Evaluation of Grading Estrogen Receptors in Breast Cancer Using Fully Automated Rapid Immunohistochemistry Based on Alternating-Current Electric Field Technology.

To investigate the relationship between hypermethylation of Runx3 gene promoter and estrogen receptor (ER) and the implications of Runx3 gene promoter hypermethylation in ER positive breast cancer. Western blot and RT-PCR were used to detect the protein and mRNA expression of Runx3 gene in breast cancer cell lines (MCF7 and SKBR3) and normal breast epithelium cell line (MCF10A). Immunohistochemical SP method was used to detect the expression of ER and Runx3 proteins in 113 tissue samples of breast cancer. Moreover, methylation specific PCR was used to detect RUN3 promoter methylation in cell lines MCF7, SKBR3, MCF10A and 113 tissue samples of breast cancer. Of the 3 cell lines, Runx3 protein and mRNA were detectable in MCF10A, but were absent in MCF7 and SKBR3. MCF7 had a high methylation status at Runx3 promoter, in contrast, MCF10A and SKBR3 showed unmethylated RUN3 promoter. Among the 113 cases of breast cancer, 68 cases were ER positive and 45 were negative. The positive rates of Runx3 protein expression in ER positive and negative tumors were 26.5% (18/68) and 66.7% (30/45), respectively (P<0.05). Runx3 promoter hypermethylation was seen in 82.4% (56/68) of ER positive breast cancer cases and 22.2% (10/45) of ER negative ones (P<0.05). Among 68 cases of ER positive cases, Runx3 promoter hypermethylation was positively correlated with the clinical tumor stage (OR=5.84, P<0.05). Runx3 gene promoter hypermethylation is present mainly in the ER positive breast cancers. Testing of Runx3 promoter methylation may provide additional reference for clinical stage and prognosis of breast cancer patients, especially in those with ER positive tumors.

This study aims to predict expression of estrogen receptor (ER) in breast cancer by radiomics. Firstly, breast cancer images are segmented automatically by phase-based active contour (PBAC) method. Secondly, high-throughput features of ultrasound images are extracted and quantized. A total of 404 high-throughput features are divided into three categories, such as morphology, texture and wavelet. Then, the features are selected by R language and genetic algorithm combining minimum-redundancy-maximum-relevance (mRMR) criterion. Finally, support vector machine (SVM) and AdaBoost are used as classifiers, achieving the goal of predicting ER by breast ultrasound image. One hundred and four cases of breast cancer patients were conducted in the experiment and optimal indicator was obtained using AdaBoost. The prediction accuracy of molecular marker ER could achieve 75.96% and the highest area under the receiver operating characteristic curve (AUC) was 79.39%. According to the results of experiment, the feasibility of predicting expression of ER in breast cancer using radiomics was verified.

e11535 Background: : About 50% of ER positive (ERpos) BC are resistant to hormone treatment. In absence of ligand, ERs are evenly distributed in nuclei in normal tissue. Upon ligand binding, ERs dimerizes and form a discrete focal subnuclear distribution pattern (FDP), which are associated with transcriptional activation of ER. This ER FDP is observed in BC. We hypothesized that in BC that the presence/absence of ER in the FDP could predict antiestrogen activity. This study describes an immunohistochemistry (IHC) method, by which a biomarker could be developed to investigate this hypothesis. Methods: 37 paraffin embedded, formalin fixed archived BC samples were processed using 4 ER-alpha antibodies (Ab) under different IHC conditions to develop and refine this biomarker, the ER nuclear morphology was analyzed with standard microscopy at x1000. Interpretation of the IHC slides was done by an experienced pathologist. Standard ER, progesterone receptor (PR), and Ki67 testing were done. Tumor grade was obtained from the patients’ records. Analysis of the different Ab was with the kappa statistics. Comparison of the IHC technique to immunofluorescence (IHF) was done on 8 samples. Results: Consistent with prior research observations, tumors had two ER nuclear morphologies: 1. Diffuse pattern (D) where the ER was distributed evenly in a fine granular pattern, 2. Aggregate pattern (A) where the ER is distributed in distinct clumps or aggregates. This defined 3 tumor phenotypes: A cells only (A) 1% (4/37), D cells only (D) 65% (24/37), and a heterogenous mix of A + D cells (AD) 18% (7/37), ER Neg 14% (5/37). IHC and IHF analysis for ER nuclear morphology was concordant. The ER activation status (A/AD or D) was not correlated with staining ER intensity, ERpos %, PRpos%, but was correlated with % Ki67 staining (p=0.09) and tumor grade (0.035). Conclusions: ERpos early BC biopsies can be grouped in two categories based on ER nuclear morphology: A group with diffuse and homogenous nuclear staining, and a different group with heterogeneous area of cells having a nuclear pattern consistent with a functional or activated ER. These observations warrant clinical pathological studies to determine if this biomarker is potentially predictive of clinical outcomes.

