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Background The mechanistic target of rapamycin (mTOR) pathway promoted by positive energy imbalance and insulin-like growth factors can be a mechanism by which obesity influences breast cancer risk. We evaluated the associations of body fatness with the risk of breast cancer varied with phosphorylated (p)-mTOR protein expression, an indication of the pathway activation. Methods Women with newly diagnosed breast cancer (n = 715; 574 [80%] Black and 141 [20%] White) and non-cancer controls (n = 1983; 1280 [64%] Black and 713 [36%] White) were selected from the Women’s Circle of Health Study. Surgical tumor samples among the cases were immunostained for p-mTOR (Ser2448) and classified as p-mTOR-overexpressed, if the expression level ≥ 75th percentile, or p-mTOR-negative/low otherwise. Anthropometrics were measured by trained staff, and body composition was determined by bioelectrical impedance analysis. Odds ratios (ORs) of p-mTOR-overexpressed tumors and p-mTOR-negative/low tumors compared to controls were estimated using polytomous logistic regression. The differences in the associations by the p-mTOR expression status were assessed by tests for heterogeneity. Results Cases with p-mTOR-overexpressed tumors, but not cases with p-mTOR-negative/low tumors, compared to controls were more likely to have higher body mass index (BMI), percent body fat, and fat mass index (P-heterogeneity P-heterogeneity = 0.27 and 0.48, respectively). Conclusions Our findings suggest that in this population composed of predominately Black women, body fatness is associated with breast cancer differently for p-mTOR overexpression and p-mTOR negative/low expression. Whether mTOR plays a role in the obesity and breast cancer association warrants confirmation by prospective studies.

Background African American/Black women with breast cancer have poorer survival than White women, and this disparity persists even after adjusting for non-biological factors. Differences in tumor immune biology have been reported between Black and White women, and the tumor immune milieu could potentially drive racial differences in breast cancer etiology and outcome. Methods We examined the association of CD8+ cytotoxic T cells with clinical-pathological variables in the Women’s Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We evaluated 688 invasive breast tumor samples (550 Black, 138 White) using immunohistochemical staining of tissue microarray slides. CD8+ T cells were scored for each patient tumor sample with digital image analysis. Results Black women had a significantly higher percentage of high-grade, estrogen receptor (ER)-negative, and triple-negative tumors than White women and significantly higher CD8+ T cell density (median 87.6/mm2 vs. 53.1/mm2; p + T cell density was significantly higher in younger patients and patients with high-grade and ER/PR-negative tumors. No significant associations were observed between CD8+ T cell density and overall survival or breast cancer-specific survival in the overall population, or when Black patients were analyzed as a separate group. However, when stratified by subtype, Black women with triple-negative breast cancer and high CD8+ T cell density showed a trend towards better overall survival in comparison with patients with low CD8+ T cell density (HR = 0.51; 95% CI 0.25–1.04). Conclusions Our data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups.

Background: Forkhead box protein A1 (FOXA1) promotes luminal differentiation, and hypermethylation of the gene can be a mechanism of developing estrogen receptor–negative (ER−) breast cancer. We examined FOXA1 in breast tumor and adjacent normal tissue in relation to reproductive factors, particularly higher parity and no breastfeeding, that are associated with ER− tumors. Methods: We performed IHC for FOXA1 in breast tumors (n = 1,329) and adjacent normal tissues (n = 298) in the Women's Circle of Health Study (949 Blacks and 380 Whites). Protein expression levels were summarized by histology (H) scores. Generalized linear models were used to assess FOXA1 protein expression in relation to reproductive factors by ER status. Results: ER-positive (ER+) versus ER− tumors had higher FOXA1 protein expression (P < 0.001). FOXA1 expression was higher in tumor versus paired adjacent normal tissue in women with ER+ or non-triple–negative cancer (both P < 0.001), but not in those with ER− or triple-negative cancer. Higher number of births (1, 2, and 3+) was associated with lower FOXA1 protein expression in ER+ tumors [differences in H score, or β = −8.5; 95% confidence interval (CI), −15.1 to −2.0], particularly among parous women who never breastfed (β = −10.4; 95% CI, −19.7 to −1.0), but not among those who breastfed (β = −7.5; 95% CI, −16.9 to 1.8). The associations for ER− tumors were similar, although they were not statistically significant. Conclusions: In this tumor-based study, higher parity was associated with lower FOXA1 expression in ER+ tumors, and breastfeeding may ameliorate the influence. Impact: These findings contribute to our understanding of FOXA1 methylation and breast cancer etiology.

Cigarette smoking has been consistently associated with increased risk of overall mortality, but the importance of smoking for patients with prostate cancer (CaP) who are candidates for curative radical prostatectomy (RP) has received less attention. This retrospectively designed cohort study investigated the association of smoking history at RP with subsequent CaP treatment outcomes and overall mortality. A total of 1981 patients who underwent RP at Roswell Park Cancer Institute (RPCI) between 1993 and 2014 were studied. Smoking history was considered as a risk factor for overall mortality as well as for currently accepted CaP treatment outcomes (biochemical failure, treatment failure, distant metastasis, and disease‐specific mortality). The associations of smoking status with these outcomes were tested by Cox proportional hazard analyses. A total of 153 (8%) patients died during follow‐up. Current smoking at diagnosis was a statistically significant predictor of overall mortality after RP (current smokers vs. former and never smokers, hazards ratio 2.07, 95% confidence interval [CI]: 1.36–3.14). This association persisted for overall mortality at 3, 5, and 10 years (odds ratios 2.07 [95% CI: 1.36–3.15], 2.05 [95% CI: 1.35–3.12], and 1.8 [95% CI: 1.18–2.74], respectively). Smoking was not associated with biochemical failure, treatment failure, distant metastasis, or CaP‐specific mortality, and the association of smoking with overall mortality did not appear to be functionally related to treatment or biochemical failure, or to distant metastasis. Smoking is a non‐negligible risk factor for death among CaP patients who undergo RP; patients who smoke are far more likely to die of causes other than CaP.

