in the SDG arm (median of -1.8% vs -1.2%), there was no statistically significant difference between the two arms (Mann-Whitney, p=0.72). Since luteal phase progesterone affects proliferation, we excluded 35 women that by serum progesterone levels could not be confirmed to be in the same phase of the menstrual cycle at baseline and off-study. Analyzing the remaining 117 for Ki-67 (42 placebo, 75 SDG), there was no significant change for the placebo arm (Wilcoxon, p=0.14) but the significant change in the SDG arm persisted (p=0.002). As an exploratory analysis, assessment of gene expression was performed by RT-qPCR on 77 pairs of non-bloody RPFNA specimens. 22 had significant ERα gene expression changes (defined 2.0 fold changes). There was no significant change over time for the placebo group (7/10 increases, Wilcoxon, p=0.16), but there was significant change for the SDG group (10/12 decreases, p=0.027). There was also a significant difference between the groups (Mann-Whitney, p=0.018). While the primary trial result is null, there is supportive evidence SDG may favorably affect cell proliferation and estrogen signaling in premenopausal women at high risk for development of breast cancer. Supported by Susan G. Komen Promise Grant KG101039. Study agent (Brevail®, placebo) provided by Lignan Research Inc. (later Barlean's Oils) which was otherwise not involved in the design, conduct, or analysis of the study. Citation Format: Carol J. Fabian, Seema A. Khan, Judy E. Garber, William C. Dooley, Lisa D. Yee, Carola M. Zalles, Trina Metheny, Teresa A. Phillips, Jinxiang Hu, Brian K. Petroff, Stephen D. Hursting, Bruce F. Kimler. Randomized clinical trial of a flaxseed lignan in pre-menopausal women at high risk for development of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1120.

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.

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.

Background: We conducted a pilot study of high dose omega-3 fatty acid (FA) supplementation in post-menopausal women to determine if risk biomarkers for breast cancer in benign breast tissue sampled by random peri-areolar aspiration (RPFNA) could be favorably modulated and to acquire preliminary data on possible mechanism of action. Methods: 35 post-menopausal women at increased risk for breast cancer were accrued to a trial of 6-month intervention with 4 g daily of omega-3-acid ethyl esters [1.86 g eicosapentaenoic acid (EPA), 1.5 g docosahexaenoic acid (DHA)]. Subjects had RPFNA performed pre- and post-intervention and specimens evaluated for cytomorphology and proliferation (Ki-67). FA composition was determined in plasma, red blood cells, and RPFNA specimens. Additional specimens were frozen for assessment of hormones, a panel of 11 adipokines and cytokines by Luminex, and gene expression. Results: 34 subjects completed study with specimens evaluable for change in biomarkers. The ratio of (EPA+DHA):Arachidonic Acid (AA) levels in erythrocyte phospholipid increased significantly by a median of 2.7-fold. Although there was a significant decrease in blood EPA+DHA between discontinuation at 6 months and 2 weeks later when RPFNA was performed, all ratios were above the baseline value (median 1.6-fold). There was favorable but not statistically significant modulation for cytologic evidence of atypia (53% at baseline to 41% at off-study). However, favorable modulation was exhibited for Masood score (medians of 15 to 14; p = 0.014), number of epithelial cells recovered (p = 0.019) and Ki-67 expression (medians of 1.7% to 0.75%, p = 0.036, despite 8 subjects having no Ki-67 expression at baseline). Luminex assay of serum indicated a statistically significant increase (p = 0.003) for adiponectin and decrease (p = 0.016) for TNF-alpha between baseline and off-study. For RPFNA specimens, there was a significant decrease (P = 0.001) in MCP-1 levels adjusted for protein content. By ELISA, serum high molecular weight adiponection increased (p = 0.046) and molar ratio of IGF-1:IGFBP3 decreased (p = 0.006). Note that all analyses were exploratory and without correction for multiple analyses. Conclusion: Favorable modulation of a variety of blood and tissue risk biomarkers, including cytomorphology and proliferation, along with good tolerability suggests that high dose omega-3 FA esters should be tested further in a placebo-controlled trial. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-10-01.