Background: Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are the most important predictive and prognostic biomarkers in breast cancer. The American Society of Clinical Oncology/College of American Pathologists recommends that the time from tumor removal to fixation (cold ischemic time) should be kept within 1 hour. Through this study we mean to review the actual cold ischemic time in real practice and analyze whether delayed formalin fixation effects immunohistochemical (IHC) testing results. Methods: Patients, who received surgery for invasive or in situ breast cancer in Seoul National University Hospital, Seoul, Korea between February and December 2013, were retrospectively reviewed. Cold ischemic time was calculated by extracting the time of formalin fixation from the time when surgery ended. All patients were equally divided into two groups (short ischemic group, long ischemic group) according to median cold ischemic time. Chi-square test was done for ER and PR positive/negative (0% negative, ≥1% positive) and student t-test was done for ER and PR percentage. Also χ2 test was done for HER2 positive/negative and scoring system ranging from 0 to 3+. Results: A total of 615 patients were included in this study. The median cold ischemic time was 2h 43min 4sec (range 6min 36sec – 84h 26min 20sec). Only 48 patients had a cold ischemic time shorter than 1 hour. No association between ER, PR expression and cold ischemic time was found in the χ2 test (p=0.581, p=0.954) and student t-test (p=0.648, p=0.978). As for HER2 expression, in the long ischemic group, there were significantly more patients with positive immunohistochemical testing results (χ2 test, p=0.016), and significantly higher grades in HER2 scoring system (χ2 test, p=0.022). Compared to IHC results, FISH testing for HER2 amplification showed no significant difference (χ2 test, n=145, p=0.500). This tendency was persisted when patients were divided into four groups by 25 quartile of cold ischemic time. Conclusions: Our findings show that the actual cold ischemic time in practice is longer than recommended guidelines. Despite that, ER, PR expression was not associated with cold ischemic time. As for HER2 expression, longer cold ischemic time was associated with more HER2 positive and higher HER2 score. But this tendency was not showed in FISH testing for HER2 amplification. Citation Format: Tae-Kyung Yoo, Hyeong-Gon Moon, Jisun Kim, Jun Woo Lee, Min Kyoon Kim, Eunshin Lee, Jongjin Kim, Wonshik Han, In-Ae Park, Dong-Young Noh. Effect of prolonged cold ischemic time on immunohistochemical testing of estrogen receptor, progesterone receptor and HER2 expression in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-10.

Identification of molecular signatures that allow detection of the transition from normal breast epithelial cells to malignant invasive cells is a critical component in the development of diagnostic, therapeutic, and preventative strategies for human breast cancer. Substantial efforts have been devoted to deciphering breast cancer etiology at the genome level, but only a limited number of studies have appeared at the proteome level. In this work, we compared individual in situ proteome profiles of nonpatient matched nine noncancerous, normal breast epithelial (NBE) samples with nine estrogen receptor (ER)-positive (luminal subtype), invasive malignant breast epithelial (MBE) samples by combining laser capture microdissection (LCM) and quantitative shotgun proteomics. A total of 12,970 unique peptides were identified from the 18 samples, and 1623 proteins were selected for quantitative analysis using spectral index (SpI) as a measure of protein abundance. A total of 298 proteins were differentially expressed between NBE and MBE at 95% confidence level, and this differential expression correlated well with immunohistochemistry (IHC) results reported in the Human Protein Atlas (HPA) database. To assess pathway level patterns in the observed expression changes, we developed protein set enrichment analysis (PSEA), a modification of a well-known approach in gene expression analysis, Gene Set Enrichment Analysis (GSEA). Unlike single gene-based functional term enrichment analyses that only examines pathway overrepresentation of proteins above a given significance threshold, PSEA applies a weighted running sum statistic to the entire expression data to discover significantly enriched protein groups. Application of PSEA to the expression data in this study revealed not only well-known ER-dependent and cellular morphology-dependent protein abundance changes, but also significant alterations of downstream targets for multiple transcription factors (TFs), suggesting a role for specific gene regulatory pathways in breast tumorigenesis. A parallel GOMiner analysis revealed both confirmatory and complementary data to PSEA. The combination of the two annotation approaches yielded extensive biological feature mapping for in depth analysis of the quantitative proteomic data.

