Progesterone Receptor Expression Levels Research Articles

Abstract P1-02-02: Concordance of microarray based determination of ER, PR and HER2 receptor status and local IHC/FISH assessment in the prospective neo-adjuvant breast registry symphony trial (NBRST)

Abstract Background The level of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression is predictive for prognosis and/or treatment response in breast cancer patients. However, differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of the results. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2. Previously TargetPrint was shown to be strongly correlated with high quality IHC/FISH assessment, especially for ER and HER2. Concordance rates were 98% (k = 0.90) for ER; 85% (k = 0.62) for PR and 96% for HER2 (k = 0.78) in 619 patients (Viale et al., SABCS 2011). This study compares results from the microarray-based TargetPrint with IHC and FISH conducted according to local standard procedures in the prospective NBRST study. Methods The NBRST study includes women aged 18–90 with histologically proven breast cancer, who are scheduled to start neo-adjuvant chemotherapy (CT) or neo-adjuvant endocrine therapy (ET), and who provide written informed consent. The mRNA level of ER, PR and HER2 (TargetPrint) was assessed at the Agendia laboratory (Agendia Inc, Irvine, CA) in fresh and formalin fixed paraffin embedded tumor samples submitted from 40 institutes in the US. The results of the IHC/FISH assessments conducted according to local standard procedures were compared to the quantitative gene expression readouts. Results There were 355 eligible patients enrolled. 67% of patients are IHC ER positive and 25% Her2 IHC/FISH positive. 11 patients were IHC/FISH HER2 equivocal (all TargetPrint HER2 negative). Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 88% (k = 0.75)for ER; 83% (k = 0.66) for PR and 89% (k = 0.70) for HER2. The discordance range for institutes who submitted more than 10 samples was 0-30% for ER, 0-47% for PR and 0-28% for HER2. 16% of all IHC ER+ samples were classified negative by microarray. In contrast, 4% of IHC ER- samples were classified positive by microarray. However for HER2, as many as 33% of IHC/FISH HER2+ samples were classified negative by microarray; 3% of IHC/FISH HER2- samples were classified positive by microarray. Conclusions Microarray based readout of ER, PR and HER2 status using TargetPrint is comparable to local IHC and FISH analysis in 355 analyzed samples from 40 US institutes but the discordance range for individual institutes was up to 30% for ER and 28% for Her2. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-02.

Real-time fluorescent quantitative reverse transcription polymerase chain reaction in detecting expression of ERα, PR and Her2 mRNA in breast cancer tissues

Objective To evaluate the clinical value of real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR) in detecting expression of estrogen receptor alpha(ERα),progesterone receptor(PR) and human epidermal growth factor receptor 2(Her2) genes in breast cancer tissues.Methods Totally 48 breast cancer tissues and 28 benign breast tumor tissues(control) were obtained from patients undergoing surgery in our hospital during Mar.2010 and Oct.2010.The expression of ERα,PR and Her2 protein was examined by immunohistochemistry(IHC) in breast cancer tissues and the expression levels of ERα,PR and Her2 mRNA were detected by real-time qRT-PCR in breast cancer tissues and benign breast tumor tissues.The values of these two methods in diagnosis of breast cancer were evaluated. Results The expressions of ERα and Her2 mRNA were significantly higher in the breast cancer tissues than in the controls(P0.05,P0.01),while the expression of PR mRNA had no significant difference between controls and breast cancer tissues.ERα,PR and Her2 protein expressions were associated with the TNM stage of breast cancer, with the former two being negatively associated with TNM stages(P0.05) and Her2 being positively associated with TNM stages(P0.05).The expressions of ERα and Her2 mRNA were significantly higher in breast cancer tissues of patients with lymph node metastasis than in those without lymph node metastasis(P0.05).The expression of PR mRNA was not significantly different in breast cancer tissues between patients with and without lymph node metastasis(P0.05).Real-time qRT-PCR in detecting ERα,PR and Her2 mRNA expression had similar capability with IHC method in evaluating the sensitivity,specificity of endocrine therapy;moreover,the two methods also had a consistent pathological diagnosis rates(P0.05).Conclusion ERα,PR and Her2 genes are important predictive markers for endocrine therapy or targeted therapy of breast cancer.Real-time qRT-PCR method can be used for clinical detection and research of ERα,PR and Her2 mRNA.