Uptake of anti-hormonal agents for primary prevention of breast cancer is poor due to concern about side effects, especially induction of menopausal symptoms. A combination of 20 mg bazedoxifene plus 0.045 mg conjugated estrogen is FDA approved (as Duavee®) for treatment of hot flashes and prevention of osteoporosis in postmenopausal women with a uterus. We undertook a pilot study to assess the feasibility of using this formulation as a breast cancer prevention agent in women at increased risk for development of breast cancer. Feasibility was to be assessed by accrual, retention, and documentation of a lack of enhanced proliferation in benign breast tissue acquired by random periareolar fine needle aspiration (RPFNA). Eligibility criteria included risk >2X that of average risk woman for age group, postmenopausal having hot flashes or night sweats and not on systemic hormone replacement, and at least 500 cells on a baseline RPFNA. Women were ineligible if they had LCIS or DCIS, a BRCA1/2 germline mutation, had had a hysterectomy, or had >4% Ki-67 positive cells by immunocytochemistry. Fasting blood draw, digital mammography with Volpara software, and DXA scan for body composition was performed at baseline along with QOL questionnaires. Women then received Duavee® daily for 6 months, followed by repeat of baseline tests. We accrued the first 20 subjects in 14 months. Many of the women followed in our cohort and interested in the trial were not eligible due to prior hysterectomy, prior LCIS or a high penetrance gene mutation. Thus accrual was slower than anticipated. All women have reported improvement in hot flash frequency and intensity. None have discontinued prematurely or had a study related serious adverse event. Fourteen women have completed the 6-month intervention and are evaluable for modulation of biomarkers. There have been no protocol-defined increases in proliferation (to >2% Ki-67 for baseline Ki-67 Citation Format: Carol J. Fabian, Kandy R. Powers, Jennifer L. Nydegger, Amy L. Kreutzjans, Trina Metheny, Teresa A. Phillips, Lauren Nye, Carola M. Zalles, Bruce F. Kimler. Pilot study of the combination of bazedoxifene and conjugated estrogen to modulate risk biomarkers in women with hot flashes at increased risk for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3261.

Higher intakes of the omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) have been variably associated with reduced risk of premenopausal breast cancer. The purpose of this pilot trial was to assess feasibility and explore the effects of high-dose EPA and DHA on blood and benign breast tissue risk biomarkers before design of a placebo-controlled phase IIB trial. Premenopausal women with evidence of hyperplasia ± atypia by baseline random periareolar fine needle aspiration were given 1860 mg of EPA + 1500 mg of DHA ethyl esters daily for 6 months. Blood and benign breast tissue were sampled during the same menstrual cycle phase prestudy and a median of 3 weeks after last dose. Additional blood was obtained within 24 hours of last dose. Feasibility, which was predefined as 50% uptake, 85% retention, and 70% compliance, was demonstrated with 46% uptake, 94% completion, and 85% compliance. Cytologic atypia decreased from 77% to 38% (P = 0.002), and Ki-67 from a median of 2.1% to 1.0% (P = 0.021) with an increase in the ratio of EPA + DHA to AA in erythrocyte phospholipids but no change in blood hormones, adipokines, or cytokines. Exploratory breast proteomics assessment showed decreases in several proteins involved in hormone and cytokine signaling with mixed effects on those in the AKT/mTOR pathways. Further investigation of EPA plus DHA for breast cancer prevention in a placebo-controlled trial in premenopausal women is warranted. Cancer Prev Res; 8(10); 912–21. ©2015 AACR. See related article, p. 922.

Estrogen receptor (ER) expression in breast epithelial cells has potential as a risk marker for development of breast cancer and as a response marker for preventive interventions.The purpose of this study was to determine if ER expression in benign cytologic specimens acquired by random periareolar fine needle aspiration (RPFNA) increases with morphologic abnormality as has been reported for histologic preparations.ER expression was assessed in 122 women at high risk for development of breast cancer who had RPFNA hyperplasia +/- atypia and were being screened for entry into one of two chemoprevention trials. ER was assessed using antigen retrieval at 90 degrees C for 2 min and the DAKO ER monoclonal antibody (Clone number 1D5). The proportion of cells with definitive staining at each intensity level (0-4) was recorded as a percentage of the total cells counted, to give a weighted intensity score (IS).Of 122 women, 65% exhibited hyperplasia and 35% exhibited hyperplasia with atypia in their RPFNA specimens. A majority (66%) of subjects had at least 10% of ductal cells exhibiting nuclear staining for ER. Median percent of cells withor =1+ staining was 20% and the median ER IS was 0.23. There was a strong correlation between ER IS and percentage of ER positive cells (R(2) = 0.88). By univariate analysis ER IS was statistically significantly higher in women older than median age of 48 years (P = 0.025), in postmenopausal women on HRT (P0.017), and in women with a Masood cytomorphology index score ofor =14 (P = 0.005). On multivariable analysis, ER IS was significantly associated with postmenopausal status (P = 0.038) and cytomorphology as measured by Masood score (P = 0.043).ER can be readily measured in cytologic specimens obtained by RPFNA with the use of antigen retrieval method. Further, ER expression in cytologic specimens is influenced by postmenopausal status and morphologic abnormality.