Introduction: Breast cancer incidence in African American women (AAW) in the U.S. is now equal to that in European American women (EAW). Despite having the highest survival rates of all breast cancer subtypes, AAW are nearly twice as likely to die from hormone receptor-positive (HR+) disease compared to EAW. ER+/HER2- breast cancer is a heterogeneous subtype, with tumors showing anywhere from 1-100% ER staining by immunohistochemistry. AAW are also more likely to have a lower proportion of HR+ cells within their breast tumor compared to EAW, and tumors with lower proportions of HR+ cells are associated with worse survival. While this is consistent with the fact that AAW are more likely to have aggressive breast cancer subtypes like triple negative breast cancer, our understanding of epidemiologic risk factors associated with various levels of ER staining is limited particularly among scientifically underrepresented populations. Therefore, our objective was to identify predictors associated with estrogen receptor staining levels in AAW. Methods: Questionnaire and population-based registry data from 748 female HR+ breast cancer survivors enrolled in the Detroit Research on Cancer Survivors (ROCS) study were analyzed. Risk factors of interest included self-reported demographics, socioeconomic characteristics, and health behaviors. Individual and combined estrogen receptor (ER) and progesterone (PR) percent staining levels were obtained from medical record review. A combined hormone report (HR) staining level variable was created based on the maximum percent staining among ER and PR. Associations between risk factors and HR staining levels were evaluated using chi-squared tests, and univariable and multivariable linear regression. An alpha value of 0.05 was set to determine statistical significance. Results: In our cohort, a majority of patients (86.6%), had tumors that were characterized as high HR staining (&amp;gt;90% of tumor cells HR+). A small percentage of tumors were found to be in the low (1-10% HR+: 6.6%) or medium (11-90% HR+: 6.8%) staining categories. When looking at each variable individually, we saw that demographic and medical history variables were associated with percent HR staining levels, including, age at diagnosis (beta=0.038,p&amp;lt;0.001), diabetes (beta=0.052,p= 0.03), high blood pressure (beta=0.060,p= 0.0037) and high cholesterol (beta=0.052,p= 0.028). When stratified by HER2 status, these associations remained similar in effect size and statistical significance only among HER2- patients. In a multivariable model, percent HR staining was associated with age at diagnosis (beta=0.002,p= 0.031), HER2+ status (beta= -0.15, p&amp;lt;0.001), marital status (beta= -0.016,p= 0.084) and nulliparity (b=0.073,p=0.012). Conclusions: Our findings identify multiple risk factors that are associated with weakly positive ER tumors. As AAW die more frequently from ER+/HER- weak breast cancers, these findings have potential application in improving future screenings and providing optimal therapeutics, resulting in a better prognosis among this cohort. Citation Format: Abigail M. Fielder, Jaclyn Kyko, Mrudula Nair, Julie Ruterbusch, David Gorski, Michael Simon, Lauren Hamel, Ann G. Schwartz, Kristen S. Purrington. Predictors of estrogen receptor staining levels in African American women with ER+ breast cancer in the Detroit Research on Cancer Survivors study [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B068.

