Random Periareolar Fine Needle Aspiration Research Articles

Abstract P2-11-17: Feasibility of microbiome analysis from random periareolar fine needle aspiration in premenopausal women at increased risk for breast cancer

Abstract 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.

Abstract P2-10-02: 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 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.

Abstract P1-10-04: AGR2, an estrogen response gene associated with tamoxifen resistance, is modulated by acolbifene in premenopausal women at high risk for development of breast cancer

Abstract Anterior Gradient 2 is an estrogen early response gene (AGR2) and protein (AGR-2). High expression is associated with endoplasmic reticulum stress and endocrine therapy resistance. Developmentally, AGR2 is associated with estrogen-mediated epithelial proliferation and lobuloalveolar development. In the adult, AGR2 is upregulated by inflammatory and metabolic stress signaling from NFKB, HSP90, IGFR-1, and FOXA1, in addition to estrogen bound to its receptor. The Selective Estrogen Receptor Modulator (SERM) Full dose tamoxifen has been reported to upregulate AGR2 in breast cancer but Selective Estrogen Receptor Down Regulators (SERDs) and aromatase inhibitors do not. AGR-2 is also a secreted protein, with increased tumor levels associated with metastases and decreased disease-free survival in tamoxifen-treated patients. We assessed AGR2 expression in reserved benign breast tissue from baseline and off-study specimens acquired by random periareolar fine needle aspiration (RPFNA) of premenopausal women at high risk for development of breast cancer who had participated in an early phase prevention trial of the SERM acolbifene (20 mg daily for 6 months). This pilot had demonstrated reduced Ki-67 and down-regulation of several estrogen response genes (Fabian et al. Ca Prev Res 2015). Methods Total RNA was extracted from aliquots of frozen benign breast tissue using TRIzol LS (Life Technologies) and purified using RNeasy MinElute Cleanup Kit (Qiagen). RNA was amplified using MessageAmpII aRNA amplification kit (Life Technologies) and reverse transcribed to cDNA using SMARTScribe Reverse Transcriptase (Takara Bio USA, Inc.) and random nonamer primers. Real-time quantitative PCR (qPCR) was performed using hydrolysis probes with baseline and postintervention specimens assessed together. PCR reactions were run on an Applied Biosystems Prism 7000 Sequence Detection System. The cycle threshold mean value for each transcript (from duplicate assays per specimen) was normalized using three reference transcripts, plus two epithelial cell transcripts. Relative levels of each transcript were calculated using the ΔΔCt method. The ratio (postintervention: baseline) of final values indicated upregulation (value > 1) or downregulation (value < 1). Results There were 17 available paired specimens for analysis for AGR2 gene expression which was downregulated in 13 with nine by more than a 50% reduction; for the four cases of up-regulation, three were increased by more than two-fold (p=0.076, Wilcoxon signed ranks test). Several estrogen response genes (TFF1, PGR, and the ratio of ESR1:ESR2) exhibited significant down-regulation (p≤0.007). Further, there was no evidence of linear correlation (p>0.28; Spearman’s rho) between change in AGR2 and any of these other estrogen response genes. Lastly, when an Estrogen Response Gene Index (ERGI) was constructed using the average log2(fold change) for TFF1, PGR, and the ratio of ESR1:ESR2, down regulation was observed in 16 of the 17 paired specimens with only one increase. ERGI and AGR2 change indicate favorable modulation by acolbifene and appear to provide independent information. Conclusions These results suggest that change in expression of benign breast AGR2 might be a useful addition to change in expression of other classic estrogen response genes in evaluating the potential of novel SERMs such as acolbifene compared to standard SERMs like tamoxifen in early phase prevention trials. Citation Format: Carol J. Fabian, Teresa A. Phillips, Bruce F. Kimler. AGR2, an estrogen response gene associated with tamoxifen resistance, is modulated by acolbifene in premenopausal women at high risk for development of 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 P1-10-04.

Abstract PD11-02: 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 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.

Abstract 1120: Randomized clinical trial of a flaxseed lignan in pre-menopausal women at high risk for development of breast cancer

Abstract 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 <0.5 or >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.