// James R. Marshall 1 , Raymond F. Burk 2 , Rochelle Payne Ondracek 1 , Kristina E. Hill 2 , Marjorie Perloff 3 , Warren Davis 1 , Roberto Pili 4 , Saby George 1 , Raymond Bergan 5 1 Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 2 Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University Medical Center, C2104 Medical Center North, Nashville, TN 37232, USA 3 National Cancer Institute, Bethesda, MD 20892, USA 4 Department of Medicine, Indiana University School of Medicine, R3 C516, Indianapolis, IN 46202, USA 5 Knight Cancer Institute, Oregon Health Sciences University, Portland, OR 97239, USA Correspondence to: James R. Marshall, email: james.marshall@roswellpark.org Keywords: selenium, selenomethionine, methyl selenocysteine, chemoprevention, pharmacokinetics Received: December 01, 2016 Accepted: February 06, 2017 Published: February 17, 2017 ABSTRACT According to the Nutritional Prevention of Cancer (NPC) trial, a selenized yeast supplement containing selenium, 200 mcg/day, decreased the incidence of total cancer, cancers of the prostate, colon and lung, and cancer mortality. The active agent in the selenized yeast supplement was assumed to be selenomethionine (SEMET), although the supplement had not been well speciated. The SELECT study, largely motivated by the NPC trial, enrolling nearly 40 times as many subjects, showed unequivocally that selenium 200 mcg/day, with selenium in the form of SEMET, does not protect selenium-replete men against prostate or other major cancer. The agent tested by SELECT, pure SEMET, could have been different from the selenized yeast tested in NPC. One of the selenium forms suspected of having chemopreventive effects, and which may have been present in the NPC agent, is methyl selenocysteine (MSC). This study, with 29 selenium-replete patients enrolled in a randomized, double-blind trial, compared the multiple-dose toxicity, pharmacokinetics and pharmacodynamics of MSC and SEMET. Patients were on trial for 84 days. No toxicity was observed. Although SEMET supplementation increased blood selenium concentration more than MSC did, neither form had a more than minimal impact on the two major selenoproteins: selenoprotein P(SEPP1) and glutathione peroxidase(GPX).

Energy imbalance has an important role in breast cancer prognosis. Hyperactive mechanistic Target of Rapamycin (mTOR) pathway is associated with breast tumor growth, but the extent to which body fatness is associated with mTOR pathway activities in breast cancer is unclear. We performed immunostaining for mTOR, phosphorylated (p)-mTOR, p-AKT, and p-p70S6K in tumor tissue from 590 women (464 African Americans/Blacks and 126 Whites) with newly diagnosed invasive breast cancer in the Women’s Circle of Health Study. Anthropometric measures were taken by study staff, and body composition was measured by bioelectrical impedance analysis. Linear regressions were used to estimate percent differences in protein expression between categories of body mass index (BMI), waist circumference, waist/hip ratio, fat mass, fat mass index, and percent body fat. We observed that BMI ≥ 35.0 vs. 2 was associated with 108.3% (95% CI = 16.9%–270.9%) and 101.8% (95% CI = 17.0%–248.8%) higher expression in p-mTOR and normalized p-mTOR, i.e., p-mTOR/mTOR, respectively. Quartiles 4 vs. 1 of waist/hip ratio was associated with 41.8% (95% CI = 5.81%–89.9%) higher mTOR expression. Similar associations were observed for the body fat measurements, particularly in patients with estrogen receptor-negative (ER−) tumors, but not in those with ER+ tumors, although the differences in associations were not significant. This tumor-based study found positive associations between body fatness and mTOR pathway activation, evident by a p-mTOR expression, in breast cancer. Our findings suggest that mTOR inhibition can be a treatment strategy to prevent the recurrence of these tumors in obese individuals.

e13608 Background: Literature regarding racial differences in tumor immune responses in breast cancer remains sparse. To address this research gap, we assessed CD8+ T-cells in breast tumor samples from the Women’s Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We characterized and compared the density of CD8+ T cells, their prognostic value and their association with pharmacologic beta-blockers. Prior studies suggest that reducing adrenergic signaling through beta blockers can stimulate CD8+ T-cells. Methods: Tumor-infiltrating CD8+ T-cells were assessed by IHC staining of tissue microarray cores from 688 breast cancer patients, including 550 Blacks and 138 Whites. CD8+ T cells were scored with digital image analysis. Comparisons of demographic and clinical variables (including CD8+ T cell density) were made using the Mann-Whitney U or Kruskal-Wallis and Fisher’s exact tests. Associations with overall survival (OS) and disease-specific (DSS) survival were evaluated using the log-rank test of Cox regression. Analyses were performed in the overall sample and by disease sub-type. Results: Higher CD8+ T cell density was seen in Black women compared to White, with mean values of 756.2/mm2 and 292.4/mm2 respectively (p < 0.001). Within the overall population and in black women, CD8+ T cell density was significantly higher in younger patients, patients with high grade, and ER negative tumors. No significant associations were observed between CD8+ T cell density and OS or DSS. However, when stratified by subtype, Black patients with triple negative breast cancer and high CD8+ T cell density showed a trend towards improved OS in comparison to patients with low CD8+ T cell density (p = 0.065). 170 patients with information on beta blocker usage were analyzed. No significant associations were noted between CD8+ T cell density and beta blocker use. Conclusions: We observe a significantly higher CD8+ T cell density in Blacks compared to Whites, but this does not confer a survival advantage. Additionally, beta-blockers did not seem to enhance CD8+ T cell infiltration in tumors from Black patients. Our data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups. Future studies are needed to determine the functional properties of CD8+ T cells in Black women and to characterize additional immune cell subtypes that may also play a role.

