Estrogen Receptor Protein Content Research Articles

Estrogen receptor protein content is different in abdominal than gluteal subcutaneous adipose tissue of overweight-to-obese premenopausal women

ObjectivePremenopausal women demonstrate a distinctive gynoid body fat distribution and circulating estrogen status is associated with the maintenance of this adiposity patterning. Estrogen's role in modulation of regional adiposity may occur through estrogen receptors (ERs), which are present in human adipose tissue. The purpose of this study was to determine regional differences in the protein content of ERα, ERβ, and the G protein-coupled estrogen receptor (GPER) between the abdominal (AB) and gluteal (GL) subcutaneous adipose tissue of overweight-to-obese premenopausal women. Materials/MethodsBiopsies of the subcutaneous AB and GL adipose tissue were performed in 15 premenopausal women (7 Caucasian/8 African American, 25.1±1.8years, BMI 29.5±0.5kg/m2). Adipose tissue protein content was measured by western blot analysis and correlation analyses were conducted to assess the relationship between ER protein content and anthropometric indices/body composition measurements. ResultsWe found that ERα protein was higher in AB than GL (AB 1.0±0.2 vs GL 0.67±0.1 arbitrary units [AU], P=0.02), ERβ protein was higher in GL than AB (AB 0.78±0.12 vs GL 1.3±0.2AU, P=0.002), ERα/ERβ ratio was higher in AB than GL (AB 1.9±0.4 vs GL 0.58±0.08AU, P=0.007), and GPER protein content was similar in AB and GL (P=0.80) subcutaneous adipose tissue. Waist-to-hip ratio was inversely related to gluteal ERβ (r2=0.315, P=0.03) and positively related to gluteal ERα/ERβ ratio (r2=0.406, P=0.01). ConclusionsThese results indicate that depot specific ER content may be an important underlying determinant of regional effects of estrogen in upper and lower body adipose tissue of overweight-to-obese premenopausal women.

ESR1 gene status correlates with estrogen receptor protein levels measured by ligand binding assay and immunohistochemistry

The Estrogen Receptor (ER) is an established predictive marker for the selection of adjuvant endocrine treatment in early breast cancer. During the 1990s Immunohistochemistry (IHC) replaced cytosol based assays for determination of ER status. This study examined the association between ER protein level determined by two different methods and ESR1 gene copy number. From 289 primary high-risk breast cancer patients, randomized in the Danish Breast Cancer Cooperative Group (DBCG) 77C trial, results from cytosolic ER levels were available from ligand binding assays. Archival tumor tissue was retrieved from 257 patients. ESR1/CEN-6 ratio was analyzed successfully by Fluorescence In Situ Hybridization (FISH) in 220 (86%) patients. ESR1 amplification (ESR1/CEN-6 ≥ 2.00) was observed in 23% of the patients and ESR1 deletion (ESR1/CEN-6 < 0.80) was observed in 32%. Further, we identified ESR1 gain (ratio ESR1/CEN-6 from 1.30 to 1.99) in 19% of the patients. A positive correlation of ESR1 FISH with both ER-cytosol and ER IHC was found (p < 0.0001). Amplification and gain of the ESR1 gene are associated with higher ER protein content measured by ligand binding assay and a more intense nuclear staining by IHC compared to tumors with normal ESR1 gene status. Major variations in ER measured by ligand binding assay and IHC are observed within all ESR1 copy number subgroups and other mechanisms than gene copy number seem to contribute to the ER protein content in the tumors.

A Quantitative Immunofluorescence Assay (AQUA) Suggests Significant Misclassification (15%) of Estrogen Receptor Status in Breast Cancer.

Abstract Introduction: Estrogen Receptor (ER) is arguably the most powerful predictive marker in breast cancer. However, a recent incident in Canada revealed a strikingly high false-negative rate and raised awareness of the current limitations in our measurement of ER. The current clinical standard is both subjective and qualitative. Even though guidelines are about to be issued to standardize this assay, there is very little data on the misclassification rate in current practice in the US.Hypothesis: Our hypothesis is that the use of a quantitative assay for ER on a series of retrospective collections may reveal both the level and significance of the misclassification rate.Method: Cell lines with a range of ER expression levels were analyzed by quantitative western blotting in parallel with IF/AQUA analysis (r2 = 0.865), in order to create standard curves for assessment of absolute ER protein concentration in tissue. The optimized assay was then used to quantify ER protein expression in a large cohort of archival breast cancer samples from Yale (1962-1982, n =617).Results: Using a set of standard curves with recombinant ER and cell line controls, we developed a standardized method for quantifying ER as an absolute concentration (pg ER per μg total protein) in formalin-fixed tissue on TMAs. This ER AQUA assay has a range in sensitivity from 50 pg/μg to 1500pg/μg total protein. Quantification of ER protein expression on the Yale archival cohort revealed a unimodal distribution with 49.8% of cases above the 50pg/ug threshold and thus defined as positive. When compared to pathologist performed conventional ER classification, we found a false negative rate of 6.65% and a false positive rate of 10.2%, for a total misclassification rate of 16.6%. Although no response data is available on that cohort, data is under analysis on 4 other cohorts with endocrine therapy treatment information, including an independent Yale cohort, SWOG 9313, NSABP B14, and the TEAM trial.Conclusion: We have developed a quantitative method to measure absolute levels of ER in breast tissue. Use of this assay on a series of cohorts suggests a misclassification rate in the 15% range. The significance of this level of misclassification with respect to response to endocrine therapy is currently under study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4068.

