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.

Histologic features and decreased lung FOXF1 gene expression in severe bronchopulmonary dysplasia without a genetic diagnosis of alveolar capillary dysplasia.

We report the case of a preterm infant who died at 10 months of age with severe bronchopulmonary dysplasia (sBPD) with refractory pulmonary hypertension and respiratory failure who had striking histologic features compatible with the diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) but without genetic confirmation of the diagnosis. We further demonstrate dramatic reductions in lung FOXF1 and TMEM100 content in sBPD, suggesting common mechanistic links between ACDMPV and sBPD with impaired FOXF1 signaling.

Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways.

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

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.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

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

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.

Reproducibility of random periareolar fine needle aspiration in a multi-institutional Cancer and Leukemia Group B (CALGB) cross-sectional study.

BACKGROUND: Random periareolar fine needle aspiration (RPFNA) is a research technique developed to assess short-term breast cancer risk in women at increased risk of breast cancer. Although there is increasing acceptance of RPFNA, neither the reproducibility nor the inter-institutional compatibility of RPFNA has been established. To address these key limitations, the Cancer and Leukemia Group B (CALGB) Prevention Group tested the reproducibility of RPFNA in a multi-institutional cross-sectional study. METHODS: Sixty-three high-risk women from five CALGB institutions (Duke, Ohio State, Roswell Park, Dana Farber, and Vermont) underwent RPFNA from July 1, 2007 to June 30, 2008. Duplicate bilateral RPFNA was performed on each woman by a single investigator on a single day. Masood Cytology Index score was assessed by a single blinded cytopathologist. RESULTS: There was a high degree of statistical agreement in the Masood Cytology Index scores of duplicate RPFNA samples from the same breast, with a Spearman correlation coefficient of 0.8312 (P < 0.0001). Importantly, although there was agreement in duplicate samples from the same breast, there was lack of agreement between duplicate samples from the opposite breast. CONCLUSIONS: This multi-institutional study shows that RPFNA is a highly reproducible measure of breast cytology in a cooperative group cross-sectional trial. RPFNA did not show a high degree of agreement between breasts, suggesting that breast cancer risk and progression may occur at different rates in individual breasts from a single woman. These studies provide proof-of-principle for future RPFNA-based cooperative group prevention studies.

CpG island tumor suppressor promoter methylation in non-BRCA-associated early mammary carcinogenesis.

BACKGROUND: Only 5% of all breast cancers are the result of BRCA1/2 mutations. Methylation silencing of tumor suppressor genes is well described in sporadic breast cancer; however, its role in familial breast cancer is not known. METHODS: CpG island promoter methylation was tested in the initial random periareolar fine-needle aspiration sample from 109 asymptomatic women at high risk for breast cancer. Promoter methylation targets included RARB (M3 and M4), ESR1, INK4a/ARF, BRCA1, PRA, PRB, RASSF1A, HIN-1, and CRBP1. RESULTS: Although the overall frequency of CpG island promoter methylation events increased with age (P<0.0001), no specific methylation event was associated with age. In contrast, CpG island methylation of RARB M4 (P=0.051), INK4a/ARF (P=0.042), HIN-1 (P=0.044), and PRA (P=0.032), as well as the overall frequency of methylation events (P=0.004), was associated with abnormal Masood cytology. The association between promoter methylation and familial breast cancer was tested in 40 unaffected premenopausal women in our cohort who underwent BRCA1/2 mutation testing. Women with BRCA1/2 mutations had a low frequency of CpG island promoter methylation (15 of 15 women had

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.

ESR1 promoter hypermethylation does not predict atypia in RPFNA nor persistent atypia after 12 months tamoxifen chemoprevention.

PURPOSE: Currently, we lack biomarkers to predict whether high-risk women with mammary atypia will respond to tamoxifen chemoprevention. EXPERIMENTAL DESIGN: Thirty-four women with cytologic mammary atypia from the Duke University High-Risk clinic were offered tamoxifen chemoprevention. We tested whether ESR1 promoter hypermethylation and/or estrogen receptor (ER) protein expression by immunohistochemistry predicted persistent atypia in 18 women who were treated with tamoxifen for 12 months and in 16 untreated controls. RESULTS: We observed a statistically significant decrease in the Masood score of women on tamoxifen chemoprevention for 12 months compared with control women. This was a significant interaction effect of time (0, 6, and 12 months) and treatment group (tamoxifen versus control) P = 0.0007. However, neither ESR1 promoter hypermethylation nor low ER expression predicted persistent atypia in Random Periareolar Fine Needle Aspiration after 12 months tamoxifen prevention. CONCLUSIONS: Results from this single institution pilot study provide evidence that, unlike for invasive breast cancer, ESR1 promoter hypermethylation and/or low ER expression is not a reliable marker of tamoxifen-resistant atypia.

Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF.

PURPOSE: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. EXPERIMENTAL DESIGN: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. RESULTS: INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-beta2, estrogen receptor-alpha, and breast cancer-associated 1 genes (P = 0.001). CONCLUSIONS: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.

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.

Mammographic density does not correlate with Ki-67 expression or cytomorphology in benign breast cells obtained by random periareolar fine needle aspiration from women at high risk for breast cancer.

BACKGROUND: Ki-67 expression is a possible risk biomarker and is currently being used as a response biomarker in chemoprevention trials. Mammographic breast density is a risk biomarker and is also being used as a response biomarker. We previously showed that Ki-67 expression is higher in specimens of benign breast cells exhibiting cytologic atypia that are obtained by random periareolar fine needle aspiration (RPFNA). It is not known whether there is a correlation between mammographic density and Ki-67 expression in benign breast ductal cells obtained by RPFNA. METHODS: Included in the study were 344 women at high risk for developing breast cancer (based on personal or family history), seen at The University of Kansas Medical Center high-risk breast clinic, who underwent RPFNA with cytomorphology and Ki-67 assessment plus a mammogram. Mammographic breast density was assessed using the Cumulus program. Categorical variables were analyzed by chi2 test, and continuous variables were analyzed by nonparametric test and linear regression. RESULTS: Forty-seven per cent of women were premenopausal and 53% were postmenopausal. The median age was 48 years, median 5-year Gail Risk was 2.2%, and median Ki-67 was 1.9%. The median mammographic breast density was 37%. Ki-67 expression increased with cytologic abnormality (atypia versus no atypia; P 50 years; P

Atypia in random periareolar fine-needle aspiration affects the decision of women at high risk to take tamoxifen for breast cancer chemoprevention.

Random periareolar fine-needle aspiration (RPFNA) is a research procedure designed to (a) evaluate short-term breast cancer risk in women at high risk for developing breast cancer, and (b) track response to chemoprevention. Of import, cellular atypia in breast RPFNA is prospectively associated with a 5.6-fold increase in breast cancer risk in women at high risk. Among 99 women attending a clinic for high-risk breast cancer, we explored the effects of RPFNA cytology results on decision making pertaining to the use of tamoxifen for breast cancer chemoprevention. No patient with nonproliferative or hyperplastic cytology subsequently elected to take tamoxifen. Only 7% of subjects with borderline atypia elected to take tamoxifen. In contrast, 50% with atypia elected to take tamoxifen. These results suggest that the provision of a biomarker of short-term risk can affect the motivation to take tamoxifen for chemoprevention. This conclusion is informative given that tamoxifen, due to its side effects, is often underused by women at high risk of developing breast cancer. Further research is needed to determine the mechanisms through which RPFNA results affect the decision to use tamoxifen, or any other breast cancer chemopreventive agent.

Overweight and obese perimenopausal and postmenopausal women exhibit increased abnormal mammary epithelial cytology.

High body mass index (BMI >or= 25 kg/m2) is associated with increased postmenopausal breast cancer incidence and mortality. However, few studies have explored associations between BMI and direct measures on target tissue. Epithelial cytology was assessed in 62 high-risk perimenopausal and postmenopausal women using random periareolar fine needle aspiration. Masood cytology index scores were significantly higher among women with BMIs >or=25 kg/m2 than in women with BMIs <25 kg/m2 (13.9 +/- 0.42 versus 12.7 +/- 0.29 kg/m2; P = 0.017). Overweight or obese women also had significantly higher random periareolar fine needle aspiration epithelial cell counts compared with those who were normal weight (1,230 +/- 272 versus 521 +/- 185; P = 0.028). These data suggest that overweight in perimenopausal and postmenopausal women is associated with direct cytologic abnormalities within the breast. Further research is needed to confirm these findings and to determine if this potential biomarker is responsive to changes in body weight resulting from diet and/or exercise interventions.