The effect of letrozole with misoprostol for medical termination of pregnancy on the expression of steroid receptors in the placenta

What is the effect of letrozole on the expression of steroid receptors in the placentae in cases of termination of pregnancies? The expression of estrogen receptor-α (ERα) and progesterone receptor (PR) transcripts, as well as ERα protein, in placentae was suppressed by letrozole pretreatment in second trimester termination of pregnancy. There have been no data in the literature on the effect of letrozole in termination of human pregnancies. This study is part of a clinical randomized trial in which 50 subjects were recruited and 44 placentae were collected. Women (n = 50) requesting second trimester abortion between 12 and 20 gestational weeks were randomized to receive either letrozole or placebo pretreatment for 3 days before administration of vaginal misoprostol. Placentae were collected from both groups of women after the abortion. Total RNA from the frozen placenta samples was extracted and subjected to real-time RT-PCR analysis of ERα and estrogen receptor-β (ERβ), PR and glucocorticoid receptor (GR) transcripts. Immunohistochemical studies of ERα, ERβ, PR and GR expression, as well as Ki67 and PCNA staining for proliferation, were performed. TUNEL assays were performed to determine the extent of apoptosis. Real-time RT-PCR demonstrated that the median ERα {3.900 [95% confidence interval (CI): -0.643-8.443] in the letrozole group versus 4.714 (95% CI: 1.776-7.652) in the control group; P = 0.005} and the median PR [0.701 (95% CI: 0.333-1.069) in the letrozole group versus 1.774 (95% CI: 1.07-2.478) in the control group; P = 0.003] were significantly lower in the letrozole group compared with the control group. Furthermore, ERα protein levels, in both syncytiotrophoblasts and cytotrophoblasts but not in villous stromal cells, were significantly reduced [H-score of 113 (95% CI: 103-119) in the letrozole group versus 217 (95% CI: 214-290) in the control group, in syncytiotrophoblasts; 100 (95% CI: 98-105) in the letrozole group versus 210 (95% CI: 200-286) in the control group, in cytotrophoblasts; P = 0.004], while the expression levels of ERβ, PR, GR, PCNA, Ki67 and TUNEL were not significantly different between the two groups. Only the placentae from the second trimester termination of pregnancy were collected in this study. Information from first trimester terminations is still lacking. The results shed some light on the mechanism of action of letrozole pretreatment in termination of pregnancies. This study was funded by the GRF/RGC and CRCG grants of the University of Hong Kong. HKClinicalTrials.com with trial number HKCTR-695.

Influence of body mass index on clinicopathological factors including estrogen receptor, progesterone receptor, and Ki67 expression levels in breast cancers

High body mass index (BMI) is associated not only with a higher incidence of breast cancers but also with poorer prognosis. It is speculated that both enhanced production of estrogens and other factors associated with obesity are involved in these associations, but the biological characteristics associated with high BMI have yet to be thoroughly identified. We studied 525 breast cancers, focusing on biological differences between tumors associated with high and low BMI and by immunohistochemically defined intrinsic subtype. Ki67 expression levels were used to differentiate luminal A from luminal B estrogen receptor (ER)+/human epidermal growth factor receptor 2 (HER2)-breast cancers. Premenopausal patients with high BMI showed a significantly higher frequency of lymph node metastasis (46.4 % vs. 22.9 %, P = 0.005) and tended to have a larger tumor size (P = 0.05) and higher nuclear grade (P = 0.07) than those with low BMI. These differences were not observed among postmenopausal patients. BMI was not associated with distribution of breast cancer subtypes, and ER, progesterone receptor (PR), and Ki67 expression levels of each subtype showed no differences between high and low BMI among premenopausal patients. Higher BMI might influence aggressive tumor characteristics among premenopausal patients, but its influence on ER, PR, and Ki67 expression levels seems to be limited.