Abstract P1-14-01: Feasibility study of moderate dose omega 3 fatty acid supplementation in premenopausal women at high risk for breast cancer considering future pregnancy

Abstract 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.

Abstract 3261: 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 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 <1% or doubling if baseline Ki-67 ≥1%), with 10 of 14 paired specimens exhibiting a decrease. Ten women had Volpara fibroglandular assessments pre- and post-study with a median relative decrease of 11% (8 decreased and 2 increased). For the first ten subjects where serum hormones and growth factors were assessed in paired assays, favorable modulation was observed for estradiol, testosterone, SHBG, bioavailable testosterone, IGF-1, and the molar ratio of IGF1:IGFBP3. A primary prevention trial in symptomatic women appears feasible given the favorable initial results. The current pilot will continue to accrue so as to inform the design of a randomized, placebo-controlled Phase II trial of Duavee® in women at risk for breast cancer. Financial support provided by grants from the Breast Cancer Research Foundation (BCRF-16-049, BCRF-17-049). Duavee® provided by Pfizer, Inc. which was not involved in design, conduct, or analysis of the study. 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.

Abstract 3255: 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 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<0.050), and women with visceral fat mass greater than 50%. With limited numbers, there was no apparent association of CASC frequency with cytology or proliferation (Ki-67) of benign breast epithelial cells acquired by RPFNA. 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

Abstract 4230: 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 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; p<0.01) and leptin (decrease, p<0.001), as well as the ratio of adiponectin to leptin (A:L, increase, p<0.001). The same effect for all three measures was observed in serum collected 2 hours after a standard meal; and for leptin and the A:L ratio in breast tissue also collected postprandial. When dichotomized to relative weight losses of <10% vs >10%, women with >10% loss had greater favorable modulations for leptin and the A:L ratio for fasting and non-fasting serum (<0.0001 for all) as well as breast tissue (p<0.004). There were no significant differences between groups of women dichotomized by whether they exhibited (or not) an increase in the ratio of EPA:DHA to arachidonic acid in erythrocyte phospholipids at 6 months. Conclusions These results demonstrate that the cytokines adiponectin and leptin, and the ratio of the two, are robust biomarkers for modulation in serum and breast tissue of women achieving a successful weight loss on a structured intervention trial.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: 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

Gene expression in breast and adipose tissue after 12 months of weight loss and vitamin D supplementation in postmenopausal women

Adipose tissue is involved in the etiology of postmenopausal breast cancer, possibly through increased sex steroid hormone production, inflammation, and altered adipokines. Vitamin D may affect these pathways but its effect on gene expression in different tissues has not been examined. Within a double-blind, 12-month placebo-controlled randomized trial, we compared 2000 IU/day oral vitamin D3 supplementation (N = 39) vs. placebo (N = 40) on the expression of 5 genes in breast and adipose tissue in overweight/obese postmenopausal women (50–75 years). All participants had serum 25-hydroxyvitamin D (25(OH)D) levels ≥ 10–<32 ng/mL (“insufficient”) and concurrently completed a behavioral weight loss program. Random periareolar fine needle aspiration (RPFNA) and abdominal subcutaneous adipose tissue biopsies were performed at baseline and 12 months. Changes in expression of aromatase (CYP19A1), peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), monocyte-chemoattractant protein 1 (MCP-1), and vitamin D receptor (VDR) were analyzed by qRT-PCR. Compared to placebo, 2000 IU vitamin D did not show significant effects on gene expression in breast or adipose tissue. Replete women (i.e., 25(OH)D ≥ 32 ng/mL; N = 17) showed a small decrease in MCP-1 expression compared to an increase among women who remained ‘insufficient’ despite supplementation (N = 12) (Replete:−1.6% vs. Non-replete: 61.2%, p = 0.015) in breast, but not adipose tissue. No statistically significant differences in gene expression were detected according to degree of weight loss. Vitamin D repletion during weight loss may have different effects on gene expression in breast and adipose tissue. Further research on the localized effects of vitamin D is needed to determine its effect on breast cancer risk.