A semi-automated imaging system is described to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer. The system works for any conventional method of image acquisition using microscopic slides that have been processed for immunohistochemical analysis of the estrogen receptor and progesterone receptor. Estrogen receptor and progesterone receptor immunohistochemical staining produce colorimetric differences in nuclear staining that conventionally have been interpreted manually by pathologists and expressed as percentage of positive tumoral nuclei. The estrogen receptor and progesterone receptor status of human breast cancer represent important prognostic and predictive markers of human breast cancer that dictate therapeutic decisions but their subjective interpretation result in interobserver, intraobserver and fatigue variability. Subjective measurements are traditionally limited to a determination of percentage of tumoral nuclei that show positive immunoreactivity. To address these limitations, imaging algorithms utilizing both colorimetric (RGB) as well as intensity (gray scale) determinations were used to analyze pixels of the acquired image. Image acquisition utilized either scanner or microscope with attached digital or analogue camera capable of producing images with a resolution of 20 pixels /10 mu. Areas of each image were screened and the area of interest richest in tumour cells manually selected for image processing. Images were processed initially by JPG conversion of SVS scanned virtual slides or direct JPG photomicrograph capture. Following image acquisition, images were screened for quality, enhanced and processed. The algorithm-based values for estrogen receptor and progesterone receptor percentage nuclear positivity both strongly correlated with the subjective measurements (intraclass correlation: 0.77; 95% confidence interval: 0.59, 0.95) yet exhibited no interobserver, intraobserver or fatigue variability. In addition the algorithms provided measurements of nuclear estrogen receptor and progesterone receptor staining intensity (mean, mode and median staining intensity of positive staining nuclei), parameters that subjective review could not assess. Other semi-automated image analysis systems have been used to measure estrogen receptor and progesterone receptor immunoreactivity but these either have required proprietary hardware or have been based on luminosity differences alone. By contrast our algorithms were independent of proprietary hardware and were based on not just luminosity and colour but also many other imaging features including epithelial pattern recognition and nuclear morphology. These features provide a more accurate, versatile and robust imaging analysis platform that can be fully automated in the near future. Because of all these properties, our semi-automated imaging system 'adds value' as a means of measuring these important nuclear biomarkers of human breast cancer.

Background: The expression of ER, PR and HER2 in breast cancer is determined by routine pathology assessment to indicate systemic treatment. Here, we investigated the ability of a standardized mRNA-based assay to predict ER, PR and HER2 status and treatment response. Methods: ESR1, PGR and ERBB2 expression was analyzed from the standardized nCounter-based PAM50 assay in 1,544 FFPE breast tumors obtained from 13 independent studies (NeoEribulin, GEICAM 2012-09, TBCRC023/006, LPT109096, EGF117165, PAMELA, PerELISA, GEICAM 2003-11, VENTANA, IBIMA and HCB). All immunohistochemical and in situ hybridization analyses followed ASCO/CAP criteria and were performed in central labs except for Neoeribulin and VENTANA studies. To explore the best cutoff for each gene, we used Monte Carlo cross validation (repeated 1000 times, 2/3 training, 1/3 testing) to achieve the highest kappa values when gene expression was compared to pathology assessment. Receiver operating characteristic analysis and area under the ROC curve (AUC) was used to evaluate the performance of each gene to predict ER, PR and HER2 status. Finally, the association of each gene (using the pre-established cutoffs) with pathological complete response (pCR) was evaluated using univariate logistic regression analyses in 2 neoadjuvant cohorts: 1) A combined HER2+ cohort using 191 tumor samples from PAMELA/PerELISA phase II trials, where patients received neoadjuvant dual HER2 blockade without chemotherapy for 15-18 weeks and 2) a combined cohort of hormone receptor-positive/HER2-negative using 205 tumor samples from IBIMA/HCB consecutive series, where patients received anthracycline/taxane-based chemotherapy. Results: Concordance between ESR1 and ER was 95.3% (95% confidence interval [95CI] 94.0-96.4%; AUC=0.98). ER+ and ER- cases were classified as ESR1- and ESR1+ in 5.5% and 4.8% of the cases, respectively. Concordance between PR and PGR was 85.2% (95CI 83.1-87.1%; AUC=0.95), and between ERBB2 and HER2 was 92.8% (95CI 91.4-94.1%; AUC=0.95). HER2+ and HER2- cases were classified ERBB2- and ERBB2+ in 16.7% and 2.9% of the cases, respectively. In the neoadjuvant HER2+ cohort, the pCR rates were 44.6% in ESR1- and 18.8% in ESR1+ (odds ratio [OR] 3.9; 95CI 1.9-8.4; p&amp;lt;0.001), 38.5 in PGR- and 15.8% in PGR+ (OR 3.2; 95CI 1.6-6.7; p=0.001), 2.6% in ERBB2- and 35.3% in ERBB2+ (OR 19.7; 95CI 2.6-149.1; p&amp;lt;0.001). In the neoadjuvant HR+/HER2- cohort, the pCR rates were 40% in ESR1- and 5.12% in ESR1+ (OR 11.5; 95IC 2.7-48.5; p&amp;lt;0.001), 15.5% in PGR- and 4.3% in PGR+ (OR 3.8; 95IC 1.3-11.7; p=0.019), 7.36% in ERBB2- and 0% in ERBB2+ (OR NA). Finally, ESR1, PGR and ERBB2 expression as continuous variables were found significantly associated with pCR in both cohorts. Conclusion: ESR1, PGR and ERBB2 standardized mRNA levels show high concordance with ER, PR and HER2, and might provide a more objective and quantitative prediction of response to chemotherapy and anti-HER2 therapy. The level of concordance between GE and pathology-based assessments is similar to 2 FDA 510(k) cleared pathology-based ER assays (e.g. SP1 versus 6F11 antibody clones), local versus central HER2 testing, and ER or PR status when determined by the ligand-binding assay versus immunohistochemistry. Citation Format: Pascual T, Tsai YS, Martín M, Lluch A, Cortes J, Llombart A, Conte P, Guarneri V, Rimawi MF, Alba E, Ruiz-Borrego M, Rojo F, de la Haba J, Schiff R, Adamo B, Vidal M, Paré L, Chic N, Muñoz M, Galvan P, Gonzalez-Farre B, Brauer HA, Sullivan A, Nuciforo P, Parker JS, Prat A. Standardized nCounter-based determination of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor status in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-06.