Prognostic relevance of Ki-67 in the primary tumor for survival after a diagnosis of distant metastasis

Prediction of the prognosis for metastatic breast cancer patients depends on molecular subtypes similar to those found in patients with primary breast cancer. Several studies have shown that estrogen receptor (ER) and progesterone receptor (PR) status determine the course of the disease and the prognosis. As Ki-67 helps to differentiate molecular subtypes in patients with primary breast cancer, the aim of this study was to assess the prognostic relevance of Ki-67 in the primary tumor in relation to its prognostic relevance for patients with metastatic breast cancer. A total of 467 patients with invasive breast cancer were identified in the database of a single breast cancer center, in whom Ki-67 had been assessed in tumor material from the breast at the time of the primary diagnosis and who had developed a metastasis at any time during the subsequent course. For these patients, tumor and patient characteristics were used to determine prognostic factors relative to overall survival after the diagnosis of distant metastases. Ki-67 was added to this model to investigate whether this might improve the prediction of overall survival. In the multivariate Cox model, age at diagnosis, body mass index, nodal status, tumor size, ER and PR status, and time from diagnosis to metastasis were identified as relevant prognostic factors. Adding Ki-67 to the model improved the prediction of overall survival. There was also a significant and relevant interaction with the PR status. In patients with a low-proliferation primary tumor, a high level of PR expression would indicate an extraordinarily good prognosis (HR 0.39; 95 % CI, 0.23-0.66). In patients with higher-proliferation primary tumors, PR status was not capable of differentiating prognostic groups. Ki-67 is useful in addition to known prognostic factors for breast cancer. It is able to indicate a group of women with a poorer prognosis, specifically in the group of patients with PR-positive breast cancer.

Dienogest increases the progesterone receptor isoform B/A ratio in patients with ovarian endometriosis

BackgroundThe resistance of endometriotic tissue to progesterone can be explained by alterations in the distribution of progesterone receptor (PR) and estrogen receptor (ER) isoforms. The aims of this study were to examine the expressions of PR-A, PR-B, ERα and ERβ in endometrioma and assess whether these expressions are affected by dienogest or leuprolide acetate (LA) treatment.MethodsWe enrolled 60 females, including 43 patients with endometriosis (14 who received no medical treatment, 13 who received dienogest and 16 who received LA before undergoing laparoscopic surgery) and 17 patients with leiomyoma. The expression levels of PR and ER isoforms in eutopic and ectopic endometrium were assayed with quantitative real-time PCR, and confirmed with immunohistochemistry.ResultsA decreased PR-B/PR-A ratio and an increased ERβ/ERα ratio were demonstrated in ectopic endometrium derived from females with endometriosis compared with the ratios observed in eutopic endometrium obtained from females without endometriosis. Although LA treatment did not affect the PR-B/PR-A and ERβ/ERα ratios, dienogest treatment increased the PR-B/PR-A ratio and decreased the ERβ/ERα ratio in patients with endometriomas.ConclusionsDienogest may improve progesterone resistance in endometriotic tissue by increasing the relative expressions of PR-B and PR-A, and decreasing the relative expressions of ERβ and ERα.

Central review of discordant samples for microarray-based ER, PR, and HER2 and local IHC/FISH assessment worldwide from 827 patients.

11 Background: Differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and is 98% concordant with centrally assessed ER as presented by Viale et al, SABCS 2011. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. Methods: Fresh tumor samples (core needle biopsies or surgical) were collected for 831 patients diagnosed with breast cancer stage I to IV (Feb 2008 - Jan 2011) from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts with TargetPrint. Discordant cases were centrally reviewed for IHC/FISH assessment. Results: Of the 831 samples, IHC assessment was unknown for 4 ER/ PR samples; HER2 was unknown for 12 samples. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 83% for PR and 94% for HER2. In this study, 3% of all IHC ER positive samples were classified negative by microarray, and 11% of IHC PR positive samples were classified negative by microarray. For HER2, 4% of IHC/FISH HER2 positive samples were classified negative by microarray and 2% of IHC/FISH HER2 negative samples were classified positive by microarray. Most notably, all available 5 ER IHC negative/TargetPrint positive samples turned out to be positive with central re-assessment. HER2 IHC2+ samples with discordant classifications for TargetPrint and local assessment are currently being reviewed for FISH/SISH assessment. Conclusions: Microarray based readout of ER, PR and HER2 status using TargetPrint is fairly comparable to local IHC and FISH analysis in 827 analyzed samples in various hospitals worldwide. However, re-assessment of discordant cases–especially IHC ER-/TargetPrint ER+ cases- confirms TargetPrint to be a useful high quality second opinion for local IHC/FISH assessment.