Dietary Associations with a Breast Cancer Risk Biomarker Depend on Menopause Status

ABSTRACTWe investigated how timing influences the role of diet in breast cancer risk with a cross-sectional study of pre-malignant change in breast tissue. Women with an elevated risk of developing breast cancer (33 premenopausal and 32 postmenopausal) completed the National Cancer Institute's food frequency questionnaire and underwent random periareolar fine-needle aspiration for evaluation of cytologic atypia, an established risk biomarker. Fatty acid composition of breast adipose was measured in 32 (49%) subjects. We found that premenopausal and postmenopausal women had similar diets, but the associations between atypia and intake of total n-3 polyunsaturated fatty acids (PUFA) and soy differed by menopause status (both Pinteraction < 0.001). Total n-3 PUFA intake was inversely associated with atypia among premenopausal women (P < 0.0001), but not among postmenopausal women (P = 0.91); associations were similar for soy (P = 0.0003 and P = 0.48, respectively). This pattern of dietary interaction with menopause was mirrored in tissue fatty acids (Pinteraction < 0.05), wherein 1) higher levels of linolelaidic acid (an industrially-produced trans fat) and 2) lower levels of docosahexaenoic acid (the predominant long-chain n-3 PUFA) in breast adipose were associated with atypia in premenopausal (both P < 0.05) but not postmenopausal women (both P > 0.37). Dietary associations with breast cancer risk are stronger prior to menopause.

Abstract 1789: Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene

Abstract Background We conducted a pilot study to assess the feasibility of a 4th generation selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women at high risk for development of breast cancer [Cancer Prev Res 8, December 2015]. Favorable modulation of biomarkers was observed in benign breast tissue acquired before and after intervention. This included reduction in proliferation (Ki-67 immunocytochemistry) and decreases in expression of genes that code for ERα, pS2, and PgR. While ERα is over-expressed and stimulatory in breast carcinogenesis, ERβ which would be inhibitory is down-regulated. Thus, a change in the ratio of gene expression might be most informative. We have now analyzed gene expression of ERβ so as to examine whether it, or the ratio of ERα to ERβ, may contribute to assessment of response. 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. Eligible women (n = 25) received 6-8 months of open-label acolbifene (20 mg/d), followed by repeat RPFNA. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed on 16 paired specimens for several genes associated with estrogen activity: estrogen receptor 1 (ESR1) for ERα, trefoil factor 1 (TFF1) for pS2, GREB1 for growth regulation by estrogen in breast cancer 1, PGR for progesterone receptor, and estrogen receptor 2 (ESR2) for ERβ. The quantity of each biomarker transcript was expressed relative to the reference transcript HPRT1. Further normalization by epithelial cell markers (KRT19 for cytokeratin 19 or CDH1 for E-Cadherin) did not materially alter the results. Results Significant changes had been previously shown for transcripts that code for pS2 (15 decreases, 1 increases; p = 0.002), ERα (11↓, 5↑; p = 0.013), and PgR (15↓, 1↑; p = 0.001); plus a suggestion of effect of GREB-1α (10↓, 6↑; p = 0.074). ERβ exhibited a general increase, but this was not statistically significant (5↓, 11↑; p = 0.24). However, the reduction of the ratio of ERα to ERβ was statistically significant (15↓, 1↑; p = 0.002). Conclusions Assessment of the ratio of gene expression for ERα to ERβ, and possibly their protein products by immunocytochemistry, may provide a useful surrogate endpoint biomarker by which to monitor response to SERMs used for breast cancer chemoprevention in premenopausal women. Supported by NO1-CN-35135. Citation Format: Bruce F. Kimler, Teresa A. Phillips, Carol J. Fabian. Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1789.