Evaluation of the false-negative rate of standardized and quantitative measurement of estrogen receptor (ER) in tissue using AQUA technology

567 Background: The discovery of an astoundingly high false negative rate for estrogen receptor (ER) testing in Canada has raised questions about the accuracy and reproducibility of ER testing. One solution would be the introduction of a standardized and reproducible diagnostic test that is easily adaptable in the clinical setting. Here, we have tested the AQUA method of quantitative immunofluorescence for the standardized and reproducible quantification of ER protein expression in tissue. Methods: Quantitative Western blotting was used in conjunction with AQUA analysis to create standard curves for assessment of absolute ER protein concentration in tissue (n = 118). We used standard scoring methods and AQUA analysis to quantify ER protein expression in a large cohort of breast cancer samples (n =669). Results: Using a series of standard curves, we determined that the range of the ER AQUA assay is between 100 pg/μg and 1500 pg/μg total protein. ER protein concentration in breast cancer samples showed an expected unimodal distribution for quantitative assessment of ER. Reproducibility studies of AQUA analysis demonstrated significant instrument-to-instrument (laboratory-to-laboratory) reproducibility for 3 instruments across the range of AQUA scores (average %CV = 1.34; R2&gt;0.99; ANOVA p = 0.67). The same cases were then read and classified by 3 pathologists using the Allred scoring system. Although their concordance is similar to that seen in the literature (Kappa = 0.81, 0.88, and 0.89), pathologist concordance rate is lower than for that observed with AQUA analysis (Kappa = 0.95, 0.96, and 0.97). Importantly, 9.0% of cases showed a change of diagnosis (positive/negative) across 3 pathologists whereas only 2.8% of cases changed classification using AQUA analysis. Additionally, misclassified cases occurred across the entire range of Allred scores, but were restricted to a narrow region defined by the cut-point for AQUA scoring. Conclusions: We have demonstrated that AQUA technology can provide for the standardized and reproducible quantification of ER with a 3 fold reduction in misclassification. This approach has the potential to decrease the problem of false negative tests for ER. [Table: see text]

Evidence of Gender- and Tissue-Specific Promoter Methylation and the Potential for Ethinylestradiol-Induced Changes in Japanese Medaka (Oryzias Latipes) Estrogen Receptor and Aromatase Genes

In order to explore the potential of DNA methylation to serve as a biomarker of toxicity, thus establishing a link between exposure to environmental contaminants and physiologically significant changes in gene expression, tissue- and gender-specific methylation patterns in the promoter regions of estrogen receptor (ER) and aromatase genes of Japanese medaka (Oryzias latipes) were determined. Adult male and female medaka were exposed to either 0 or 500 ng/L 17 α-ethinylestradiol (EE) for 14d via a waterborne exposure. Livers, gonads, and brains were removed and genomic DNA was extracted. Samples of genomic DNA were then analyzed by bisulfite-mediated methylation-specific polymerase chain reaction (PCR) of an ∼300-bp region containing suspected methylation sites from the two genes, amplified, cloned, and sequenced. ER protein content in exposed medaka was significantly induced in all male and female tissues compared to controls. Aromatase activity in exposed medaka was significantly increased in the male brain, testes, and female brain as compared to controls. The methylation changes described by these studies indicate the potential for anthropogenic alteration of the mechanisms controlling gene expression, as well as gender- and tissue-specific sensitivity. While methylation differences were not paralleled by changes in protein expression in this study, changes in methylation have the potential to impact the regulation of normal gene expression and these changes could be transmitted to offspring.