Association of response to neoadjuvant chemotherapy (NAC) in premenopausal patients with hormone receptor-positive early breast cancer with chemotherapy-induced ovarian function suppression by NAC.

e11017 Background: The purpose of this study is to assess the additive endocrine effect by chemotherapy-induced ovarian function suppression (CIOS) for premenopausal women with early breast cancer in neoadjuvant chemotherapy (NAC). Methods: We retrospectively compared the clinical efficacy to NAC between premenopausal and postmenopausal patients with early breast cancer to evaluate the endocrine effect by CIOS. One-hundred twenty two patients received NAC with anthracycline-containing regimen and/or taxane in Kumamoto University Hospital between April 2004 and March 2011. Results: Objective response rate by NAC in premenopausal ER-positive/HER2-negative patients is greater than that in postmenopausal ER-/HER2+ patients (premenopausal 93% vs. postmenopausal 63%, p=0.0121). Reduction rate of primary tumor by NAC on imaging studies in premenopausal ER+/HER2- patients is greater than that in postmenopausal ER-/HER2+ patients (premenopausal 54% vs. postmenopausal 40%, p=0.006). However, no significant difference in response rate between in premenopausal patients with other subtypes and postmenopausal patients with those was found (ER+/HER2+; premenopausal 50% vs. postmenopausal 48%, ER-/HER2+; premenopausal 43% vs. postmenopausal 77%, Triple-negative; premenopausal 86% vs. postmenopausal 65%). All of premenopausal women were suppressed ovarian function within two months after initiation of NAC. Expression levels of progesterone receptor on primary tumor in premenopausal ER+ cancer were significantly decreased after NAC compared with that in postmenopausal ER+ cancer (premenopausal -40.3% vs. postmenopausal +3.6%, p<0.001). Conclusions: These data suggests that CIOS by NAC contributes the additive effects through the endocrine fashion in premenopausal patients with ER+/HER2- breast cancer.

Central review of discordant samples for microarray based on ER, PR, and HER2 and local IHC/FISH assessment worldwide from 827 patients.

10554 Background: Differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and is 98% concordant with centrally assessed ER as presented by Viale et al, SABCS 2011. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. Methods: Fresh tumor samples (core needle biopsies or surgical) were collected for 831 patients diagnosed with breast cancer stage I to IV (Feb 2008 - Jan 2011) from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts with TargetPrint. Discordant cases were centrally reviewed for IHC/FISH assessment. Results: Of the 831 samples, IHC assessment was unknown for 4 ER/ PR samples; HER2 was unknown for 12 samples. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 83% for PR and 94% for HER2. In this study, 3% of all IHC ER positive samples were classified negative by microarray, and 11% of IHC PR positive samples were classified negative by microarray. For HER2, 4% of IHC/FISH HER2 positive samples were classified negative by microarray and 2% of IHC/FISH HER2 negative samples were classified positive by microarray. Most notably, all available 5 ER IHC negative/TargetPrint positive samples turned out to be positive with central re-assessment. HER2 IHC2+ samples with discordant classifications for TargetPrint and local assessment are currently being reviewed for FISH/SISH assessment. Conclusions: Microarray based readout of ER, PR and HER2 status using TargetPrint is fairly comparable to local IHC and FISH analysis in 827 analyzed samples in various hospitals worldwide. However, re-assessment of discordant cases –especially IHC ER-/TargetPrint ER+ cases- confirms TargetPrint to be a useful high quality second opinion for local IHC/FISH assessment.