Background: Multi-gene assays have informed the treatment of patients (pts) with estrogen receptor positive (ER+) and lymph node negative (LN-) breast cancer (BC). For pts with tumors in the 21-gene RS intermediate range (RS18-30), the benefit of adjuvant chemotherapy (chemo) added to endocrine therapy is uncertain. The 70-GS provides prognostic information by classifying LN+ and LN-, ER+ and ER- tumors as low or high risk though lacks predictive validation in phase III trials. Our primary objective was to perform the 70-GS on a large consecutive series of intermediate RS with known adjuvant treatment recommendations to evaluate the distribution of treatment recommendations by 70-GS risk category. Other objectives were to identify how many pts with intermediate RS are classified by the 70-GS as low or high risk; compare microarray-based assessment of ER, PR, and HER2 with ER, PR and HER2 by conventional immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and the 21-gene RS assay; and to compare clinical subtyping based on ER, PR, HER2 and Ki-67 with the 80-gene molecular subtyping signature. Methods: All consecutive ER+LN- BC cases with an intermediate 21-gene RS obtained at Loyola University Medical Center from 1/2005 - 9/2012 were analyzed. All adjuvant treatment recommendations were made after obtaining the 21-gene RS. The 70-GS, micro-array assessment of ER, PR and HER2, and molecular subtyping were performed on paraffin embedded tumor blocks. Standard descriptive statistical methods were employed. Results: 102 samples were analyzed. The 3 assays were successfully performed in 89 cases. Median RS was 21, range 18-30. 50 (56.2%) samples were 70-GS high risk; 39 (43.8%) were low risk. The table below shows the distribution of adjuvant chemotherapy recommendations made after obtaining the 21-gene RS. Of the 39 pts with low risk 70-GS results, 14 had been recommended chemo. Of the 50 high risk pts, 34 had been recommended no chemo. Concordance of microarray-based assessment of ER with the 21-gene ER test was 54/57 and 85/89 with ER by IHC. Concordance of microarray-based assessment of HER2 with the 21-gene HER2 test was 49/50, 87/89 with HER2 by IHC, and 87/89 by FISH. The 89 ER+HER2- clinical subtypes were re-categorized as 39 luminal A, 48 luminal B, 1 basal and 1 HER2 molecular subtypes. Conclusions: Within the 21-gene RS intermediate range, there is a mix of 70-GS high and low risk results. There is good concordance of microarray-based assessment of ER and HER2 with the 21-gene ER and HER2 test; with ER by IHC, and HER2 by IHC and FISH. The majority of the clinical ER+HER2- cases were categorized as molecular subtypes luminal A and B. The optimal adjuvant treatment recommendation for pts with intermediate RS but either 70-GS high risk or low risk is uncertain. Results of completed phase III prospective studies (TAILORx and MINDACT) will clarify this critical treatment dilemma for patients and their physicians. Adjuvant Chemotherapy Recommendations in pts with intermediate RS by 70-GS Risk Category Chemotherapy RecommendedChemotherapy Not RecommendedTotal21-gene Intermediate RS30598970-GS Low Risk14253970-GS High Risk16345070-GS Total305989 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-13.

