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.

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.

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.

Reduction in Ki-67 in benign breast tissue of high-risk women with the lignan secoisolariciresinol diglycoside.

Preclinical and correlative studies suggest reduced breast cancer with higher lignan intake or blood levels. We conducted a pilot study of modulation of risk biomarkers for breast cancer in premenopausal women after administration of the plant lignan secoisolariciresinol given as the diglycoside (SDG). Eligibility criteria included regular menstrual cycles, no oral contraceptives, a >3-fold increase in 5-year risk, and baseline Ki-67 of ≥2% in areas of hyperplasia in breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) during the follicular phase of the menstrual cycle. SDG (50 mg/d) was given for 12 months, followed by repeat RPFNA. The primary end point was change in Ki-67. Secondary end points included change in cytomorphology, mammographic breast density, serum bioavailable estradiol and testosterone insulin-like growth factor-I and IGF-binding protein-3, and plasma lignan levels. Forty-five of 49 eligible women completed the study with excellent compliance (median = 96%) and few serious side effects (4% grade 3). Median plasma enterolactone increased ∼9-fold, and total lignans increased 16-fold. Thirty-six (80%) of the 45 evaluable subjects showed a decrease in Ki-67, from a median of 4% (range, 2-16.8%) to 2% (range, 0-15.2%; P < 0.001, Wilcoxon signed rank test). A decrease from baseline in the proportion of women with atypical cytology (P = 0.035) was also observed. Based on favorable risk biomarker modulation and lack of adverse events, we are initiating a randomized trial of SDG versus placebo in premenopausal women.

Reduction in proliferation with six months of letrozole in women on hormone replacement therapy.

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

Optimization of estrogen receptor analysis by immunocytochemistry in random periareolar fine-needle aspiration samples of breast tissue processed as thin-layer preparations.

Immunostaining of estrogen receptor alpha (ER) in samples of benign breast tissue obtained by random periareolar fine-needle aspiration (RPFNA) is a practical tool for breast cancer chemoprevention trials. The authors report an optimized method of ER immunostaining for use with thin-layer preparations of modified Cytolyt-fixed benign breast tissue acquired by RPFNA. Samples of benign breast tissue and MCF-7 controls processed as thin-layer preparations were tested for the effects of antibody titer, antigen retrieval temperature (90 degrees or 115 degrees C), buffer (20% nuclear decloaker, pH 9.3; 10 mM citrate buffer, pH 6), and blocking solution (0.01% glucose oxidase or 0.3% H2O2) on ER immunostaining. The prevalence of positively stained breast epithelial cells, mean intensity of ER staining, and composite immunostaining score were evaluated for effect of immunostaining protocol. RPFNA samples and MCF-7 cells processed using nuclear decloaker and low-temperature antigen retrieval had more ER-positive cells (P<0.0001) and increased mean staining intensity and weighted staining indices (P<0.05) compared with samples prepared with citrate buffer and high-temperature antigen retrieval. Glucose oxidase increased ER-positive cells in comparison to hydrogen peroxide (P<0.04) when combined with low-temperature antigen retrieval and the use of nuclear decloaker. Staining was negative for all non-immune controls regardless of protocol. The combination of low-temperature antigen retrieval, diluted commercial nuclear decloaker solution, and a glucose oxidase blocking step yielded optimal ER immunostaining for thin-layer preparations of benign breast tissue harvested by RPFNA.

Estrogen receptor expression in benign breast ductal cells obtained from random periareolar fine needle aspiration correlates with menopausal status and cytomorphology index score.