Abstract P3-10-02: Differential response of inflammatory cytokines to omega-3 fatty acid supplementation based on menopausal status

Abstract Background: High dose marine omega-3 fatty acid supplementation is currently undergoing assessment in early phase primary prevention trials. A potential mechanism of action is thought to be inflammation resolution. Since post-menopausal women may have higher systemic levels of pro-inflammatory cytokines, and hormones may affect response to fatty acids, we conducted two parallel pilot trials of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as Lovaza® supplementation in women at high risk for development of breast cancer. This post-hoc analysis examines whether five cytokines involved in inflammatory processes (HGF, MCP-1, NGF, PAI-1, TNF-alpha) are impacted by menopausal status. Methods: Separate trials were conducted in pre-menopausal (N=36) or post-menopausal (N=35) women. Blood (fasting) and benign breast tissue (non-fasting, sampled by random periareolar fine needle aspiration) was acquired before and after intervention with 4 g Lovaza® daily for 6 months. In addition to standard risk and response biomarkers, panels of cytokines associated with inflammatory processes were assayed by Luminex® technology: HGF, insulin, MCP-1, NGF, PAI-1, resistin, TNF-alpha. For breast tissue, values were normalized to protein content. Results: Thirty-four women in each trial completed the intervention and provided pre-study and post-study specimens. At baseline, higher values for post-menopausal compared to pre-menopausal women were observed for serum levels of MCP-1 (p=0.002, non-parametric Mann-Whitney test), PAI-1 (p=0.001), and TNF-alpha (p=0.019). The same was observed for off-study specimens. There was no statistically significant (non-parametric Wilcoxon signed rank test) effect of the intervention in either cohort for MCP-1 or PAI-1. However, for TNF-alpha, while there was no modulation observed in pre-menopausal women, there was a modest decrease (medians of 3.7 to 3.4 pg/ml; p=0.016) for post-menopausal women. An effect on TNF-alpha would not have been detected if the two cohorts had been combined. For breast tissue, higher values for post-menopausal women at baseline were observed for HGF (p=0.029), MCP-1 (p=0.013), NGF (p=0.001), and TNF-alpha (p=0.001), but not PAI-1 (p=0.79). Off-study values were also typically higher in post-menopausal women. As with serum levels, there was a significant decrease in TNF-alpha values over the course of the study (p=0.042) in post-menopausal women but not pre-menopausal women. A decrease was also observed for HGF (p=0.002) and MCP-1 (p=0.001) for post-menopausal women but not pre-menopausal women. Conclusion: This post-analysis suggests caution when admixing pre-menopausal and post-menopausal women in small clinical trials of omega-3 fatty acid supplementation, especially when assessing biomarkers of inflammatory processes. For trials with both, sufficient subjects should be enrolled that stratification prior to randomization is possible. Citation Format: Kimler BF, Fabian CJ. Differential response of inflammatory cytokines to omega-3 fatty acid supplementation based on menopausal status. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-10-02.

Abstract P5-06-04: Synergistically elevated hallmarks of cancer induced in non-malignant human breast cells by mixtures of environmental chemicals

Abstract BACKGROUND: Estrogenic overexposure is a well-accepted risk factor in the etiology of breast cancer. Synthetic chemicals that simulate natural estrogens, i.e., xenoestrogens (XEs), occur widely in the environment entering the human body through multiple routes. Consequently, mixtures of XEs are known to circulate in the blood at any given time. Current protocols to evaluate chemical safety rely primarily on testing chemicals individually, ignoring the fact that the general population is exposed daily to mixtures of XEs. We asked whether mixtures of low dose XEs would intensify the cellular effects of individual XEs at the same concentrations. METHODS: We exposed non-malignant breast epithelial cell cultures (HRBECs), obtained from high-risk human donors by random periareolar fine needle aspiration (RPFNA), to the high volume chemicals - bisphenol-A (BPA), methylparaben (MP) and perflourooctanoic acid (PFOA). Cells were exposed to individual XEs or mixtures of all three at a range of concentrations encompassing environmentally relevant levels. Breast cancer associated phenotypes were quantitated as test endpoints using fluorescence-activated cell sorting (FACS) and/or Western blotting. The endpoints included: total and phosphorylated estrogen receptor (ER)α, ERβ, S-phase fraction, and 4-hydoxytamoxifen (OHT) induced apoptotic fraction. We fit a log-linear dose-response model to the data generated from 3 doses for each chemical, individually and as a mixture. Evidence of synergism was tested by comparing the observed data for mixtures with that predicted by an additive-in-dose model for single doses. RESULTS: Our data demonstrates that the degree of perturbation induced by the chemical mixture tested here is not merely an additive effect of each chemical; instead it reflects considerable synergism in the mode of action. In 3/3 SNP-authenticated non-malignant HRBEC cell lines exposed to the chemical mixture at the lowest test dose, total and phosphorylated ERα levels were consistently elevated. Concurrently, ERβ level was reduced. A dose-dependent increase was observed in the S-phase fraction, together with a marked reduction of the OHT-induced apoptotic fraction in HRBECs exposed to BPA, MP, and PFOA individually (p&amp;lt;0.001 for each exposure). A significantly greater effect, or synergism, was detected for these endpoints upon exposure to a mixture of the chemicals at the same concentrations (p&amp;lt;0.001 for the combination vs. the predicted sum of individual doses). Primary HRBEC cultures from 7 additional human volunteers displayed a similar pattern of response. CONCLUSION: Routine tests conducted to assess the carcinogenic potential of chemicals overlook possible synergism among different chemicals. We show that the combined effects of low doses of various chemicals can be much stronger than predicted by addition of their effects as single agents. Despite being cost, time, and labor-intensive, current testing of individual chemicals fails to inform regulators about the safe dose range for human exposure. It is thus imperative to expand present methods to include combinatorial approaches pertaining to test chemicals, endpoints, and target cells. Citation Format: Dairkee SH, Luciani-Torres G, Moore DH, Goodson WH. Synergistically elevated hallmarks of cancer induced in non-malignant human breast cells by mixtures of environmental chemicals. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-04.