Biological characteristics of luminal subtypes in pre- and postmenopausal estrogen receptor-positive and HER2-negative breast cancers

Estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancers can be divided into luminal A and luminal B subtypes based on Ki67 expression levels. However, the biological differences in ER and progesterone receptor (PR) expression levels between these luminal subtypes are not clear. We examined immunohistochemical expression levels of ER, PR, and Ki67 in 180 ER-positive/HER2-negative breast cancers while taking menopausal status into account. Breast cancers were divided according to ER and PR levels (H: >50%, L: ≤ 50%), and luminal A and B were classified by the Ki67 labeling index (A: Ki67 <14%, B: Ki67 ≥ 14%). When breast cancers were classified based on ER and PR levels, the distribution of pre- and postmenopausals was significantly different for luminal A (P < 0.0001), but not for luminal B cancers. As for luminal A, ER-H/PR-L cancers were rare among premenopausals (8%), but frequent among postmenopausals (54%). Correlation between ER and PR levels among luminal A cancers was strong in premenopausals but weak in postmenopausals. Since crosstalk with growth factor signaling is unlikely in luminal A, we speculate that intratumoral estrogen insufficiency contributed to the characteristics of postmenopausal ER-H/PR-L cancers. We speculate that the biological characteristics of luminal A cancers are influenced by the estrogen environment, but its influence on luminal B cancers may be limited. We believe these considerations constitute useful information for a better understanding of the biology of ER-positive-HER2-negetive breast cancers.

Abstract IA11: Control of gene expression in the nucleus by small RNAs

Abstract Double-stranded RNAs are widely used to control gene expression and are a promising class of therapeutic agent. Most studies have focused on recognition of mRNA in the cytoplasm and inhibition of translation. My presentation focuses on the action of small RNAs in the nucleus to modulate transcription or redirect alternative splicing. In 2005, my laboratory observed that synthetic RNAs complementary to the promoter region at the transcriptional start site of human progesterone receptor (PR) could block PR expression [1]. Inhibition was potent and robust, consistent with the suggestion that the synthetic RNAs were exploiting an endogenous mechanism for controlling gene expression. Promoter-targeted synthetic miRNAs were also capable of robust gene silencing [2], further supporting the existence of endogenous regulatory pathways. Modulation required complete seed sequence complementarity to the PR promoter, consistent with a mechanism that involves RNA-RNA recognition through Watson-Crick base-pairing. In 2007, we observed that small RNAs could also target the PR gene promoter and activate PR expression [3]. Activation was observed in cells that had a low, but detectable, level of PR expression, while gene silencing had been observed in cells with a high level of PR expression. Inactive and active RNAs compete for recognition, demonstrating that small changes in target site location affect activity. These results are reminiscent of protein transcription factors, which bind to specific sequences, are sensitive to target site location or small conformation changes, and can activate gene expression in some contexts but not others. Our promoter-targeted RNAs bind to long noncoding RNA transcripts at the PR promoter [4]. Use of antisense oligonucleotides to reduce transcript levels also reduces RNA-mediated modulation of gene expression. The small RNAs recruit AGO2 to the noncoding transcript, and inhibition of AGO2 expression reverses gene silencing or activation [5]. While AGO2 is normally expected to cleave fully complementary targets in the cytoplasm, our data suggests that AGO2 does not cleave transcripts associated with gene promoters in the nucleus. A noncoding RNA transcript also overlaps the 3′ terminus of the PR gene [6]. Small RNAs complementary to this noncoding transcript modulate PR expression, depending on the cell line used. The PR gene loops, juxtaposing its 3′ and 5′ termini and providing a direct path for a signal transmitting a signal from the 3′ terminus of the gene to the promoter. We have investigated whether RNAs can modulate other nuclear events. We designed small RNAs to be complementary to sequence within exons or introns near splice sites. These RNAs redirect alternative splicing and AGO2 was recruited to pre-mRNA [7]. These data provide more support for the action of small RNA in cell nuclei and for the conclusion that AGO2 may not always cause RNA cleavage when it encounters complementary targets in the nucleus. Our data suggest that small RNAs can bind noncoding transcripts and modulate gene expression. Binding occurs in proximity to the gene promoter, in cis relative to the target gene. The RNA-AGO complex acts like a transcription factor to affect the transcription machinery and tip the balance of inducible genes like PR towards repression or activation. Citation Format: David Corey. Control of gene expression in the nucleus by small RNAs [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA11.

Expression of Progesterone Receptor Membrane Component 1, Serpine mRNA Binding Protein 1 and Nuclear Progesterone Receptor Isoforms A and B in the Bovine Myometrium During the Estrous Cycle and Early Pregnancy

The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

P5-11-09: High Concordance for Microarray Based Determination of ER, PR and HER2 Receptor Status and Local IHC/FISH Assessment Worldwide in 749 Patients.