Hypermethylation of the breast cancer-associated gene 1 promoter does not predict cytologic atypia or correlate with surrogate end points of breast cancer risk.

Mutation of the breast cancer-associated gene 1 (BRCA1) plays an important role in familial breast cancer. Although hypermethylation of the BRCA1 promoter has been observed in sporadic breast cancer, its exact role in breast cancer initiation and association with breast cancer risk is unknown. The frequency of BRCA1 promoter hypermethylation was tested in (a) 14 primary breast cancer biopsies and (b) the initial random periareolar fine-needle aspiration (RPFNA) cytologic samples obtained from 61 asymptomatic women who were at increased risk for breast cancer. BRCA1 promoter hypermethylation was assessed from nucleotide -150 to nucleotide +32 relative to the transcription start site. RPFNA specimens were stratified for cytologic atypia using the Masood cytology index. BRCA1 promoter hypermethylation was observed at similar frequency in nonproliferative (normal; Masood

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.

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.

Retinoic acid receptor-beta2 promoter methylation in random periareolar fine needle aspiration.

Methylation of the retinoic acid receptor-beta2 (RARbeta2) P2 promoter is hypothesized to be an important mechanism for loss of RARbeta2 function during early mammary carcinogenesis. The frequency of RARbeta2 P2 methylation was tested in (a) 16 early stage breast cancers and (b) 67 random periareolar fine needle aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either (a) 5-year Gail risk calculation > or = 1.7%; (b) prior biopsy exhibiting atypical hyperplasia, lobular carcinoma in situ, or ductal carcinoma in situ; or (c) known BRCA1/2 mutation carrier. RARbeta2 P2 promoter methylation was assessed at two regions, M3 (-51 to 162 bp) and M4 (104-251 bp). In early stage cancers, M4 methylation was observed in 11 of 16 (69%) cases; in RPFNA samples, methylation was present at M3 and M4 in 28 of 56 (50%) and 19 of 56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARbeta2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in (a) 0 of 10 (0%) of RPFNAs with Masood scores of < or = 10 (nonproliferative), (b) 3 of 20 (15%) with Masood scores of 11 to 12 (low-grade proliferative), (c) 3 of 10 (30%) with Masood scores of 13 (high-grade proliferative), and (d) 7 of 14 (50%) with Masood scores of 14 of 15 (atypia). Results from this study indicate that the RARbeta2 P2 promoter is frequently methylated (69%) in primary breast cancers and shows a positive association with increasing cytologic abnormality in RPFNA.

A phase II breast cancer chemoprevention trial of oral alpha-difluoromethylornithine: breast tissue, imaging, and serum and urine biomarkers.

PURPOSE: A double-blind randomized Phase II chemoprevention trial of alpha-difluoromethylornithine (DFMO) was conducted in a group of women at high risk for development of breast cancer. DFMO is an irreversible inhibitor of ornithine decarboxylase, the limiting enzyme of polyamine synthesis that is often up-regulated in breast cancer. EXPERIMENTAL DESIGN: Study entrants were required to have random periareolar fine-needle aspiration cytology prior to entry that exhibited hyperplasia or hyperplasia with atypia, as well as a mammogram and clinical breast exam judged as not suspicious for breast cancer and no clinical hearing loss. Subjects were randomized to 6 months of oral DFMO (0.5 g/m(2)/day) or placebo, followed by repeat fine-needle aspiration and biomarker assessment. The main study end point was an improvement in cytologic pattern. RESULTS: Of 119 subjects entered, 96% completed the study and were evaluable for the main study end point. A modest reduction (28%) in average total urine polyamines was obtained in the DFMO group, but there was no reduction in the spermidine:spermine ratio. There was no difference in cytologic improvement between DFMO and placebo. Likewise, there was no difference between DFMO and placebo for the secondary end points of breast molecular marker changes (immunocytochemical expression of proliferating cell nuclear antigen, p53, and epidermal growth factor receptor), mammographic breast density, serum insulin-like growth factor I: insulin-like growth factor binding protein 3 ratio, adverse events, quality of life indices, or subsequent cancer development. CONCLUSIONS: DFMO at a dose level of 0.5 g/m(2)/day administered for 6 months does not modulate breast risk biomarkers tested in this study.