Estrogen and progesterone are critically involved in the development of breast cancer and their effects are mediated by the estrogen receptor (ER) and progesterone receptor (PR) respectively. The activities of ER and PR are regulated at multiple steps of steroid hormone receptor (SHR) signaling pathway. Post-translational modifications (PTMs) play pivotal roles in regulating SHR signaling, enable quick responses of these receptors to stimuli without the need for new protein synthesis. Initial mass spectrometry analysis of PTMs on ER identified multiple methylated arginines, suggesting that arginine methylation is an important player regulating ER activity. The objectives of the first part of the thesis were to identify protein arginine methyltransferases (PRMTs) that methylate ER and PR and to determine the functional relevance of the methylation. This study showed that PRMT6 binds to ER, PR and androgen receptor (AR), and methylates them independent of ligand. The site of the interaction and mode of regulation by PRMT6 seems to vary among the receptors but the role of PRMT6 in regulating ER activity has been further defined. PRMT6 binds to ER at the C-terminus domains (LBO-region F) and proximity ligation assays indicated that PRMT6 interacts with ER exclusively in the nucleus. PRMT6 overexpression enhances ER ligand-independent and ligand-induced activity via different mechanisms. PRMT6 significantly enhances the ligand-independent activity of ER without the need for its methyltransferase activity. We present evidences suggesting that the ligand-independent activation of ER by PRMT6 is mediated through its binding to ER which may lead to dissociation of chaperone heat shock protein 70 (Hsp70) and Hsp90. On the other hand, enhancement of ligang-induced ER activity requires enzymatically active PRMT6 and possible arginine residues targeted by PRMT6 have been identified. In light of the recent report that PRMT6 dysfunction is associated with worse prognosis in ER-positive breast cancers, our results suggest that PRMT6 may promote the mitogenic effect of ER both in the presence and absence of estrogen. The increase in ligand-independent ER signaling with high PRMT6 expression level may result in hormone-independent tumor growth which is resistant to the inhibitory effect of tamoxifen. In contrast to the mitogenic role of ER in breast cancer, PR can exert inhibitory effect on breast cancer growth. Progesterone-induced growth inhibition in PR-transfected MDAMB-231 cells was associated with a remarkable upregulation of tripartite motif-containing 22 (TRIM22), a p53 and interferon (lFN) target gene. The objective of the second part of the thesis work was to understand the regulation of TRIM22 gene expression and its clinical significance. TRIM22 protein level is consistently downregulated in breast cancer cell lines and breast tumor tissues as compared to non-malignant mammary epithelial cells and normal breast tissues, making it a potential marker for breast malignancy. TRIM22 expression also correlates with p53 expression in normal breast tissue and it can be induced upon p53 activation in non-malignant cell. However, in breast cancer cells, TRIM22 expression is not inducible in response to p53 activation by genotoxic drugs, suggesting that regulation of TRIM22 by p53 is specifically disrupted during tumorigenesis. In addition, TRIM22 is also a regulator of PR activity. It upregulates both the expression level and ligand induced activity of full length PR but inhibits ligandindependent activity of LBO truncation PRo Together with the reported growth inhibitory role of TRIM22 for leukemic cells, this study presents evidence that TRIM22 may inhibit the growth of breast cancer cells via p53 and PR signaling pathway. Further correlation of TRIM22 expression in clinical breast cancer samples with clinicopathological parameters will shed light on the prognostic value of TRIM22 in breast cancer.

