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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.

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 anthropomorphic variables. Supplementary Table 3: Changes in levels of cytokines and adipokines in RPFNA specimens, by Luminex. Supplementary Table 4: Changes in levels of mRNA in RPFNA specimens, by RT-qPCR. Supplementary Table 5: 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.

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.

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

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.

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, p5000 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%, p5000 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.

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.

We sought to demonstrate that older, sedentary, obese breast cancer survivors could achieve > 200 minutes per week of moderate to vigorous physical activity (MVI PA) as part of a weight loss intervention; and to assess modulation of risk biomarkers. This level of PA in combination with moderate calorie restriction is associated with weight losses of >10% in women without cancer, which in turn is associated with significant modulation of cancer risk biomarkers. Eleven participants with BMI > 30 kg/m2 enrolled in a 12-week program that consisted of moderate caloric restriction, weekly phone group behavioral sessions, and individualized exercise plans based on measured heart rate reserve. Women were provided an accelerometer with heart rate monitor linked to GarminConnect, membership to a YMCA, twice weekly supervised exercise sessions with a personal trainer, and weekly feedback regarding weight and physical activity progress. The goal was to increase MVI PA (≥45% heart rate reserve) gradually from 200 minutes per week. The median age was 61, 5/11 women had received prior chemotherapy, and 7/11 were currently taking aromatase inhibitors. Median values of baseline anthropomorphic measures acquired by dual energy x-ray absorptiometry (GE Lunar iDXA) included BMI, 37.3 kg/m2; total mass, 97.5 kg; fat mass, 47.6 kg; visceral fat, 1.7 kg (range 1.4-3.0); and fat mass index, 17.6 kg/m2. The majority had a baseline VO2 peak in the poor range for their age. All 11 participants completed the intervention, with no reported serious adverse events. Median MVI PA achieved over weeks 5-12 was 161 minutes/week (range 48-320). VO2 peak was increased in 10/11 with a median relative change of 12% from baseline. All but one lost weight with an overall median of 8% total mass loss, which was associated with 13% total fat mass loss and 21% visceral fat mass loss. For those with MVI PA above the median, values were 11%, 17%, and 40%, respectively. Visceral fat mass loss was linearly correlated with minutes per week of MVI PA (p=0.032); these parameters in turn were associated with changes in a number of serum biomarkers, including adiponectin-leptin ratio, TNF-alpha, as well as circulating adipose stromal cells, a potential marker for metastasis. Insulin and hs-CRP were favorably modulated in almost all participants but change was not linearly correlated with activity or mass loss parameters; thus these may not be ideal biomarkers to document a dose response to level of MVI PA. Conclusion: These results demonstrate that older, sedentary, obese breast cancer survivors can safely achieve a high level of MVI PA when provided a structured program that includes an exercise trainer. It is feasible to design a clinical trial for such breast cancer survivors to examine biomarker modulation as a function of level of physical activity. Citation Format: Fabian CJ, Klemp JR, Burns JM, Vidoni ED, Nydegger JL, Kreutzjans AL, Phillips TL, Baker HA, Hendry B, John C, Amin AL, Khan QJ, Mitchell MP, O'Dea AP, Sharma P, Wagner JL, Hursting SD, Kimler BF. Feasibility and biomarker modulation due to high levels of moderate to vigorous physical activity as part of a weight loss intervention in older, sedentary, obese breast cancer survivors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-12-11.

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.