Abstract Background The level of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression is predictive for prognosis and/or treatment response in breast cancer patients. However, differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of the results. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and provides high quality second opinion for local IHC/FISH assessment. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. Methods Prospective tumor samples were collected for 749 patients diagnosed with breast cancer stage I to IV between February 2008 and January 2011. The mRNA level of ER, PR and HER2 (TargetPrint) was assessed in the Agendia laboratories (Agendia Inc, Irvine, CA; Agendia BV, Amsterdam, the Netherlands) in fresh tumor samples submitted from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts. Results Of the 749 samples, IHC assessment was unknown for 5 ER samples and 4 PR samples; FISH was unknown for 24 samples. TargetPrint read out was not assessed for HER2 for 11 samples. Median age of these patients was 61 years. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 82% for PR and 91% for HER2. In this study, only 4% of all IHC ER positive samples were classified negative by microarray. In contrast, 14% of IHC ER negative samples were classified positive by microarray. However for HER2, 28% of IHC/FISH HER2 positive samples were classified negative by microarray and 5% of IHC/FISH HER2 negative samples were classified positive by microarray. Samples with discordant classifications for TargetPrint and local assessment are being reviewed in greater detail by a central pathologist. Conclusions Microarray based readout of ER, PR and HER2 status using TargetPrint is highly comparable to local IHC and FISH analysis in 749 analyzed samples in various hospitals worldwide. The results indicate mRNA expression read out for ER, PR and HER2 by TargetPrint provides high quality second opinion for local IHC/FISH assessment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-09.

Association Between Hormone Receptor Expression and Epidermal Growth Factor Receptor Mutation in Patients Operated on for Non-Small Cell Lung Cancer

Estrogen receptor (ER) and progesterone receptor (PR) play important roles in breast cancer. Similarly, there have been several reports of ER and PR expression in lung cancers, but the results have not been consistent. The aim of this study was to investigate the association between hormone receptor expression and clinicopathologic factors and epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). We evaluated 316 resected NSCLC specimens for ER-α, PR, human epidermal growth factor receptor 2, and aromatase (n=272) expression using immunohistochemical methods. EGFR mutations were evaluated with polymerase chain reaction (PCR). ER-α and PR were detected in 36.1% and 44.9% of all patients, respectively. The expression of ER-α was observed mostly in cytoplasm (94.7%) and the expression of PR was observed mostly in nucleus (95.8%). Aromatase was detected in the cytoplasm of 64.0% of patients. ER-α expression was significantly associated with female gender (p=0.004). The expression of PR was significantly associated with better clinicopathologic features. ER-α expression showed a positive correlation with PR (r=0.275; p<0.001) and aromatase (r=0.244; p<0.001) expression levels. EGFR mutation was independently associated with the female gender (p=0.001), negative expression of PR (p<0.001), and negative expression of aromatase (p=0.027). The expression of ER-α and PR distinguish a subset of NSCLC that has defined clinicopathologic features. Negative expression of PR and aromatase correlate with EGFR mutation.

Abstract 2286: Medroxyprogesterone acetate induces expression of 17βHSD1 and 17βHSD5 in the progesterone receptor positive breast cancer cell lines T47D and MCF7