Background Breast cancer subtype can be classified using standard clinical markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)), supplemented with additional markers. However, automated biomarker scoring and classification schemes have not been standardized. The aim of this study was to optimize tumor classification using automated methods in order to describe subtype frequency in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Methods Using immunohistochemistry (IHC), we quantified the expression of ER, PR, HER2, the proliferation marker Ki67, and two basal-like biomarkers, epidermal growth factor receptor (EGFR) and cytokeratin (CK)5/6, in 1381 invasive breast tumors from African American women. RNA-based (prediction analysis of microarray 50 (PAM50)) subtype, available for 574 (42%) cases, was used to optimize classification. Subtype frequency was calculated, and associations between subtype and tumor characteristics were estimated using logistic regression. Results Relative to ER, PR and HER2 from medical records, central IHC staining and the addition of Ki67 or combined tumor grade improved accuracy for classifying PAM50-based luminal subtypes. Few triple negative cases (

Author(s): Cheng, Ting-Yuan David; Darke, Amy K; Redman, Mary W; Zirpoli, Gary R; Davis, Warren; Payne Ondracek, Rochelle; Bshara, Wiam; Omilian, Angela R; Kratzke, Robert; Reid, Mary E; Molina, Julian R; Kolesar, Jill M; Chen, Yuhchyau; MacRae, Robert M; Moon, James; Mack, Philip; Gandara, David R; Kelly, Karen; Santella, Regina M; Albain, Kathy S; Ambrosone, Christine B | Abstract: Background:To what extent steroid hormones contribute to lung cancer in male and female never smokers and smokers is unclear. We examined expression of hormone receptors in lung tumors by sex and smoking. Methods:Patients with primary non-small cell lung cancer were recruited into an Intergroup study in the United States and Canada, led by SWOG (S0424). Tumors from 813 cases (450 women and 363 men) were assayed using immunohistochemistry for estrogen receptor (ER)-α, ER-β, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Linear regression was used to examine differences in expression by sex and smoking status. Cox proportional hazard models were used to estimate survival associated with the receptors. All statistical tests were two-sided. Results:In ever smokers, postmenopause and oral contraceptive use were associated with lower nuclear ER-β (P = .02) and total (nuclear + cytoplasmic) PR expression (P = .02), respectively. Women had lower cytoplasmic ER-α (regression coefficient [β], or differences in H-scores = -15.8, P = .003) and nuclear ER-β (β = -12.8, P = .04) expression than men, adjusting for age, race, and smoking. Ever smokers had both higher cytoplasmic ER-α (β = 45.0, P l .001) and ER-β (β = 25.9, P l .001) but lower total PR (β = -42.1, P l .001) than never smokers. Higher cytoplasmic ER-α and ER-β were associated with worse survival (hazard ratio = 1.73, 95% confidence interval [CI] = 1.15 to 2.58, and HR = 1.59, 95% CI = 1.08 to 2.33, respectively; quartiles 4 vs 1). Conclusions:Lower expression of nuclear ER-β in women supports the estrogen hypothesis in lung cancer etiology. Increasing cytoplasmic ER-α and ER-β and decreasing PR protein expression may be mechanisms whereby smoking disrupts hormone pathways.

Background Minimizing the time between tissue devascularization in robot-assisted laparoscopic radical prostatectomy (RALP) and tissue procurement should produce the highest quality tissue for research study. This study examines the relationship between intra-operative time and two indicators of tissue integrity: number of epithelial cells per gram of tissue and RNA integrity numbers (RINs). The study also compares the RIN values of tissue obtained intra-operatively by biopsy, before and after devascularization, to those from RALP specimen tissue, obtained through the routine research tissue procurement process. Methods Prostate tissues from two series of patients were analyzed. In the first, tissue from 18 patients undergoing RALP was analyzed for number of epithelial cells per gram of tissue. In the second, RIN values of tissue from 46 patients involved in a clinical study were analyzed. RIN values were assessed from RALP specimen tissue as well as tissue removed intra-operatively by biopsy, before and after devascularization. Results Time from RALP to tissue procurement was not significantly associated with number of epithelial cells per gram of tissue or with RIN values. RINs of biopsy tissue obtained intra-operatively before and after devascularization were similar. However, the RIN values of tissue from RALP specimens were significantly higher than those of biopsy tissue obtained either before or after devascularization. Conclusions Tissue quality, defined by number of epithelial cells or RIN values, was not affected by time between devascularization and procurement. Obtaining tissue from intra-operative biopsies, either before or after devascularization, is not necessary and actually produced lower RINs than found in tissue from RALP specimens, obtained through the routine research tissue procurement process. Prostate 75:1910–1915, 2015. © 2015 Wiley Periodicals, Inc.

