Change in Blood and Benign Breast Biomarkers in Women Undergoing a Weight-Loss Intervention Randomized to High-Dose ω-3 Fatty Acids versus Placebo.

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.

Randomized Phase IIB Trial of the Lignan Secoisolariciresinol Diglucoside in Premenopausal Women at Increased Risk for Development of Breast Cancer.

We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was -1.8% in the SDG arm (P = 0.001) and -1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = -2.2%; P = 0.002) but not placebo (median = -1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (<0.5 or >2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.

Effect of Bazedoxifene and Conjugated Estrogen (Duavee) on Breast Cancer Risk Biomarkers in High-Risk Women: A Pilot Study.

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.

Breast Hormone Concentrations in Random Fine-Needle Aspirates of Healthy Women Associate with Cytological Atypia and Gene Methylation.

Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cytomorphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman r = 0.34, Padj = 0.001 and r = 0.69, Padj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (Padj < 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (P = 0.026 and Padj = 0.208). Breast androgens were significantly correlated with breast density (Spearman r = 0.27, Padj = 0.02 for testosterone) and CMI (Spearman r = 0.3, Padj = 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study. Cancer Prev Res; 11(9); 557-68. ©2018 AACR.

Gene Methylation and Cytological Atypia in Random Fine-Needle Aspirates for Assessment of Breast Cancer Risk.

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

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

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.

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

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

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

Associational studies suggest higher intakes/blood levels of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) are associated with reduced breast cancer risk. We performed a pilot study of high-dose EPA + DHA in postmenopausal women to assess feasibility before initiating a phase IIB prevention trial. Postmenopausal women with cytologic evidence of hyperplasia in their baseline random periareolar fine needle aspiration (RPFNA) took 1,860 mg EPA +1500 mg DHA ethyl esters daily for 6 months. Blood and breast tissue were sampled at baseline and study conclusion for exploratory biomarker assessment, with P values uncorrected for multiple comparisons. Feasibility was predefined as 50% uptake, 80% completion, and 70% compliance. Trial uptake by 35 study entrants from 54 eligible women was 65%, with 97% completion and 97% compliance. Favorable modulation was suggested for serum adiponectin (P = 0.0027), TNFα (P = 0.016), HOMA 2B measure of pancreatic ß 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.

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.

Favorable modulation of benign breast tissue and serum risk biomarkers is associated with > 10 % weight loss in postmenopausal women.

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