Ductal 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.

We conducted a phase II feasibility study of a 6-month behavioral weight loss intervention in postmenopausal overweight and obese women at increased risk for breast cancer and the effects of weight loss on anthropomorphic, blood, and benign breast tissue biomarkers. 67 women were screened by random peri-areolar fine-needle aspiration, 27 were registered and 24 participated in the interventional phase. The 24 biomarker evaluable women had a median baseline BMI of 34.2 kg/m(2) and lost a median of 11 % of their initial weight. Significant tissue biomarker modulation after the 6-month intervention was noted for Ki-67 (if restricted to the 15 women with any Ki-67 at baseline, p = 0.041), adiponectin to leptin ratio (p = 0.003); and cyclin B1 (p = 0.001), phosphorylated retinoblastoma (p = 0.005), and ribosomal S6 (p = 0.004) proteins. Favorable modulation for serum markers was observed for sex hormone-binding globulin (p0.001), bioavailable estradiol (p0.001), bioavailable testosterone (p = 0.033), insulin (p = 0.018), adiponectin (p = 0.001), leptin (p0.001), the adiponectin to leptin ratio (p0.001), C-reactive protein (p = 0.002), and hepatocyte growth factor (p = 0.011). When subdivided by10 or10 % weight loss, change in percent total body and android (visceral) fat, physical activity, and the majority of the serum and tissue biomarkers were significantly modulated only for women with10 % weight loss from baseline. Some factors such as serum PAI-1 and breast tissue pS2 (estrogen-inducible gene) mRNA were not significantly modulated overall but were when considering only those with10 % weight loss. In conclusion, a median weight loss of 11 % over 6 months resulted in favorable modulation of a number of anthropomorphic, breast tissue and serum risk and mechanistic markers. Weight loss of 10 % or more should likely be the goal for breast cancer risk reduction studies in obese women.

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.

The objective of this study was to determine if 6 months of the aromatase inhibitor letrozole, administered to postmenopausal women taking a stable dose of hormone replacement remedy, would be safe and would modulate biomarkers of breast cancer risk. The intent was to reduce the proliferation marker Ki-67 while maintaining adequate systemic levels of estradiol so as to avoid perimenopausal symptoms. Postmenopausal women at high risk for development of breast cancer and taking a stable dose of estrogen or estrogen plus progestin were screened by random periareolar fine needle aspiration (RPFNA). To be eligible, the acquired breast epithelial cells had to be characterized as cytologic atypia or borderline atypia withor =1,000 epithelial cells on the cytomorphology slide; plusor =500 epithelial cells on a slide processed for Ki-67 immunocytochemistry. Forty-two women were enrolled in the one arm study and received 2.5 mg letrozole per day for 6 months, followed by repeat assessment of biomarkers. Ki-67 was reduced by a median relative value of 66%. There was no significant change in breast cell cytomorphology; ER weighted index score; serum estradiol, testosterone, or IGF-1:IGFBP-3 ratio; mammographic breast density, or frequency or severity of perimenopausal symptoms. Given the dramatic reduction in proliferation, the effect of letrozole on risk and response biomarkers should be explored further in a randomized, placebo-controlled Phase IIB breast cancer chemoprevention trial.

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 degrees or 115 degrees 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

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

1515 Background: We conducted a pilot study of high dose omega-3 fatty acid (FA) supplementation in pre-menopausal women to determine if risk biomarkers for breast cancer in benign breast tissue sampled by random peri-areolar aspiration (RPFNA) could be favorably modulated and to acquire preliminary data on possible mechanism of action. Methods: 36 pre-menopausal women at increased risk for breast cancer were accrued to a trial of 6-month intervention with 4 g daily of omega-3-acid ethyl esters [1.86 g eicosapentaenoic acid (EPA), 1.5 g docosahexaenoic acid (DHA)]. To date, 31 subjects have completed study with RPFNA performed pre- and post-intervention in the follicular phase of the menstrual cycle and specimens evaluated for cytomorphology and proliferation (Ki-67). FA composition was determined in plasma, red blood cells, and RPFNA specimens. Additional specimens were frozen for assessment of hormones, a panel of 11 adipokines and cytokines, and gene expression. Results: The ratio of (EPA+DHA):Arachidonic Acid (AA) levels increased significantly in plasma and erythrocytes by a median of three-fold. There was a significant decrease in blood EPA+DHA between discontinuation at 6 months and 2 weeks later when RPFNA was performed. Despite that, there was favorable modulation for cytologic evidence of atypia (81% at baseline to 42% at off-study; p=0.003), Masood score (medians of 15 to 14; p=0.001), number of epithelial cells recovered (p=0.004) and Ki-67 expression (p=0.025 for 30 women with any Ki-67 at baseline, medians of 1.9% to 0.9%). Serum assays have been completed for 24 subjects. No statistically significant changes were observed for estradiol, testosterone, progesterone, SHBG, IGF-1, IGFBP-3. There was a trend (p=0.056) towards a decrease in resistin. To date, only two subjects have discontinued the study early; grade 2 or greater gastrointestinal side effects have been reported by only two subjects. Conclusions: Favorable modulation of tissue risk biomarkers, cytologic atypia and proliferation along with good tolerability suggests that high dose omega-3 FA esters should be tested further in a placebo controlled trial. Clinical trial information: NCT01252277.

