Figure S1: Performance of a minimal 5-gene marker panel.

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.

Table S1: DNA methylation in rFNA samples Table S2: Association between methylation and menopause as measured by LH and FSH- Univariate analysis Table S3: Association between Methylation and Menstrual Cycle as Measured by Estradiol and Progesterone - Univariate Analysis Table S4: Association between DNA methylation and lifetime Gail risk score - multivariate analysis Table S5: Association between DNA Methylation and percent breast density Table S6: Association between DNA Methylation and Masood cytology score Table S7: Gene methylation versus standard cytology Table S8: Spearman correlation: relationship between biomarkers within the panel Table S9: Agreement between baseline and 6 month methylation levels in rFNA among individuals (n=15).

PDF file - 44K, Hormone and protein concentrations in nipple aspiration fluid (NAF) samples

PDF file - 42K, Changes in cellular parameters for equol producers compared to control women, shown as the differences in post-intervention and baseline values

Supplementary Figures and Tables

PDF file - 65K, The correlation between Ct values of unamplified cDNA (assayed by qRT-PCR) and post-amplification samples (using TLDA) was examined in seven RNA samples with sufficient amount available for both methods.

Supplemental Tables 1-5. Supplemental Table 1: comparison of fatty acid composition by cytologic atypia or not. Supplemental Table 2: Comparison of fatty acid composition of plasma phospholipids (PLs) by evidence of cytology atypia. Supplemental Table 3: Comparison of fatty acid composition of plasma triacylglycerides (TAGs) by evidence of cytology atypia. Supplemental Table 4: Comparison of fatty acid composition of breast phospholipids (PLs) by evidence of cytology atypia. Supplemental Table 5: Comparison of fatty acid composition of breast triacylglycerides (TAGs) by evidence of cytology atypia.

Table S1. Correlations between parameters that influence the quality of the rFNA sample (119 women) Table S2. Correlation between Breast and serum hormone concentrations. Table S3. Cytology according to menopausal status and menstrual cycle.

PDF file - 45K, Adverse events in soy and placebo groups

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% or >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. -

Introduction: In 2000 we published initial observations from a high-risk cohort of 480 women that cytologic evidence of hyperplasia with atypia in benign breast tissue obtained by random periareolar fine-needle aspiration (RPFNA) was associated with a five-fold increased risk of developing DCIS or invasive breast cancer at a median follow-up of 45 months [Fabian, JNCI 2000]. Few women in the initial cohort had any exposure to prevention agents, as NSABP-P1 was not reported until 1998. We began a new high-risk cohort in 2002 as the tissue processing changed from a filter technique to Thin PrepTM. The superior nuclear detail permitted assessment of cytologic atypia in the absence of high cellularity. Women in the second cohort were told that RPFNA atypia was a risk factor for developing breast cancer and in addition to standard prevention options were offered participation in clinical trials, if applicable. The purpose of this subsequent analysis was to determine if the predictive value of RPFNA atypia was maintained with change in tissue processing and availability of prevention options. Methods: A total of 1,135 high-risk women, eligible on the basis of family history, BRCA1/2 mutation status, prior biopsy indicating LCIS or atypical hyperplasia (AH), prior contralateral breast cancer and/or high mammographic breast density, underwent baseline RPFNA and were enrolled in our second cohort between 2002 and 2015. As in the earlier study published in 2000, if women had 2 aspirations without an intervening intervention within 21 months the worse result was utilized as baseline. In addition to cytomorphology, breast tissue was also categorized by cellularity of the aspirate (10-100, 100-1000, 1000-5000, and >5000 cells per cytology slide). The primary aspirator (CJF) and cytopathologist (CMZ) were the same for both cohorts. Women were censored at the time of prophylactic mastectomy, development of other site cancer, or death. Kaplan-Meier hazard plots and Cox-regression analysis were used to analyze time to development of DCIS or invasive breast cancer and effect of joint variables, respectively. Results: At a median follow-up of 86 months, 79 cases of DCIS or invasive breast cancer had been diagnosed. By univariate analysis, RPFNA atypia at entry was not predictive of subsequent breast cancer (p=0.58; log-rank test). However, breast cancer risk was increased by a prior breast biopsy with LCIS or AH (HR 2.6, 95% CI 1.6-2.4, p<0.001) or by high RPFNA cellularity, defined as >5000 epithelial cells per RPFNA cytomorphology slide (HR 2.1, 95% CI 1.3-3.5, p=0.0034). Thirty-six percent of women subsequently chose to undergo a prevention intervention, including a standard drug, enrollment in a prevention clinical trial of 6-12 months duration, or a prophylactic bilateral salpingo-oophorectomy (BSO) prior to age 45. Prevention interventions were more prevalent in women with RPFNA atypia (49% vs 30%, p<0.001). Not only was uptake of a prevention intervention associated with reduced breast cancer risk on univariate analysis (p=0.043), it was retained on multivariate analysis. The final Cox Regression risk model included prior breast biopsy with LCIS or AH (HR 2.4, 95% CI 1.5-3.9; p<0.001), high RPFNA cellularity (HR 2.2, 95% CI 1.3-3.7; p=0.003) and prevention intervention (HR 0.54, 95% CI 0.33-0.89; p=0.015). Conclusions: In a second large cohort of high-risk women for whom RPFNA acquired cells were processed to slides via ThinPrep™, high RPFNA cellularity (>5000 epithelial cells/slide) but not cytologic atypia predicted short-term breast cancer risk. However, women with atypia were significantly more likely than women without atypia to participate in a prevention intervention, which in turn was associated with reduced breast cancer risk Citation Format: Whitney L Hensing, Carol J Fabian, Carola M Zalles, Priyanka Sharma, Amy L Kreutzjans, Kandy R Powers, Lynn Chollet-Hilton, Bruce F Kimler. Random periareolar fine-needle aspiration (RPFNA) cell number, prior precancerous breast disease and subsequent uptake of a prevention intervention predict short-term breast cancer risk [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 P2-10-02.