Reproductive factors, particularly parity, have differential effects on breast cancer risk according to estrogen receptor (ER) status, especially among African American (AA) women. One mechanism could be through DNA methylation, leading to altered expression levels of genes important in cell fate decisions. Using the Illumina 450K BeadChip, we compared DNA methylation levels in paraffin-archived tumor samples from 383 AA and 350 European American (EA) women in the Women’s Circle of Health Study (WCHS). We combined 450K profiles with RNA-seq data and prioritized genes based on differential methylation by race, correlation between methylation and gene expression, and biological function. We measured tumor protein expression and assessed its relationship to DNA methylation. We evaluated associations between reproductive characteristics and DNA methylation using linear regression. 410 loci were differentially methylated by race, with the majority unique to ER− tumors. FOXA1 was hypermethylated in tumors from AA versus EA women with ER− cancer, and increased DNA methylation correlated with reduced RNA and protein expression. Importantly, parity was positively associated with FOXA1 methylation among AA women with ER− tumors (P = 0.022), as was number of births (P = 0.026), particularly among those who did not breastfeed (P = 0.008). These same relationships were not observed among EA women, although statistical power was more limited. Methylation and expression of FOXA1 is likely impacted by parity and breastfeeding. Because FOXA1 regulates a luminal gene expression signature in progenitor cells and represses the basal phenotype, this could be a mechanism that links these reproductive exposures with ER− breast cancer.

BACKGROUND Due to the indolent nature of prostate cancer, new prognostic measures are needed to identify patients with life threatening disease. SAM pointed domain-containing Ets transcription factor (SPDEF) has been associated with good prognosis and demonstrates an intimate relationship with the androgen receptor (AR), however its role in prostate cancer progression remains unclear. METHODS A tissue microarray constructed from cores of 713 consecutive radical prostatectomy specimens were immunohistochemically stained for SPDEF and correlated with progression free and metastatic free survival. In vitro studies assessed growth rate, migration, and sensitivity to bicalutamide to explore mechanisms behind the tissue microarray observations. RESULTS Patients with high SPDEF demonstrate longer metastases free survival after receiving the standard of care (HR = 9.80, P = 0.006). SPDEF expression corresponded with bicalutamide growth inhibition and apoptosis induction in all cell lines studied. In addition, a feedforward loop of AR-SPEF expression regulation is observed. CONCLUSIONS SPDEF may be clinically useful to identify patients who will have extended benefits from androgen deprivation therapy. In vitro observations suggest SPDEF mediates initial sensitivity to androgen deprivation therapy through both AR regulation and downstream events. Prostate 74:509–519, 2014. © 2013 Wiley Periodicals, Inc.

Background The Kattan postoperative radical prostatectomy (RP) nomogram is used to predict biochemical recurrence-free progression (BCRFP) after RP. However, external validation among contemporary patients using modern outcome definitions is limited. Methods A total of 1,931 patients who underwent RP at Roswell Park Cancer Institute (RPCI) between 1993 and 2014 (median follow-up, 47 months; range, 0-244 months) were assessed for NCCN-defined biochemical failure (BF) and RPCI-defined treatment failure (TF). Actual rates of biochemical failure-free survival (BFS; defined as 1 - BF) and treatment failure-free survival (TFS; defined as 1 - TF) were compared with Kattan BCRFP nomogram predictions. Results The Kattan BCRFP nomogram predictions at 5 and 10 years were predictive of BFS (area under the receiver operating characteristic curve [AUC], 0.772) and TFS (AUC, 0.774). The Kattan BCRFP nomogram tended to underestimate BFS and TFS compared with actual outcomes. The Kattan 5-year BCRFP predictions consistently overestimated actual 5-year BFS outcomes among subgroups of high- and intermediate-risk patients with at least 5-year outcomes. Conclusions The Kattan BCRFP nomogram is a robust predictor of NCCN-defined BF in a large sample of patients with RP with substantial follow-up and modern, standardized failure definitions.

Background: Women of African ancestry (AA) have higher breast cancer mortality than other US racial/ethnic groups, attributed to a number of social and economic factors. However, breast tumors in AA women are more likely to present with higher grade and lacking estrogen receptor (ER) expression compared to those from women of European ancestry (EA), suggesting the importance of tumor factors in breast cancer disparities. Emerging evidence suggests that stromal tumor-infiltrating lymphocytes (TIL-str) have a role in immune response to therapy and prognosis of breast cancer. The objective of this study was to examine associations between risk factors for breast cancer and TIL-str, with consideration of tumor characteristics. Methods: We reviewed the hematoxylin and eosin slides from surgical or biopsy tumor blocks of invasive breast cancer from 1,385 cases (986 AA and 399 EA women, aged 20-75 years) in the Women's Circle of Health Study, a multicenter case-control study in New York and New Jersey. In-person interviews were conducted at diagnosis to obtain data on demographics, family histories, hormone use, and reproductive and menstrual histories. Anthropometric measurements were taken by trained staff. TIL-str were scored as 0%, 1%, and 10% to 100% in increments of 10%. Linear regressions were performed to estimate differences in TIL-str scores (β). Results: In univariate analysis, TIL-str was higher in tumors from AA than those from EA women overall (mean score = 21.5% vs. 12.5; P Conclusion: Self-reported African compared to European ancestry is independently associated with higher TIL-str scores in breast tumors. Our findings suggest that immune response may play a role in the differences in breast cancer biology and outcomes between AA and EA women. (Funding: The Breast Cancer Research Foundation; NIH P01CA151135, R01CA100598, R01CA185623, P30CA072720, K07CA201334; US Army Medical Research and Material Command DAMD-17-01-1-0334) Citation Format: Ting-Yuan D. Cheng, Rochelle Payne Ondracek, Song Yao, Warren Davis, Angela Omilian, Mateusz Opyrchal, Elisa V. Bandera, Michael J. Higgins, Christine B. Ambrosone, Thaer Khoury. Differences in stromal tumor-infiltrating lymphocytes in breast cancer from women of African and European ancestry after accounting for tumor characteristics [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 3242.