Background: Breast cancer exhibits familiar aggregation. Such susceptibility is found to be largely polygenic-with susceptibility conferred by a large number of low-penetrance loci. We have been using a bioinformatics approach to identify potential functional genetic variants, reducing the test complexities and costs compared to Genome Wide Association Study. Several single nucleotide polymorphisms (SNPs) in human histone deacetylase (HDAC) 9 were identified via such approach to test for associations with breast cancer risk and prognosis. It is well known that estrogen receptor (ER) and progesterone receptor (PR) expression are important markers for prognosis and prediction of response to endocrine therapy. Recent studies and clinical trials suggest that inhibition of HDAC may represent a novel strategy to modulate ER expression through epigenetic regulation of ER-a. Methods: This study evaluated the association of human HDAC9 SNPs at three loci (rs2239926, rs2240279 and rs2286003) with age of onset and recurrence of breast cancer in a cohort of 1101 patients enrolled prospectively from 2004 to 2009 at The Cancer Institute of New Jersey. Genotyping was performed using an Applied Biosystems TaqMan assay on the ABI 7900HT Fast Real-Time PCR System. A permutation test was performed to determine the statistical significance of differences in mean age at diagnosis between different genotypes. Logistic regression model was used to determine odds ratios for recurrence. Results: To limit potential differences due to the ethnicity and breast cancer subtypes, the association between the HDAC9 genotypes and the age at diagnosis was evaluated in Caucasian women with ductal carcinomas. Of the 1101 patients, 516 patients were Caucasians with ductal carcinomas; among which 74.8% were ER(+) with a mean age of diagnosis of 53.7 years; 25.2% were ER(-) with a mean age of diagnosis of 48.4 years. For patients with ER(+) breast cancer, the homozygous variant CC at HDAC9 rs2239926 developed breast cancer at an earlier age (49.3 years) than G allele carriers (GG+CG) (54.1 years) such that the age at diagnosis was accelerated by 4.8 years (P = 0.043). Conversely, for patients with ER(-) breast cancer, the CC variant appears to develop breast cancer at a later age (53.7 years) than G allele carriers (48.0 years) such that the age at diagnosis was delayed by 5.7 years, although this comparison was statistically limited due to the fewer number of cases. SNPs at the two other HDAC9 loci (rs2240279 and rs2286003) were not shown to be associated with age of onset of breast cancer. However, the homozygous variant CC at rs2240279 exhibits a strong association with recurrence in Caucasians with PR(+) breast cancer: OR 4.1, 95% CI (1.18-7.85) (P = 0.01). Conclusions: A polymorphic variant of human HDAC9 rs2239926 associates with an earlier age of onset of ER positive breast cancer while rs2240279 associates with recurrence of PR positive disease. We hypothesize that such polymorphisms may modulate ER and/or PR and its downstream gene expression leading to alteration of the clinical features of breast cancer including age of diagnosis and natural biology. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-12-03.

567 Background: That tumor heterogeneity exists and evolves over time is well appreciated but how often to biopsy patients’ metastatic BC is not well established. Methods: Immunohistochemical (IHC) and in situ hybridization (ISH) analysis of estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), and HER2 in 337 cases with > 1 asynchronous primary/metastatic BC molecular profiles and in 40 cases with > 1 synchronous molecular profiles was performed at a single institution. We evaluated differences in ER, PR and HER2 status in same or contralateral breast, and in primary vs. locally recurrent or metastatic BC’s. Results: We identified a change in ER or HER2 status in 8 (31%) synchronous BCs and in 55 (16%) primary/recurrent BCs, including in biopsies of distinct tumor foci within the same breast or metastatic organ site. Of the 8 synchronous bilateral primary BC’s, 4 (50%) had discordant ER results (ER, PR, and HER2 negative [TN] vs. ER+); 5 of 18 (28%) with two or more metastatic foci ...