520 Background: Selective Estrogen Receptor Modulators (SERMs) are approved for reduction of risk for breast cancer; however, uptake and use is limited. We conducted a pilot study of a 4th generation SERM to determine tolerability and effect on tissue biomarkers in healthy women at high risk for development of breast cancer. Methods: Premenopausal women at elevated risk for breast cancer were screened by random periareolar fine needle aspiration (RPFNA) performed during the follicular phase of the menstrual cycle. Women were eligible if breast epithelial cells exhibited evidence of cytologic hyperplasia with or without atypia, as well as Ki-67 ≥2% by immunocytochemistry. Following 6-8 months of open-label acolbifene (20 mg/d), the RPFNA was repeated. The primary endpoint was modulation of the proportion of cells that expressed Ki-67. Body composition (DEXA), pelvic sonography, mammographic breast density, and serum levels of IGF-1/IGFBP3 and several bioavailable hormones were assessed pre and post intervention. Results: 76 women were screened by RPFNA, with 25 eligible and enrolled in the intervention over a 9 month period. All 25 (7 on oral contraceptives) subjects completed the study, had a second RPFNA, and were evaluable. Median Ki-67 at baseline was 4.6% (range 2.4 – 21.9%) and off study 1.4% (range 0 – 6.6%); median change was a reduction of 3.0% (range -20.2% to +2.8%; decreased in 23, increased in 2) or a relative reduction of 77%. The end-of-study Ki-67 was significantly less than baseline (p

1507 Background: The lignans enterolactone and enterodiol are derived from the action of gut bacteria on ingested secoisolariciresinol diglycoside (SDG) which is commonly found in flaxseed. Enterolactone and enterodiol are thought to impair mammary carcinogenesis via reduction in aromatase activity and the mid-cycle surge of luteinizing hormone. We assessed the modulatory activity of 1 year of SDG on a number of risk biomarkers for breast cancer in a prospective phase II pilot study. We report the effect of SDG on the primary endpoint, proliferation in benign breast tissue as measured by Ki-67 immunocytochemistry, in the first 35 women completing study. Methods: Premenopausal women age 21 to 55 at increased risk for breast cancer underwent a baseline random periareolar fine needle aspiration (RPFNA) between the first and tenth days of their menstrual cycle. Those with RPFNA evidence of hyperplasia and Ki-67 greater than or equal to 2% were invited to participate. Women taking flaxseed or oral contraceptives were ineligible. All women took one Brevail (lignan research) capsule containing 50 mg of SDG daily. Ki-67 staining was performed with DAKO M7240 antibody on hematoxylin counterstained slides and the number of cells staining positive in 500 cells within hyperplastic clusters was counted. Results: Forty-nine women were enrolled on study between February 2006 and June 2008. Of these, four stopped prematurely, 10 women have not completed, and 35 have completed study and undergone follow-up RPFNA. Baseline characteristics of the 35 women completing study are as follows: median age 44 (range 29–50), median baseline 5-year Gail model risk 1.6% (range 0.1%-5.7%), median Ki-67 4.2% (range 2.0%-16.8%). Thirty seven percent had hyperplasia without atypia, and 63% had atypia. At repeat RPFNA, Ki-67 expression was reduced (median value of 2.0%, range 0%-15.2%); with 29 of the 35 women demonstrating a decrease (median relative reduction of 0.70). Conclusions: Based upon reduction in Ki-67 expression in hyperplastic benign breast tissue after 12 months, 50 mg of SDG administered daily as Brevail appears promising as a preventive. Supported in part by grant R21 CA117847 from the National Cancer Institute. No significant financial relationships to disclose.