Minimizing the time between tissue devascularization in robot-assisted laparoscopic radical prostatectomy (RALP) and tissue procurement should produce the highest quality tissue for research study. This study examines the relationship between intra-operative time and two indicators of tissue integrity: number of epithelial cells per gram of tissue and RNA integrity numbers (RINs). The study also compares the RIN values of tissue obtained intra-operatively by biopsy, before and after devascularization, to those from RALP specimen tissue, obtained through the routine research tissue procurement process.Prostate tissues from two series of patients were analyzed. In the first, tissue from 18 patients undergoing RALP was analyzed for number of epithelial cells per gram of tissue. In the second, RIN values of tissue from 46 patients involved in a clinical study were analyzed. RIN values were assessed from RALP specimen tissue as well as tissue removed intra-operatively by biopsy, before and after devascularization.Time from RALP to tissue procurement was not significantly associated with number of epithelial cells per gram of tissue or with RIN values. RINs of biopsy tissue obtained intra-operatively before and after devascularization were similar. However, the RIN values of tissue from RALP specimens were significantly higher than those of biopsy tissue obtained either before or after devascularization.Tissue quality, defined by number of epithelial cells or RIN values, was not affected by time between devascularization and procurement. Obtaining tissue from intra-operative biopsies, either before or after devascularization, is not necessary and actually produced lower RINs than found in tissue from RALP specimens, obtained through the routine research tissue procurement process.

Background: Forkhead box protein A1 (FOXA1) plays a key role in determining estrogen receptor (ER) function and mammary ductal development and may repress the basal cell phenotype during differentiation of breast epithelium. While reproductive factors are known to influence breast cancer risk depending on the subtypes, data on the association of tumor FOXA1 protein expression and reproductive characteristics are very limited. Methods: Tissue microarrays comprising surgical tumors from 638 women (466 African-American [AA] and 172 European-American [EA], aged 20-75 years) with primary breast cancer in the Women’s Circle of Health Study (WCHS) were analyzed for FOXA1 expression by immunohistochemistry and automated image analysis. In-person interviews were conducted to obtain data on demographics, medical and family histories, and reproductive and menstrual histories. Logistic regression was performed for FOXA1 positivity (>10% cells with strong staining) with menopausal status, age at menarche, age at first live birth, parity, and breastfeeding, adjusting for age at diagnosis, family history of breast cancer, and history of benign breast disease. Results: FOXA1 expression was higher in tumors from EA compared to those from AA women (80% vs. 70% positivity, P=0.011). FOXA1 expression was highly correlated with ER positivity (88% positive in ER+ and 26% in ER- breast cancer among AA women; 90% and 29% respectively among EA women; both P Conclusion: In AA women, a higher number of live births, which has been related to greater risk of ER- breast cancer, is associated with lower FOXA1 expression. Because FOXA1 promotes luminal and represses the basal differentiation, respectively, the effects of parity on expression of FOXA1 may be the link whereby it increases risk of ER- breast cancer. (Funding: NIH P01CA151135, R01CA100598, R01CA185623, P30CA072720, K07CA201334; US Army Medical Research and Material Command DAMD-17-01-1-0334; the Breast Cancer Research Foundation; and the Philip L. Hubbell family) Citation Format: Ting-Yuan David Cheng, Rochelle Payne Ondracek, Song Yao, Wiam Bshara, Thaer Khoury, Gary R. Zirpoli, Warren Davis, Elisa V. Bandera, Michael Higgins, Christine B. Ambrosone. Breast tumor FOXA1 protein expression and reproductive characteristics among African-American and European-American women [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 4998. doi:10.1158/1538-7445.AM2017-4998

Fatty-acid synthase (FASN), selectively overexpressed in prostate cancer (PCa) cells, has been described as linked to the aggressiveness of PCa. Constitutional genetic variation of the FASN gene and the expression levels of FASN protein in cancer cells could thus be expected to predict outcome after radical prostatectomy (RP). This study evaluates the associations of malignant tissue status, neoadjuvant androgen deprivation therapy (NADT) and single-nucleotide polymorphisms (SNPs) of FASN with FASN protein expression in prostate tissue. The study then examines the associations of FASN SNPs and gene expression with three measures of post-prostatectomy outcome.Seven tagging FASN SNPs were genotyped in 659 European American men who underwent RP at Roswell Park Cancer Institute between 1993 and 2005. FASN protein expression was assessed using immunohistochemistry. The patients were followed for an average of 6.9 years (range: 0.1-20.6 years). Outcome was assessed using three end points: biochemical failure, treatment failure and development of distant metastatic PCa. Cox proportional hazards analyses were used to evaluate the associations of the tagging SNPs and FASN expression with these end points. Bivariate associations with outcomes were considered; the associations also were controlled for known aggressiveness indicators.Overall, no SNPs were associated with any known aggressiveness indicators. FASN staining intensity was stronger in malignant than in benign tissue, and NADT was associated with decreased FASN staining in both benign and malignant tissue. The relationships of FASN SNPs and staining intensity with outcome were less clear. One SNP, rs4246444, showed a weak association with outcome. FASN staining intensity also showed a weak and seemingly contradictory relationship with outcome.Additional study with longer follow-up and populations that include more metastatic patients is warranted.