Abstract B52: Pilot study: Random periareolar fine needle aspiration (RPFNA) accurately depicts biochemical phenotype displayed in breast tissues of high risk African American women

Abstract Background: There are more than 2.9 million breast cancer survivors in America today. However African American women have a 79% five-year survival while survival rates for European American women are at 92%. While early detection increases the survivability rates of most breast cancer, the standard mammogram fails to identify the earlier stages of some of the more aggressive types of breast cancer. Short-term breast cancer risks in women are increasingly being identified by Random Periareolar Fine Needle Aspiration (RPFNA). While the reproducibility of this technique is no longer the primary concern for its usage; RPFNA's small sample-sets and the inability to accurately correlate pathological and biochemical findings within the entire breast inhibits full acceptance of this technique. The purpose of this study was to determine if the utilization RPFNA accurately sampled the entire breast tissue. Methods: In this pilot study 10 high risk African American women who were high risk for breast cancer (known or suspected BRCA1 mutation carriers) had breast tissues collect by RPFNA. These samples were laser captured and the RPFNA protein expression for wide array of biomarkers (stem cell, epithelial- mesenchymal transition (EMT), proliferation, apoptosis and senescence) were assessed utilizing reverse-phase protein microarray in mammary epithelial cells. The same 10 high risk African American women either underwent prophylactic mastectomies or had a mastectomy after cancer diagnosis. Mastectomy specimens were serial sliced into 2.5 centimeter slices and 2 to 3 cubic centimeter samples were excised from a specified section of each slice of breast tissue. On average 103 samples were collected from each breast and the same (RPFNA) biomarkers were analyzed. Results: In this exploratory study, we have determined there are no significant differences between the expression of the biomarkers we have tested in RPFNA samples and the whole breast tissue sampling. Specifically we have tested for EMT markers (Vimentin and Snail), p53 and additional markers involved in proliferation and apoptosis. Conclusions: This study shows that RPFNA is an accurate measure of mammary breast epithelial and this technique directly correlates to the measurements found throughout the entire breast tissues. RPFNA did not show any significant differences in the expression of specific biomarkers tested, suggesting that RPFNA samples could be utilized to develop more robust breast cancer risk profiles. Citation Format: Christopher Sistrunk, Adrian Ambrose, Amira Carter, Victoria Seewaldt. Pilot study: Random periareolar fine needle aspiration (RPFNA) accurately depicts biochemical phenotype displayed in breast tissues of high risk African American women. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B52.

Omega-3 and omega-6 Fatty acids in blood and breast tissue of high-risk women and association with atypical cytomorphology.