AbstractThe 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.

Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Abstract 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 (P<0.05) was observed for adiponectin, leptin, adiponectin:leptin ratio, insulin, lipocalin-2, resistin, PAI-1, HGF, CRP, SHBG, and bioavailable testosterone in women randomized to ω-3 FA but only for leptin, adiponectin: leptin ratio and SHBG in those randomized to placebo. At 6 months, the 21 women who lost >10% 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 (median 6%) there was significant within-group improvement only for leptin, the adiponectin: leptin ratio, and SHBG. Little change was observed for inflammatory cytokines IL-6, TNF-alpha, MCP-1 or FABP4, or FGF-21 with ω-3 FA or >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.

AbstractMethods to determine individualized breast cancer risk lack sufficient sensitivity to select women most likely to benefit from preventive strategies. Alterations in DNA methylation occur early in breast cancer. We hypothesized that cancer-specific methylation markers could enhance breast cancer risk assessment. We evaluated 380 women without a history of breast cancer. We determined their menopausal status or menstrual cycle phase, risk of developing breast cancer (Gail model), and breast density and obtained random fine-needle aspiration (rFNA) samples for assessment of cytopathology and cumulative methylation index (CMI). Eight methylated gene markers were identified through whole-genome methylation analysis and included novel and previously established breast cancer detection genes. We performed correlative and multivariate linear regression analyses to evaluate DNA methylation of a gene panel as a function of clinical factors associated with breast cancer risk. CMI and individual gene methylation were independent of age, menopausal status or menstrual phase, lifetime Gail risk score, and breast density. CMI and individual gene methylation for the eight genes increased significantly (P < 0.001) with increasing cytological atypia. The findings were verified with multivariate analyses correcting for age, log (Gail), log (percent density), rFNA cell number, and body mass index. Our results demonstrate a significant association between cytological atypia and high CMI, which does not vary with menstrual phase or menopause and is independent of Gail risk and mammographic density. Thus, CMI is an excellent candidate breast cancer risk biomarker, warranting larger prospective studies to establish its utility for cancer risk assessment. Cancer Prev Res; 9(8); 673–82. ©2016 AACR.

Abstract 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 (<0.5 or >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.

Abstract The United States is increasingly racially and ethnically diverse. In fact, California is now a majority-minority region, with a greater percentage of its population comprised of racial minorities than whites. Yet, minorities are continually under-represented in clinical research trials, which provide crucial information on which the future of cancer treatments is built. Without representative inclusion of participants of color in clinical research, we cannot develop effective preventative and treatment approaches for everyone. This current study investigates the factors, including characteristics of study consenters, that may influence women—particularly women of color (WOC)—to accept or decline participation in breast cancer-related trials. We assess these factors through a brief survey, administered to patients immediately after they were invited to participate in a breast cancer-related clinical study. From the beginning of study accrual to the present, twenty-three patients have taken the survey. We anticipate accruing 200 participants at a rate of 25 per month. For the preliminary analyses, we split participants in two groups: white women (WW) (n = 14) and women of color (WOC) (n = 9). We conducted independent sample t-tests to compare the responses of WW and WOC. More WOC (M = 1.44, SD = 0.73) reported that it is important that their consenter is of the same ethnicity or race than WW (M = 1.00, SD = .00), t (21) = -2.32, p < .05. Similarly, WOC (M = 1.44, SD = .73) also reported that it is important that the person inviting them to participate in research look like people in their community, compared to the importance placed on this factor by WW (M = 1.00, SD = .00), t (21) = -2.32, p < .05). More WOC (M = 2.33, SD = 1.23) also cited “feeling overwhelmed” with their medical condition as influential in their decision to participate in clinical research than WW (M = 1.31, SD = .48), t (20) = -2.75, p < .05. Although both groups positively rated their interaction with the consenter, we observed marginal differences between WOC and WW. WOC (M = 7.00, SD = .00) gave higher ratings to the variable of “consenter created an atmosphere of trust and support” compared to ratings given by WW (M = 6.29, SD = 1.07), t (21) = -1.99, p = .06. Though participants are generally satisfied with their consenter interaction, different factors influence WW and WOC as they decide whether to participate in clinical research. When identified, these factors can be used to inform more inclusive consenting processes. Citation Format: Noe R. Chavez, Alan Nunez, Angela K. Wong, Tanya A. Chavez, Ellen Rippberger, Christine Thai, Angelica Sanchez, Ombeni M. Idassi, Krista M. Round, Kendall Kennedy, Margarita Robles, Jackelyn A. Alva-Ornelas, Jerneja Tomsic, Chidimma M.K. Kalu, Laura L. Kruper, Veronica C. Jones, Sharon Clancy, Amy C. Polverini, Courtney Vito, Karen Harold, Terry Hyslop, Carola M. Zalles, Daniel B. Schmolze, Christopher Sistrunk, Victoria L. Seewaldt. Influencing women's attitudes toward participation in breast cancer clinical research: Improving inclusion of women of color [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A082.