Abstract Despite the fact that the ovaries stop to produce oestrogens at menopause, high levels can be detected locally in breast tumours. There are several enzymes responsible for the local production of sex steroids such as aromatase, and the 17 beta hyroxysteroid dehydrogenase (17βHSD) family has also proven to have a significant importance in breast cancer. 17βHSD1 converts estrone to estradiol, 17βHSD2 estradiol to estrone, 17βHSD5 androstendione to testosterone and in the next step aromatase is responsible for formation of testosterone to estradiol, the function of 17βHSD14 is still not clear. The regulation of 17βHSDs needs further investigation. In several studies combined hormone replacement therapy (estrogens and progestins) has shown an increased risk to develop invasive breast cancer, compared to women treated with only estrogens. It is plausible that progestins influence the expression of members in the 17βHSD-family. Previous studies investigating the role of progestins on steroid conversion have focused on measuring steroidal levels and have not investigated changes in expression levels of enzymes responsible for this conversion. The aim of this study was to treat breast cancer cell lines with progesterone and progestins to detect possible influence on 17βHSD1, 17βHSD2, 17βHSD5 and 17βHSD14 mRNA expression. We further investigated conversion of androstendione in cultured media. The breast cancer cell lines T47D, MCF7 and ZR75-1, choosen for their different endogenous expression of progesterone receptor (PgR) were exposed for progesterone, medroxyprogesterone acetate (MPA) or levenogestrel for 48 and 72 hours at 10-7 and 10-9 M. The mRNA expression of 17βHSD1, 17βHSD2, 17βHSD5 and 17βHSD14 were measured by real time PCR using assay on demand and ACTH as endogenous control. Conversion of H3 labelled androstendione was investigated using HPLC coupled radio detector. We found that mRNA expression of 17βHSD1 increased significantly after 48 h in MPA treated T47D (10-9 M p=0.03) and MCF7 cells (10-7 p=0.001; 10-9 p=0.004). Further, mRNA expression of 17βHSD5 increased after 48 h in MPA treated T47D (10-7 M p=0.0006; 10-9 M p=0.04) and MCF7 cells (10−9 M p=0.005). No significant differences were found in cells treatment with levonogestrel or progesterone. We did neither find any differences in the mRNA expression levels of 17βHSD2 nor 17βHSD14. In the ZR75-1 cell line no significant changes were detected. We have preliminary results indicating that androstendione is converted in T47D cells treated with MPA. In conclusion, we show that reductive members of the 17βHSD-family increase (17βHSD1 and 17βHSD5) in breast cancer cells with high PgR expression levels. Our results may indicate that 17βHSD5 can be involved in the high risk for breast cancer assessed with hormone replacement treatment using combined estrogen and progestin in post menopausal women. 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 2286. doi:10.1158/1538-7445.AM2011-2286

Altered AIB1 or AIB1Δ3 Expression Impacts ERα Effects on Mammary Gland Stromal and Epithelial Content

Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments.

Abstract P3-13-05: Estrogen Receptor-Positive Breast Cancer in Japanese Women: Trends in Incidence, Characteristics and Prognosis

Abstract Background: The incidence of breast cancer in Japanese women has doubled in all age groups over the past two decades. Patients and Methods: We examined the characteristics of patients and tumors treated in three time periods between 1982 and 2010. Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status were assessed by immunohistochemistry. Correlation of hormone receptor levels with clinicopathological factors and prognosis was analyzed in ER-positive, HER2-negative breast cancer in two age groups (≥50 years vs. &amp;gt;50 years). Results: A total of 1903 women with breast cancer, who were treated at Nagoya City University Hospital, were included in this study. The frequency of ER-positive breast cancer in women aged 50 years or younger increased greatly over the interval studied (1982-1991: 52.5%, 1992-2001: 72.6%, 2002-2010: 87.1%, P &amp;lt; 0.0001). The frequency of ER-positive tumors was also significantly increased in women over 50 years of age (1982-1991: 69.4%, 1992-2001: 73.3%, 2002-2010: 78.6%, P = 0.029). In ER-positive, HER2-negative breast cancer, tumor grade was negatively correlated with expression levels of ER (P = 0.0029) and PgR (P &amp;lt; 0.0001). Interestingly, PgR levels were significantly higher in women aged 50 years or younger than in women over 50 years old (P &amp;lt; 0.0001). In both age groups, the prognosis for patients with ER-positive, HER2- negative breast cancer significantly improved over time, due to advances in adjuvant therapies. ER and PgR expression levels were not associated with disease-free survival. Lymph node status and tumor size were strong prognostic factors regardless of the biological characteristics of the tumors in ER-positive, HER2-negative breast cancer in both age groups. Conclusions: It is necessary to establish risk factors, both genetic and environmental, capable of predicting the risk of ER-positive breast cancer and thus enable the efficient selection of candidates for hormone receptor-targeted chemoprevention. Furthermore, new approach should be considered to improve survival for node-positive, ER-positive breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-13-05.