Simple Summary Aerobic exercise reduces risk for developing breast cancer or for breast cancer recurrence. In obese women exercise can significantly augment the effects of caloric restriction on visceral fat, reducing metabolic abnormalities and cancer. Women who are older, obese, and sedentary, especially those who have been treated for breast cancer, find it difficult to initiate and achieve the minimum or optimum levels of exercise. In a two-part pilot we found that by providing older, obese, sedentary breast cancer survivors 12 weeks of twice weekly personal training sessions, they could safely increase exercise to ≥200 min/week by 9 weeks during caloric restriction. At 24 weeks, high levels of exercise were still observed with continued behavioral support and study-provided exercise facility. Substantial improvement in visceral fat and breast cancer risk biomarkers were observed with this affordable intervention that is readily exportable to the community. Abstract Aerobic exercise reduces risk for breast cancer and recurrence and promotes visceral adipose tissue (VAT) loss in obesity. However, few breast cancer survivors achieve recommended levels of moderate to vigorous physical activity (MVPA) without supervision. In a two-cohort study, feasibility of 12 weeks of partially supervised exercise was started concomitantly with caloric restriction and effects on body composition and systemic risk biomarkers were explored. In total, 22 obese postmenopausal sedentary women (including 18 breast cancer survivors) with median age of 60 and BMI of 37 kg/m2 were enrolled. Using personal trainers twice weekly at area YMCAs, MVPA was escalated to ≥200 min/week over 9 weeks. For cohort 2, maintenance of effect was assessed when study provided trainer services were stopped but monitoring, group counseling sessions, and access to the exercise facility were continued. Median post-escalation MVPA was 219 min/week with median 12-week mass and VAT loss of 8 and 19%. MVPA was associated with VAT loss which was associated with improved adiponectin:leptin ratio. In total, 9/11 of cohort-2 women continued the behavioral intervention for another 12 weeks without trainers. High MVPA continued with median 24-week mass and VAT loss of 12 and 29%. This intervention should be further studied in obese sedentary women.

Background Change in the relative composition of the gut microbiome at the phyla level, particularly decreases in Bacteroidetes and increases in Firmicutes species, has been associated with both obesity and increased risk for breast cancer. It is unclear how rapidly the microbiome changes in response to a reduced calorie and fat diet during a weight loss intervention. As a planned sub-study of a clinical trial with a structured behavioral weight loss intervention with randomization to high dose omega-3 fatty acids or placebo (NCT02101970; clinical trials.gov), we evaluated changes in the gut microbiome after 2 weeks of dietary intervention. Methods 46 post-menopausal women at increased risk for breast cancer with a BMI > 27 kg/m2 had a baseline 3 day food record, DXA, and blood and breast tissue sampling for biomarkers. They were then started on a reduced fat and calorie diet (~1200 kcal/day from 2 portion-controlled entrees, 3 low calorie high protein shakes, and 5 servings of fruits/vegetables daily), recommendation to exercise 225 minutes per week, and a weekly behavioral intervention. Fecal samples were collected at baseline, after 2 weeks of diet but prior to study agent, and after 6 months of weight loss intervention. Stool samples were stored at -20°C until brought to the clinic, and then at -80°C until DNA extraction. Bacterial taxonomic classification was performed using real-time PCR and 16S pyrosequencing using specific 16S rRNA primers. Baseline Healthy Eating Index (HEI) was calculated from the 3 day food record; fruit and vegetable servings were obtained from weekly food logs. Results 42 women completed the 6 month weight loss intervention. At baseline, median BMI was 31.0 kg/m2 and HEI was 58 (range 28-90) with 12 and 23 servings of fruits and vegetables per week. Median relative weight loss at 6 months was -11.9 % (0 to -22.7 %). When dichotomized to relative losses of 10% (which we have previously shown to be associated with significant improvement in blood and breast tissue risk biomarkers [Fabian Cancer Prev Res 2013]), women with 6 month >10% loss had favorable change in the two major stool phyla at 2 weeks with a median 10% increase for Bacteroidetes and 8% decrease for Firmicutes. Conversely, women with 10% loss group had higher baseline consumption of vegetables and continued this after starting the diet. The more adherent a woman was to dietary recommendations in the first weeks of dietary intervention, the more likely she was to lose >10% weight by 6 months. Conclusions Favorable modulation of the gut microbiome early in a weight loss intervention is associated with subsequent substantial weight loss. Microbiome assessment after 6 months of weight loss intervention is in progress. Supported by a grant from the Breast Cancer Research Foundation and pilot funds from National Cancer Institute Cancer Center Support Grant P30 CA168524. Citation Format: Fabian CJ, Kimler BF, Umar S, Ahmed I, Befort CA, Nydegger JL, Kreutzjans AL, Powers KR, Klemp JR, Spaeth KR, Sullivan DK. Changes in the gut microbiome of post-menopausal women 2 weeks after initiating a structured weight loss intervention [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-13-03.