The majority of newly diagnosed prostate cancers will remain indolent, but distinguishing between aggressive and indolent disease is imprecise. This has led to the important clinical problem of overtreatment. THOC1 encodes a nuclear ribonucleoprotein whose expression is higher in some cancers than in normal tissue. The hypothesis that THOC1 may be a functionally relevant biomarker that can improve the identification of aggressive prostate cancer has not been tested.THOC1 protein immunostaining was evaluated in a retrospective collection of more than 700 human prostate cancer specimens and the results associated with clinical variables and outcome. Thoc1 was conditionally deleted in an autochthonous mouse model (n = 22 or 23 per genotype) to test whether it is required for prostate cancer progression. All statistical tests were two-sided.THOC1 protein immunostaining increases with higher Gleason score and more advanced Tumor/Node/Metastasis stage. Time to biochemical recurrence is statistically significantly shorter for cancers with high THOC1 protein (log-rank P = .002, and it remains statistically significantly associated with biochemical recurrence after adjusting for Gleason score, clinical stage, and prostate-specific antigen levels (hazard ratio = 1.61, 95% confidence interval = 1.03 to 2.51, P = .04). Thoc1 deletion prevents prostate cancer progression in mice, but has little effect on normal tissue. Prostate cancer cells deprived of Thoc1 show gene expression defects that compromise cell growth.Thoc1 is required to support the unique gene expression requirements of aggressive prostate cancer in mice. In humans, high THOC1 protein immunostaining associates with prostate cancer aggressiveness and recurrence. Thus, THOC1 protein is a functionally relevant molecular marker that may improve the identification of aggressive prostate cancers, potentially reducing overtreatment.

Due to the indolent nature of prostate cancer, new prognostic measures are needed to identify patients with life threatening disease. SAM pointed domain-containing Ets transcription factor (SPDEF) has been associated with good prognosis and demonstrates an intimate relationship with the androgen receptor (AR), however its role in prostate cancer progression remains unclear.A tissue microarray constructed from cores of 713 consecutive radical prostatectomy specimens were immunohistochemically stained for SPDEF and correlated with progression free and metastatic free survival. In vitro studies assessed growth rate, migration, and sensitivity to bicalutamide to explore mechanisms behind the tissue microarray observations.Patients with high SPDEF demonstrate longer metastases free survival after receiving the standard of care (HR = 9.80, P = 0.006). SPDEF expression corresponded with bicalutamide growth inhibition and apoptosis induction in all cell lines studied. In addition, a feedforward loop of AR-SPEF expression regulation is observed.SPDEF may be clinically useful to identify patients who will have extended benefits from androgen deprivation therapy. In vitro observations suggest SPDEF mediates initial sensitivity to androgen deprivation therapy through both AR regulation and downstream events.

BACKGROUND Epidemiological studies indicate that calcium channel blocker (CCB) use is inversely related to prostate cancer (PCa) incidence. The association between CCB use and PCa aggressiveness at the time of radical prostatectomy (RP) and outcome after RP was examined. METHODS Medication use, PCa aggressiveness and post-RP outcome were retrieved from a prospectively populated database that contains clinical and outcome for RP patients at Roswell Park Cancer Institute (RPCI) from 1993 to 2010. The database was queried for anti-hypertensive medication use at diagnosis for patients with ≥1 year follow-up. Recurrence was defined using NCCN guidelines. Chi-Square tests assessed the relationship between CCB use and PCa aggressiveness. Cox regression models compared the distribution of progression-free survival (PFS) and overall survival (OS) with adjustment for covariates. Results for association between CCB usage and PCa aggressiveness were validated using data from the population-based North Carolina-Louisiana Prostate Cancer Project (PCaP). RESULTS 48%, 37%, and 15% of RPCI's RP patients (n = 875) had low, intermediate, and high aggressive PCa, respectively. 104 (11%) had a history of CCB use. Patients taking CCBs were more likely to be older, have a higher BMI and use additional anti-hypertensive medications. Diagnostic PSA levels, PCa aggressiveness, and margin status were similar for CCB users and non-users. PFS and OS did not differ between the two groups. Tumor aggressiveness was associated with PFS. CCB use in the PCaP study population was not associated with PCa aggressiveness. CONCLUSIONS CCB use is not associated with PCa aggressiveness at diagnosis, PFS or OS. Prostate 73: 865–872, 2013. © 2012 Wiley Periodicals, Inc.

This study analyzed the nuclear stain of AR and Ki67 in a tissue microarray (TMA) using a custom-designed, fully automated ImagePro program, and correlated it with the results by the visual scorers. Using a red/green dot strategy, the positivity of nuclear Ki67 stain on 335 edited TMA core images was assessed by the automated ImagePro program and also by 3 independent visual scorers. …

Human evaluation of immuno-staining is limited by the subjectivity of the human evaluator, is time-consuming and is expensive. Semi-automated means of tissue evaluation have been proposed, but most of these have large subjective components. This study assesses semi-automated and newly-developed fully automated methods of evaluating Ki-67 staining of benign and malignant tissue from radical prostatectomy specimens. A research tissue microarray was constructed from 68 prostatectomy specimens and contained 3 core samples of benign and 3 of malignant tissue from each research subject. Tissue microarray sections were immune-stained for Ki-67. Two technicians (SG & CM) evaluated each TMA core and labeled the nuclei positive for Ki-67 with a green dot, and negative with a red dot. Images were collected and Image-Pro software was used to count the number of green and red dots. In a separate procedure, Image-Pro (IP) software was used to identify nuclei, and grade each nucleus as positive or negative, and the percentage of positive nuclei was calculated. The semi-automated results correlated with each other (0.58, P These results suggest that semi-automated and automated analysis of Ki-67 staining of prostate tissue produces comparable results, and automated analysis can be used for prostate cancer research. Citation Format: Rochelle P. Ondracek, James Marshall, Karin Kasza, Christopher Morrison, Simoya Ghajar, Carl Morrison, Diana Mehedint, James L. Mohler. Comparison of automated and a semi-automated methods for assessing Ki-67 staining. [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 4853. doi:10.1158/1538-7445.AM2013-4853