The ERα coactivator, HER4/4ICD, regulates progesterone receptor expression in normal and malignant breast epithelium

The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ERα) coactivator with activities in breast cancer and the developing mammary gland that appear to overlap with progesterone receptor (PgR). In fact, 4ICD has recently emerged as an important regulator and predictor of tamoxifen response, a role previously thought to be fulfilled by PgR. Here we investigated the possibility that the 4ICD coactivator regulates PgR expression thereby providing a mechanistic explanation for their partially overlapping activities in breast cancer. We show that 4ICD is both sufficient and necessary to potentiate estrogen stimulation of gene expression. Suppression of HER4/4ICD expression in the MCF-7 breast tumor cell line completely eliminated estrogen stimulated expression of PgR. In addition, the HER4/4ICD negative MCF-7 variant, TamR, failed to express PgR in response to estrogen. Reintroduction of wild-type HER4 but not the γ-secretase processing mutant HER4V673I into the TamR cell line restored PgR expression indicating that 4ICD is an essential PgR coactivator in breast tumor cells. These results were substantiated in vivo using two different physiologically relevant experimental systems. In the mouse mammary gland estrogen regulates expression of PgR-A whereas expression of PgR-B is estrogen independent. Consistent with a role for 4ICD in estrogen regulated PgR expression in vivo, PgR-A, but not PgR-B, expression was abolished in HER4-null mouse mammary glands during pregnancy. Coexpression of PgR and 4ICD is also commonly observed in ERα positive breast carcinomas. Using quantitative AQUA IHC technology we found that 4ICD potentiated PgR expression in primary breast tumors and the highest levels of PgR expression required coexpression of ERα and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERα coactivator and 4ICD cooperates with ERα to potentiate PgR expression in the normal and malignant breast. We propose that direct coupling of these signaling pathways may have important implications for mammary development, breast carcinogenesis, and patient response to endocrine therapy.

MicroRNA regulation of the expression of the estrogen receptor in endometrial cancer

microRNAs (miRNAs) are non-coding RNA transcripts thought to be instrumental in controlling eukaryotic cell function. This study aimed to identify miRNAs associated with the differential expression of estrogen receptor α (ERα) and progesterone receptor (PR) in uterine endometrial cancer. First, the pathology of Ishikawa and KLE cell lines was identified by transplant and biopsy. Expression levels of ERα and PR and their response to estrogen and progesterone were examined, and total RNA was isolated to identify differentially expressed miRNAs by microarray analysis. miRNAs targeting ESR1 and PGR were predicted by miRANDA and TargetScan software, and their expression levels in cell lines and in patient samples were validated by real-time RT-PCR. Ishikawa and KLE represent estrogen-dependent and estrogen-independent endometrial cancers, respectively. As identified by miRNA microarray, 126 miRNAs were differentially expressed between the two cell lines; hsa-miR-100 and hsa-miR-99a were predicted to target ESR1, and hsa-miR-378 and hsa-miR-768-3p to target PGR. The differential expression of these miRNAs between Ishikawa and KLE was consistent in vivo and in vitro. Hsa-miR-100 was significantly down-regulated in the estrogen-dependent endometrial cancer samples as compared to the estrogen-independent samples and thus has the potential to target ESR1.

Automated image analysis for high‐throughput quantitative detection of ER and PR expression levels in large‐scale clinical studies: The TEAM Trial Experience

Routine immunohistochemistry is regarded as a semiquantitative method for the evaluation of in situ protein expression. Analysis of tissue biomarkers in large clinical trials is central to the development of novel targeted approaches to therapy, requires the analysis of tens of thousands of data points, and frequently makes use of high-throughput analysis of tissue microarrays (TMAs). The aim of this study was to investigate the potential of image analysis for accurate and reproducible quantitative evaluation of biomarkers. We showed, in 397 cases of breast cancer from the Phase III TEAM clinical trial, excellent correlations between semiautomated image analysis of TMAs and manual scoring of oestrogen receptor (ER) and progesterone receptor (PR) levels (interclass correlation coefficients 0.93 and 0.96 respectively). Two or more TMA cores were excellently correlated with manual scores, and using more than three cores increased the number of cases available for analysis to >92%. TMAs are confirmed as representative of whole sections for immunohistochemical analysis of the tissue biomarkers ER and PR. Semiautomated image analysis is appropriate for the analysis of tissue biomarkers within large clinical trials. These data provide support for the use of TMAs and image analysis in translational research.