Endocrine regulation of cervical ripening in humans—potential roles for gonadal steroids and insulin-like growth factor-I

Cervical softening is crucial for a normal parturition and corresponds to remodeling of the dominating cervical extra cellular matrix (ECM). The onset of labor as well as cervical ripening is under hormonal control. To get further information about the endocrine regulation of term cervical ripening the following study was undertaken: cervical biopsies were obtained vaginally at elective caesareans, after normal vaginal delivery and after PGE 2 or antiprogestin RU486. Biopsies from non-pregnant women served as controls. The concentrations of estrogen receptor (ER) and progesterone receptor (PR) protein were quantitated by EIA and the mRNA levels by solution hybridization. The ERα and β were localized by immunohistochemistry, identified by RT–PCR and quantitated by solution hybridization. The co-localizations of CD45 (leukocyte antigen) and CD68 (macrophage antigen) were studied by immunohistochemistry. The cervical concentrations of ER and PR proteins decreased at term to 15 and 25%, respectively, compared to the non-pregnant levels. A further decrease was measured in the maximal ripened cervix at parturition. The mRNA levels were unchanged but IGF-I mRNA reached a maximum at term. ERα mRNA was significantly decreased until delivery, whereas ERβ mRNA, like IGF-I; was maximum at term. By immunostaining ERβ could be co-localized with CD45 leukocyte antigen and CD68 macrophage specific antigen. Oral administration of RU486 induced a significant increase in ER protein concentration, whereas PGE 2 and spontaneous ripening did not. These findings indicate that cervical ripening is related to significant local hormonal changes.

Regulation of the expression and bioactivation of the epidermal growth factor receptor system by estradiol in pig oviduct and endometrium

Growth factors, such as epidermal growth factor (EGF), have been suggested to mediate local effects of steroid hormones within female reproductive tissue. In the present study, the influence of estrogen on the expression and bioactivity of the EGF receptor (EGF-R) system was investigated in pigs. Oviducal and endometrial tissue from gilts was analysed either at two different time points after ovulation (Day 12 and Day 20), or from ovariectomized animals, with or without steroid-replacement treatment. Estrogen receptor protein concentrations were significantly down-regulated both in oviducal and endometrial tissue under estrogen-influence, in contrast to increased progesterone receptor concentrations. Transcript levels of EGF and transforming growth factor alpha remained unchanged in both the oviduct and endometrium during treatment. Oviducal EGF-R mRNA was found to be increased after estradiol treatment with concurrent increases in EGF-R protein. However, in endometrial tissue of estradiol-substituted ovariectomized pigs, the receptor transcript was significantly reduced, indicating a different regulation of EGF-R transcription within the endometrium. The bioactivity of the EGF-R, analysed by tyrosine kinase assays, was preserved throughout experiments in the porcine oviduct and endometrium without obvious changes caused by the steroids. In conclusion, estradiol may play a key role during the proliferation and differentiation of porcine oviducal tissue by activating the important paracrine or autocrine EGF system through its receptor. The cell-specific influence of progesterone during regulation of the EGF-R expression in the endometrium requires further investigation.

Quantitation of the immunocytochemical assay for estrogen receptor protein (ER-ICA) in human breast cancer by television imaging.

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.

Estrogen receptors in mammary cancer: correlation with age, menopausal status, and response to therapy.

The concentration of estrogen receptor protein (ER) in 113 primary or metastatic breast cancers was studied. ER was found to be correlated with the age of the patient. The ER values were generally lower in premenopausal patients (5.6 fmol/mg cytosol protein) than in postmenopausal patients (32.8 fmol/mg cytosol protein). The ER values of perimenopausal patients (0-5 years since the last menstrual period) were heterogeneous but generally closer to those of the premenopausal patients. Use of the ER determination for allocation of the patients either to hormonal (tamoxifen or nandrolonedecanoate) or to cytostatic (adriamycin-cyclophosphamide or Cooper's regimen) therapy was shown to result in highly satisfactory clinical response rates (including complete and partial remissions and stabilized disease) of 68% and 67%, respectively. The practical limit of ER concentration for selection is between 3 and 10 fmol/mg cytosol protein in breast cancer.

Estrogen receptor concentrations in 269 cases of histologically classified human breast cancer.

This report provides a histological review of 264 female primary breast cancers analyzed for estrogen receptor protein (ER). We also describe 5 cases of male breast cancer all of which bound estradiol specifically. Generally the ER concentrations were lower in the tumors of premenopausal women than in those of postmenopausal women. Three types of cancer with specific morphological features were shown to have ER concentrations that differed significantly from the other types of cancer. Medullary and comedo carcinoma had very low and papillary carcinoma very high levels of ER. No obvious trend in ER concentration was found in unspecified ductal carcinoma, colloid carcinoma, nor in lobular carcinoma. A positive correlation was demonstrated between ER content and degree of differentiation in ductal carcinoma. Cancer with lymphoid infiltration generally showed low ER levels.