Although manual scoring is generally used to evaluate the presence of androgen receptor (AR) protein as detected by immunohistochemistry in tissue, manual evaluation is tedious, inefficient and subjective. Therefore, manual scoring is not feasible for the evaluation of large numbers of patients. In this study, a technician (CP) rated AR staining in tissue sections of 3 tissue microarrays (TMAs) constructed from 3 cores each of benign and malignant tissue from 134 patients who underwent radical prostatectomy between 1993 and 2006 at Roswell Park Cancer Institute. The manual scorer randomly sampled 50 nuclei from each core and scored each nuclei as not stained (score =0), or lightly (score = 1), moderately (score=2) or darkly (score = 3) stained. The average staining intensity was recorded per patient across the benign cores, and across the tumor cores. Automated staining used the Image Pro image processing program, in which the entire core was scanned, all nuclei segmented and average intensity values per between 0 (completely black) and 255 (completely white) were determined for each nucleus. The mean staining score for each core is calculated by taking the average of all the identified nuclei in the core, and an average intensity is calculated per patient across the benign cores, and across the malignant cores. The Pearson correlation between manual and automated scoring was 0.76. When stratified by benign or malignant tissue, the Pearson correlation between manual and automated scoring was 0.75 (benign) and 0.78 (malignant). Automated scoring of AR staining is comparable to manual scoring of AR in prostate tissue. In light of the greater efficiency of automated scoring, future evaluations of immunohistochemical staining may be usefully conducted using automated scoring of nuclei. Citation Format: Rochelle P. Ondracek, James L. Mohler, Catherine Pilarz, Sarah McEvoy, Karin Kasza, Carl Morrison, James Marshall. Comparison of manual and automated scoring of androgen receptor staining in prostate tissue. [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 4852. doi:10.1158/1538-7445.AM2013-4852

e15203 Background: Body mass index (BMI) at time of surgery was determined among 715 radical prostatectomy patients. The association of BMI with a range of treatment outcomes was considered. Methods: The associations of BMI at time of radical prostatectomy (RP) with disease stage and aggressiveness and long-term outcome were evaluated among 715 patients treated with RP at Roswell Park Cancer Institute between 1993 and 2005. Clinical and pathological aggressiveness indicators included clinical Gleason sum and tumor stage (2002 TNM), highest preoperative PSA, pathological Gleason sum and tumor stage (2002 TNM) and surgical margin status. Ten post-RP recurrence definitions were considered: 1) PSA ≥ 0.2 ng/ml; 2) PSA ≥ 0.4 ng/ml (with 1 confirming value); 3) 1 or more post RP treatments (ADT, radiation, chemotherapy); 4) PSA doubling time < 12 months; 5) PSA doubling time < 9 months; 6) PSA doubling time < 6 months; 7) NCCN definition of PSA failure; 8) AUA definition of PSA failure; 9) diagnosis of metastatic CaP; and 10) death from CaP. Results: Of the 715 men, 33 developed metastatic prostate cancer, and 17 died of prostate cancer. 246 men had BMI ≥ 30. BMI was not significantly associated with clinical or pathological aggressiveness criteria. These analyses showed that there is a trend towards higher risk of the development of metastasis or death for men with BMI ≥ 30, although the association with high BMI and these failure types is not significant. With adjustment for the most significant tumor aggressiveness features (clinical Gleason sum, pathological tumor stage, pathological Gleason sum, and surgical margin status) in proportional hazards regression, men with BMI ≥ 30 had consistently lower risk for all definitions of recurrence except metastasis and death, although no hazard ratios were significant. In contrast, men with higher BMIs had higher risk for metastasis and death from prostate cancer, although neither association is statistically significant. Conclusions: Men with higher BMIs show similar to slightly reduced risk for PSA-based recurrence definitions. Men with higher BMIs had slightly higher risk, though not significant, for metastasis and death. These results seem to support theories that PSA is diluted in men with higher BMIs.

e15204 Background: Epidemiological studies indicate that the use of calcium channel blockers (CCB) is inversely related to prostate cancer (PCa) incidence. The goal of this study was to examine the association between CCB use and PCa aggressiveness at the time of radical prostatectomy (RP) or outcome after RP. Methods: Information on medication use, PCa aggressiveness and outcome after RP was retrieved from a prospective database that contains clinical and follow-up (FU) data for all men that have undergone RP at the Department of Urology at Roswell Park Cancer Institute since 1992. The database was queried for anti-hypertensive medication use at the time of diagnosis for all patients with ≥ 1 year FU. Prostate cancer aggressiveness (risk status) and recurrence were defined using NCCN guideline definitions. Cox regression models were performed to compare the distribution of progression-free survival (PFS) and overall survival (OS) with adjustment for covariates. Chi-Square test was used to assess the relationship between CCB use and PCa aggressiveness. Results: 875 men were included in the study. At diagnosis, mean age was 60 (SD ± 7) years and mean serum PSA value was 7.4 (SD ±7.4) ng/ml. 48%, 37%, and 15% of patients had low risk, intermediate risk, or high risk PCa, respectively. 104 (12%) had a history of CCB use. CCB users and non-users were similar by PSA at diagnosis (p=0.97) and tumor aggressiveness (p=0.88). Patients taking CCB were more likely to be older (p=0.023), have a higher BMI (p=0.006) and use additional anti-hypertensive medications (p