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.

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.

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.

Background Obesity is a modifiable risk factor for breast cancer in the United States. White adipose tissue is increased in obese (BMI ≥ 30 kg/m2) women, releasing estrogens and pro-inflammatory cytokines. Adipose stromal cells play a key role in releasing estrogens, while circulating adipose stromal cells (CASCs, characterized as CD34+CD31-CD45-) home to tumor sites and promote angiogenesis and vascularization. CASCs have been correlated with BMI in both cancer and non-cancer patients. However, BMI alone as a marker of adiposity has its limitations. We examined whether CASCs correlate with additional measures of adiposity in women who are at high risk for development of breast cancer. Methods Women at high risk for development of breast cancer but without prior breast cancer were recruited for random periareolar fine needle aspiration (RPFNA), DEXA body composition, anthropomorphic assessment, and non-fasting venous blood collection. Mononuclear cells were isolated and the frequency of CD34brightCD31-CD45- cells was assessed by flow cytometry. Results CASC frequency ranged from 0 to 0.018% (median 0.001%) for 13 non-obese and 20 obese women. There was no association between CASC frequency and BMI (range 19 - 46 kg/m2), either as a linear correlation or when dichotomized at a BMI of 30 kg/m2. However, there were tendencies for greater CASC frequencies in pre-menopausal women, women with greater waist circumference (p Conclusions This is the first study to investigate CASC frequency in a cohort of women at high-risk for development of breast cancer. Our results to date do not show an association between CASC frequency and obesity (BMI). However, associations with other indices of visceral fat suggest that evaluation of circulating adipose stromal cells could have value as a biomarker of response in clinical trials of obese breast cancer survivors and high risk women undergoing weight loss (especially fat mass) interventions. Citation Format: Hailey Baker, Amy L. Kreutzjans, Jennifer L. Nydegger, Teresa L. Phillips, Richard C. Hasting, Dan A. Dixon, Bruce F. Kimler, Carol J. Fabian. Circulating adipose stromal cells (CASCs) as a potential biomarker of response to weight loss interventions in obese women at high risk for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3255. doi:10.1158/1538-7445.AM2017-3255

Background Obesity is a modifiable risk factor for breast cancer in the United States. While structured interventions can achieve short-term weight loss, this does not necessarily correlate with favorable modulation of risk biomarkers at the blood or breast tissue level. We combined a proven intervention (calorie-controlled meals, moderate exercise, and weekly group behavioral session) with daily omega-3 fatty acid supplementation to examine whether we could improve either weight loss and/or biomarker modulation in women who are at high risk for development of breast cancer. Methods 46 post-menopausal high-risk women with a BMI > 27 kg/m2 (median 31 kg/m2) had baseline blood collections and random periareolar fine needle aspiration (RPFNA) benign breast tissue sampling for biomarkers. Two weeks after starting the weight loss intervention, subjects received study agent, randomized 1:1 to placebo vs. supplementation with 2100 mg Eicosapentaenoic Acid (EPA) + 1050 mg Docosahexaenoic Acid (DHA) daily. After 6 months, blood and tissue sampling were repeated. Results 42 women completed the 6 month weight loss intervention with all but one achieving a weight loss (median relative weight loss 12%; range 0 to 23%). For the entire cohort, there were substantial and favorable changes in levels in serum of blood collected fasting for adiponectin (increase; p10%, women with >10% loss had greater favorable modulations for leptin and the A:L ratio for fasting and non-fasting serum ( Citation Format: Bruce F. Kimler, Jennifer L. Nydegger, Amy L. Kreutzjans, Teresa L. Phillips, Kandy R. Powers, Jennifer R. Klemp, Debra K. Sullivan, Christie A. Befort, Susan E. Carlson, Stephen D. Hursting, Carol J. Fabian. Modulation of adiponectin and leptin levels in obese post-menopausal women after 6 months of a structured weight loss intervention, randomized to placebo or supplemental omega-3 fatty acids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4230. doi:10.1158/1538-7445.AM2017-4230

1521 Background: In 2000 we published our initial observation from a high risk cohort of 480 women that cytologic evidence of hyperplasia with atypia in tissue obtained by random periareolar fine n...