INTRODUCTION: Estrogen receptor (ER) expression in breast epithelial cells has potential as a risk marker for development of breast cancer and as a response marker for preventive interventions. AIM: The purpose of this study was to determine if ER expression in benign cytologic specimens acquired by random periareolar fine needle aspiration (RPFNA) increases with morphologic abnormality as has been reported for histologic preparations. METHODS: ER expression was assessed in 122 women at high risk for development of breast cancer who had RPFNA hyperplasia +/- atypia and were being screened for entry into one of two chemoprevention trials. ER was assessed using antigen retrieval at 90 degrees C for 2 min and the DAKO ER monoclonal antibody (Clone number 1D5). The proportion of cells with definitive staining at each intensity level (0-4) was recorded as a percentage of the total cells counted, to give a weighted intensity score (IS). RESULTS: Of 122 women, 65% exhibited hyperplasia and 35% exhibited hyperplasia with atypia in their RPFNA specimens. A majority (66%) of subjects had at least 10% of ductal cells exhibiting nuclear staining for ER. Median percent of cells with > or =1+ staining was 20% and the median ER IS was 0.23. There was a strong correlation between ER IS and percentage of ER positive cells (R(2) = 0.88). By univariate analysis ER IS was statistically significantly higher in women older than median age of 48 years (P = 0.025), in postmenopausal women on HRT (P < 0.017), and in women with a Masood cytomorphology index score of > or =14 (P = 0.005). On multivariable analysis, ER IS was significantly associated with postmenopausal status (P = 0.038) and cytomorphology as measured by Masood score (P = 0.043). CONCLUSION: ER can be readily measured in cytologic specimens obtained by RPFNA with the use of antigen retrieval method. Further, ER expression in cytologic specimens is influenced by postmenopausal status and morphologic abnormality.

Comparison of cytomorphology in specimens obtained by random periareolar fine needle aspiration and ductal lavage from women at high risk for development of breast cancer.

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) are both being used to harvest epithelial cells for risk assessment as well as response evaluation in chemoprevention trials. The magnitude of increase in relative risk has been defined in a prospective study for RPFNA but not for DL atypia. We attempted both procedures in 26 women at high risk for development of breast cancer. Median age was 43 (range 32-57); 15 women were premenopausal, with 6 of the postmenopausal women on HRT. Collection of nipple aspirate fluid (NAF) was attempted and, if successful, was followed by DL; RPFNA was then performed on all women. Both procedures were attempted the same day (follicular phase of menstrual cycle if premenopausal) in 24 subjects and within three months for two subjects. Twenty-three subjects produced NAF, 17 of the 23 (74%) had a successful duct cannulation as part of the DL procedure, with 16 yielding sufficient (10) ductal cells for morphologic assessment. Twenty-five of 26 (96%) subjects had a successful RPFNA procedure with adequate cellularity for morphology. There was concordance between DL and RPFNA specimens for traditional cytologic category assessment in 10/16 (63%), Masood index score in 13/16 (82%), and Consensus Panel assessment in 12/16 (75%) of specimens. We conclude that same day DL and RPFNA is feasible, with 62% and 96% of high-risk women having a successful procedure with evaluable cytomorphology. RPFNA was more likely to yield an evaluable specimen, but if a cellular DL specimen was obtained, morphology was generally similar.

Ki-67 expression in benign breast ductal cells obtained by random periareolar fine needle aspiration.

Ki-67 expression in ductal cells obtained by random periareolar fine needle aspiration (RPFNA) is currently being used as a response biomarker in phase II breast cancer chemoprevention trials; however, Ki-67 in RPFNA has not been well studied as a risk predictor for cancer, which would support its use as a response indicator. We examined the expression of Ki-67 in RPFNA specimens with hyperplasia +/- atypia obtained from 147 women at high risk for development of breast cancer. Median Ki-67 was 1.4% (range 0-24%). Ki-67 was higher in specimens from women < 50 versus those > or = 50 (median 2% versus 0.6%; P = 0.006) and from premenopausal women versus postmenopausal women (P = 0.037); however, hormone replacement therapy (predominately low-dose estrogen without progestins) had no effect. By univariate analysis, Ki-67 was positively correlated with ductal cell number (P = 0.001) and hyperplasia with atypia (P = 0.007). By multivariable analysis, the proportion of ductal cells expressing Ki-67 was again predicted by cell number, which, in turn, was predicted by cytologic atypia. The association of Ki-67 expression with cytologic atypia, a known risk factor for development of breast cancer, provides preliminary justification for its use as a response biomarker in phase II chemoprevention trials.