The ratio of omega-3 to omega-6 fatty acids, especially the long-chain eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) to arachidonic acid (AA) ratio, is inversely associated with breast cancer risk. We measured the association between cytologic atypia, a biomarker for short-term risk of breast cancer development, and omega-3 and omega-6 fatty acid intake and levels in blood and breast tissue. Blood and benign breast tissue, sampled by random periareolar fine-needle aspiration (RPFNA), was obtained from 70 women at elevated risk for breast cancer. Self-reported dietary intake was assessed by the NCI's Food Frequency Questionnaire. The fatty acid composition of five lipid compartments, red blood cell, plasma and breast phospholipids, and plasma and breast triaclyglycerides (TAG), was analyzed by gas chromatography as weight percent. Median daily intakes of EPA+DHA and total omega-3 fatty acids were 80 mg and 1.1 g, respectively. The median total omega-3:6 intake ratio was 1:10. Compared with women without atypia, those with cytologic atypia had lower total omega-3 fatty acids in red blood cell and plasma phospholipids and lower omega-3:6 ratios in plasma TAGs and breast TAGs (P < 0.05). The EPA+DHA:AA ratio in plasma TAGs was also lower among women with atypia. This is the first report of associations between tissue levels of omega-3 and omega-6 fatty acids and a reversible tissue biomarker of breast cancer risk. RPFNA cytomorphology could serve as a surrogate endpoint for breast cancer prevention trials of omega-3 fatty acid supplementation.

Abstract CN02-03: Altered imprinted gene DMR regulatory methylation in breast cancer

Abstract CN02-03: Altered imprinted gene DMR regulatory methylation in breast cancer

Identification of Breast Cancer DNA Methylation Markers Optimized for Fine-Needle Aspiration Samples

Random periareolar fine-needle aspiration (RP-FNA) is increasingly used in trials of breast cancer prevention for biomarker assessments. DNA methylation markers may have value as surrogate endpoint biomarkers, but this requires identification of biologically relevant markers suitable for paucicellular, lymphocyte-contaminated clinical samples. Unbiased whole-genome 5-aza-2'-deoxycytidine (5AZA)-induced gene expression assays, followed by several phases of qualitative and quantitative methylation-specific PCR (MSP) testing, were used to identify novel breast cancer DNA methylation markers optimized for clinical FNA samples. The initial 5AZA experiment identified 453 genes whose expression was potentially regulated by promoter region methylation. Informatics filters excluded 273 genes unlikely to yield useful DNA methylation markers. MSP assays were designed for 271 of the remaining genes and, ultimately, 33 genes were identified that were differentially methylated in clinical breast cancer samples, as compared with benign RP-FNA samples, and never methylated in lymphocytes. A subset of these markers was validated by quantitative multiplex MSP in extended clinical sample sets. Using a novel permutation method for analysis of quantitative methylation data, PSAT1, GNE, CPNE8, and CXCL14 were found to correlate strongly with specific clinical and pathologic features of breast cancer. In general, our approach identified markers methylated in a smaller subpopulation of tumor cells than those identified in published methylation array studies. Clinically relevant DNA methylation markers were identified using a 5AZA-induced gene expression approach. These breast cancer-relevant, FNA-optimized DNA methylation markers may have value as surrogate endpoint biomarkers in RP-FNA studies.

A clinical trial of lovastatin for modification of biomarkers associated with breast cancer risk