Women of African ancestry are more likely than women of European ancestry to be diagnosed with aggressive tumors characterized by negative staining for estrogen receptors (ER) and progesterone receptors (PR). Reasons for the disparity are unclear, and previous studies have lacked statistical power to investigate risk factors for the more aggressive subtypes. A recent report from the Carolina Breast Cancer Study indicated that basal-like breast cancer, a subtype of breast tumors with ER-, PR-, HER2-, HER1+ and/or CK5/6+ characteristics, was positively associated with increased parity, whereas other subtypes were inversely associated with parity. The increased risk of basal-like cancer was strongest for parous women who had never breastfed. The majority of breast tumors with negative staining for both ER and PR would likely be classified as basal-like. We examined the relation of parity and lactation to risk of ER-/PR- breast cancer among 59,000 African American women from the Black Women's Health Study in 12 years of follow-up data. Reproductive history was obtained by questionnaire at baseline in 1995 and updated every two years. ER and PR status were obtained from pathology reports and state cancer registry records. Included in this analysis were 809 incident breast cancer cases with complete data on ER and PR status, with 285 classified as ER-/PR-, 395 as ER+/PR+, 114 as ER+/PR-, and 15 as ER-/PR+. Incidence rate ratios (IRR) and 95% confidence intervals (CI) were derived from proportional hazard models for the relation of each reproductive factor to risk of specific tumor subtype, with control for all other reproductive factors, age, region, education, body mass index, waist-hip ratio, hormone use, alcohol, smoking, and physical activity. Risk of ER-/PR- breast cancer increased with increasing parity (p trend = 0.02), whereas risk of ER+/PR+ cancer decreased with increasing parity (p trend = 0.0002). For ER-/PR- breast cancer, the IRR for women who had 3+ births and never breastfed relative to nulliparous women was 1.66 (95% CI 1.09-2.54). The increased risk associated with higher parity was ameliorated by breast feeding: the comparable IRR for women who had 3+ births but breastfed at least one of the babies was 1.01 (95% CI 0.63-1.63). These results from a cohort of African American women support findings from the previous study of basal-like breast cancer and are consistent with the hypothesis that pregnancy and breastfeeding may influence risk of ER-/PR- cancer through inflammatory/immune system alterations linked to mammary gland involution. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5737.

Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)-positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110alpha and p110beta, in ER(+) breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110alpha RNAi inhibited growth and promoted apoptosis in all tested ER(+) breast cancer cells under estrogen deprived-conditions, whereas p110beta RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110alpha/p110beta inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER(+) breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110alpha and p110beta catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER(+) breast cancer.

Limited knowledge of the changes in estrogen receptor (ER) signaling during the transformation of the normal mammary gland to breast cancer hinders the development of effective prevention and treatment strategies. Differences in estrogen signaling between normal human primary breast epithelial cells and primary breast tumors obtained immediately following surgical excision were explored. Transcriptional profiling of normal ER+ mature luminal mammary epithelial cells and ER+ breast tumors revealed significant difference in the response to estrogen stimulation. Consistent with these differences in gene expression, the normal and tumor ER cistromes were distinct and sufficient to segregate normal breast tissues from breast tumors. The selective enrichment of the DNA binding motif GRHL2 in the breast cancer-specific ER cistrome suggests that it may play a role in the differential function of ER in breast cancer. Depletion of GRHL2 resulted in altered ER binding and differential transcriptional responses to estrogen stimulation. Furthermore, GRHL2 was demonstrated to be essential for estrogen-stimulated proliferation of ER+ breast cancer cells. DLC1 was also identified as an estrogen-induced tumor suppressor in the normal mammary gland with decreased expression in breast cancer. In clinical cohorts, loss of DLC1 and gain of GRHL2 expression are associated with ER+ breast cancer and are independently predictive for worse survival. This study suggests that normal ER signaling is lost and tumor-specific ER signaling is gained during breast tumorigenesis. Unraveling these changes in ER signaling during breast cancer progression should aid the development of more effective prevention strategies and targeted therapeutics.