PSA has ability to detect prostate cancer (CaP) at an early stage, but many CaP tumors are slow to progress, and many patients are overtreated for a disease that will not become life-threatening. Since there remains no reliable way to distinguish indolent from aggressive disease, doctors who advise and patients on active surveillance worry about progression to incurable disease. Therefore, new prognostic tools must be developed to identify patients with potentially metastatic, life threatening CaP. Prostate derived Ets transcription factor (PDEF) has shown to be a master negative regulator of transcriptional networks controlling migration and invasion in many cancer tissues, including CaP. Therefore we studied the expression of PDEF in radical prostatectomy specimens to determine retrospectively whether PDEF expression is predictive of clinical outcome. Patients’ tumor PDEF expression was determined by immunohistochemical staining of a 724 patient tissue microarray (TMA). CaP patients were stratified into groups based on PDEF expression (high, mid, low, null) and a Cox proportional hazards model was used to compare clinical outcomes. Univariate analysis showed that loss of PDEF expression significantly increased risk of metastatic disease (HR = 5.38, PDEF mid; to HR = 43.48, PDEF null; p=0.0059), although it did not correlate with incidence of biochemical failure detected using the American Urological Association (AUA) or National Comprehensive Cancer Network (NCCN) definitions. This result was confirmed in the Kaplan-Meier survival analysis that showed that patients whose CaP were PDEF low/null had significantly shorter metastases-free survival (p=0.0151). Multivariate analysis also demonstrated CaP with low/null PDEF expression were associated with increased risk of developing metastatic disease (HR = 6.21/10.53), although it was not statistically significant, in part due to the low incidence of patients with metastatic disease in the TMA. Therefore, we studied PDEF and vimentin (stem-like marker, repressed by PDEF) tumor expression in a cohort of patients matched for Gleason score, stage, age, and follow-up who developed metastatic disease versus those who did not. Patients that developed metastatic disease had a significantly lower PDEF (3.9 vs 1.7, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3637. doi:1538-7445.AM2012-3637

Neoadjuvant androgen deprivation therapy (NADT) was championed for men treated with radical prostatectomy (RP) for high risk prostate cancer (CaP) in the early 1990s. Although NADT improved neither surgical margin positivity nor progression-free survival, it continues to be part of some clinical trials. The effect of NADT on long term outcome was evaluated using modern aggressiveness and failure criteria among 720 patients treated with RP at Roswell Park Cancer Institute between 1993 and 2005. Among RP patients, 125 received NADT. NCCN pre-surgery risk categories were low in 36 (29%), intermediate in 54 (43%), and high or very high in 35 (28%). Clinical and pathological aggressiveness indicators included clinical Gleason sum, clinical tumor stage (2002 TNM), highest preoperative PSA, pathological Gleason sum, pathological tumor stage (2002 TNM) and surgical margin status. Ten post-RP recurrence definitions were considered: 1) at least 1 detectable PSA, 2) at least 2 detectable PSAs, 3) PSA>0.2 ng/ml, 4) PSA>0.4 ng/ml, 5) at least 1 post RP treatment (chemotherapy, hormone, radiation), 6) PSA doubling time Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4664. doi:10.1158/1538-7445.AM2011-4664

While early stage prostate cancer (CaP) is unique in that many patients will take decades to progress to symptomatic disease; there exists a subset of patients whose disease may quickly become life-threatening. Unfortunately, due to the high incidence of CaP and clinicians’ inability to differentiate the majority of patients with disease that will never become symptomatic from the minority with aggressive life-threatening tumors, patients with CaP are often over-treated. Prognostic biomarkers, such as prostate specific antigen (PSA), have shown great efficacy in improving patients’ chances of surviving cancer by allowing physicians to identify the disease at an earlier stage, though there does not exist a biomarker for CaP that adequately differentiates disease that will quickly become metastatic. In order to facilitate prognosis, the expression of prostate derived Ets transcription factor (PDEF), a protein of significance to the progression of CaP, was studied. PDEF is expressed in normal epithelial cells and negatively regulates transcriptional networks controlling migration and invasion. Previous findings in breast cancer have also demonstrated that loss of PDEF expression results in upregulation of the antiapoptotic protein survivin, indicating a possible connection to growth and survival. Loss of PDEF expression has also been shown to increase the metastatic potential of several cancers. Patients PDEF expression was determined via immunohistochemistry staining of tissue microarray in set of over 700 archival CaP tissues taken at radical prostatectomy; and patients were subsequently stratified based on their tumor PDEF expression. The stratified CaP patients were then analyzed for the association of PDEF expression status with clinical outcomes. Univariate analysis showed that low PDEF expression is correlated with incidence of biochemical failure as defined by American Urological Association (AUA; OR=1.30, p=0.0493) or National Comprehensive Cancer Network (NCCN; OR=1.36, p=0.0164) definitions; and with metastatic disease incidence (OR=3.61, p=0.0047). Multivariate analysis also showed that low PDEF expression was associated with metastatic disease incidence (OR = 4.02, p=0.0146). Furthermore, Kaplan-Meier survival analysis showed that stratification of patients into subgroups based on PDEF expression (high, middle, low) was able to significantly predict relative time to biochemical failure (NCCN, p=0.0241) or metastatic disease (p=0.0084). This study demonstrates the potential usefulness of PDEF as a prognostic biomarker to differentiate those patients at the highest risk of developing life-threatening disease. We propose that the status of PDEF expression in CaP tissues could be used in conjunction with PSA to distinguish patients who are suitable for watchful waiting and patients who require aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2011-4173