Pre-clinical and epidemiologic studies provide rationale for evaluating lipophilic statins for breast cancer prevention. We conducted a single-arm, biomarker modulation trial of lovastatin among women with increased risk of breast cancer. Eligibility criteria included a deleterious germline mutation in BRCA1, BRCA2, CDH1, or TP53; lifetime breast cancer risk of ≥20% as estimated by the Claus model; or personal history of estrogen receptor and progesterone receptor-negative breast cancer. Participants received 40 mg of lovastatin orally twice daily for 6months. We evaluated the following biomarkers before and after lovastatin use: breast duct cytology (primary endpoint), serum lipids, C-reactive protein, insulin-like growth factor-1, IGF binding protein-3, lipid peroxidation, oxidative DNA damage, 3-hydroxy-3-methylglutaryl CoA reductase genotype, and mammographic density. Thirty women were enrolled, and 26 (86.7%) completed the study. For the primary endpoint of changes in breast duct cytology sampled by random periareolar fine needle aspiration, most participants [57.7%, 95% confidence interval (CI) 38.9-74.5%] showed no change after lovastatin; 19.2% (CI 8.1-38.3%) had a favorable change in cytology, 7.7% (95% CI 1.0-25.3%) had an unfavorable change, and 15.4% (95% CI 5.5-34.2%) had equivocal results due to acellular specimens, usually after lovastatin. No significant changes were observed in secondary biomarker endpoints. The study was generally well-tolerated: 4 (13.3%) participants did not complete the study, and one (3.8%) required a dose reduction. This trial was technically feasible, but demonstrated no significant biomarker modulation; contributing factors may include insufficient sample size, drug dose and/or duration. The results are inconclusive and do not exclude a favorable effect on breast cancer risk.

Abstract LB-191: Cytological atypia predicts short-term breast cancer risk in women from high-risk families that lack a BRCA mutation.

Abstract Background: Women with deleterious mutations in BRCA1/2 have a 9- to 36-fold increased risk of breast cancer compared with general population rates. However, there are a significant number of women whose families have multi-generation premenopausal breast cancers and lack an identified BRCA-mutation. Currently we lack strategies to predict the development of breast cancer in women from BRCA-negative families. Random Periareolar Fine Needle Aspiration (RPFNA) is a research technique developed to test for “field effects” in high-risk women. Carol Fabian previously demonstrated that atypia in RPFNA predicted breast cancer risk, however, many women in this cohort were known BRCA mutation carriers. We investigated whether cytological atypia in RPFNA predicts the subsequent development of breast cancer in women from BRCA-negative breast cancer families. Results: From 01/01/03 to 07/01/12, we performed RPFNA in 254 high-risk BRCA-negative women. Women underwent yearly mammogram/Magnetic Resonance Imaging (MRI) and bi-annual clinical breast exam. During 6.5 women-years of follow-up, 24 new breast cancers were diagnosed. The median age of the 254 women in this study was 48, and the median BMI was 24. Eighty-six percent (218) of the women were Caucasian, 13% (32) were African-American, and 2 women were Native Americans. Sixty-eight women presented with cytologic-atypia in at least one breast; 16 of these women had cytologic-atypia in both breasts. Fourteen (21%) of the 68 women with baseline cytologic-atypia later developed cancer in a breast that had had baseline cytologic-atypia; none of the 68 women developed cancer in a breast without baseline cytologic-atypia. One hundred eighty-six women did not have cytologic-atypia at baseline; 10 (5%) of these women developed cancer. Ten women had a prophylactic mastectomy and were censored at the date of mastectomy. The median length of follow-up among the uncensored patients was 74 months. In the baseline atypic and non-atypic groups, the 48-month cumulative incidence of cancer development was 0.22 (95% confidence interval [CI], 0.13-0.34) and 0.04 (95% CI, 0.02-0.08), respectively. Atypia was significantly associated with cancer development both with and without controlling for covariates (p &amp;lt; 0.001). The covariate-adjusted hazard ratio (CV-AHR) for cytologic-atypia was 6.3 (95% CI, 1.7-24.2). The hazard ratios for the covariates were as follows: age (1.01; 95% CI, 0.89-1.16), BMI (1.02; 95% CI, 0.91-1.15), menopausal status (0.16; 95% CI, 0.01-2.60), Gail Risk (1.18; 95% CI, 1.03-1.35) and mammographic density for scattered (1.7; 95% CI, 0.27-10.9) and heterogenetic (1.10; 95% CI, 0.20-6.01) compared to the reference type of extreme. Conclusions: This is the first demonstration that in women from high-risk BRCA mutation-negative families, cytologic-atypia in RPFNA is associated with the subsequent development of breast cancer. Citation Format: Victoria L. Seewaldt, Bercedis Peterson, Gloria Broadwater. Cytological atypia predicts short-term breast cancer risk in women from high-risk families that lack a BRCA mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-191. doi:10.1158/1538-7445.AM2013-LB-191