Progesterone Receptor Assays Research Articles

Establishment of a graphene quantum dot (GQD) based steroid binding assay for the nuclear progesterone receptor (pgr)

Previously, we established a homogeneous assay for membrane progesterone receptor alpha (mPRα) ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to mPRα. When mixed with a progesterone-BSA-fluorescein isothiocyanate conjugate (P4-BSA-FITC), fluorescence occurred by fluorescence resonance energy transfer (FRET) but was reduced by the ligand-receptor binding activity. The established way showed ligand specificity as mPRα protein. In this study, we tried to establish the same way for nuclear progesterone receptor (Pgr). The ligand-binding domain (LBD) of zebrafish Pgr (zPgrLBD) was expressed as a fusion protein with glutathione S-transferase (GST) (GST-zPgrLBD). The recombinant protein was then purified and coupled with GQDs to produce GQD-conjugated GST-zPgrLBD (GQD-GST-zPgrLBD). When mixed with a P4-BSA-FITC and activated by 370 nm light, fluorescence at 520 nm appeared by FRET mechanism. Fluorescence at 520 nm was reduced by adding free progesterone to the reaction mixture. Reduction of fluorescence was induced by zPgr ligands but not by steroids or chemicals that do not interact with zPgr. The results showed the formation of a complex of GQD-GST-zPgrLBD and P4-BSA-FITC with ligand-receptor binding. The binding of the compounds was further confirmed by a radiolabeled steroid binding assay. A homogenous ligand-binding assay for nuclear progesterone receptor has been established.

Establishment of a steroid binding assay for goldfish membrane progesterone receptor (mPR) by coupling with graphene quantum dots (GQDs).

A homogeneous assay was developed to evaluate ligands that target the membrane progesterone receptor alpha (mPRα) of goldfish. This was achieved by employing graphene quantum dots (GQDs), a type of semiconductor nanoparticle conjugated to the goldfish mPRα. When progesterone-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was combined with the other agents, fluorescence was observed through Förster resonance energy transfer (FRET). However, this fluorescence was quenched by binding between the ligand and receptor. This established method demonstrated the ligand selectivity of the mPRα protein. Then, the methylotrophic yeast Pichia pastoris was used to express the goldfish mPRα (GmPRα) protein. The recombinant purified GmPRα protein was coupled with graphene quantum dots (GQDs) to generate GQD-conjugated goldfish mPRα (GQD-GmPRα). Fluorescence at a wavelength of 520nm was observed through FRET upon the combination of P4-BSA-FITC and subsequent activation by ultraviolet (UV) light. Adding free P4 to the reaction mixture resulted in a decrease in fluorescence intensity at a wavelength of 520nm. The fluorescence was reduced by the administration of GmPRα ligands but not by steroids that do not interact with GmPRα. The findings indicated that the interaction between the ligand and receptor led to the formation of a complex involving GQD-GmPRα and P4-BSA-FITC. The interaction between the compounds and GQD-GmPRα was additionally validated by a binding experiment that employed the radiolabeled natural ligand [3H]-17α,20β-dihydroxy-4-pregnen-3-one. We established a ligand-binding assay for the fish membrane progesterone receptor that is applicable for screening compounds.

Personalising cancer medicine with prognostic markers.

Personalising cancer medicine with prognostic markers.

Data on RT-qPCR assay of nuclear progesterone receptors (nPR), membrane progesterone receptors (mPR) and progesterone receptor membrane components (PGRMC) from human uterine endometrial tissue and cancer cells of the Uterine Cervix.

A previous investigation showed that the endometrium normalized in women with endometrial hyperplasia after three months treatment with high dose levonorgestrel IUS (intrauterine system) [1] . The effect was maintained even if immunohistochemical analyses of the endometrium showed that nuclear progesterone receptors (nPRs) were completely downregulated. These observations indicated that some type of non-genomic effect existed [2]. We conducted new investigations of endometrial hyperplasia, now with 6 months low dose levonorgestrel IUS treatment. Again, the growth disturbances were reversed with normalization of the endometrium [3,4]. In the context of these studies, RT-qPCR analyses of the endometrium were performed before and after treatment, to determine expression of nuclear progesterone receptors (nPRA+B and nPRB), membrane progesterone receptors (mPR, α-, β- and γ-subtypes) and progesterone receptor membrane components (PGRMC1and PGRMC2). The human cervical cell line (C-4 I) [5] with no detectable nPRs [6,7] , was included in the investigation as biological control .The gene expression of nPRs, mPRs and PGRMCs was determined in the logarithmic growth phase. Tissue and cellular mRNA was determined with RT-qPCR and used as a surrogate marker for receptor (protein) expression. The present data are connected to the related article entitled “Expression of nuclear progesterone receptors (nPRs), membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in the human endometrium after 6 months levonorgestrel low dose intrauterine therapy” [8].

Baicalein Is a Phytohormone that Signals Through the Progesterone and Glucocorticoid Receptors.

While flavonoids have been studied extensively for estrogen receptor activity, they have not been well studied for their ability to modify progesterone receptor (PR) and glucocorticoid receptor (GR) signaling. Three flavonoid compounds, tangeretin, wogonin, and baicalein, were selected for testing for PR and GR activity based on their structural similarity to known phytoprogesterone-like compounds. Each compound was docked in the binding pocket of PR and GR. Of these compounds, baicalein was predicted to be most likely to bind to both receptors. A fluorescence polarization competitive binding assay for PR and GR confirmed that baicalein binds to both the PR and GR with IC50 values of 15.30μM and 19.26μM, respectively. In Ishikawa PR-B and T47D cells, baicalein acted as a PR antagonist in a hormone response element (HRE) luciferase (Luc) assay. In OVCAR5 cells, which only express GR, baicalein was a GR agonist via an HRE/Luc assay and induced GR target genes, FKBP5 and GILZ. RU486, a PR and GR antagonist, abrogated baicalein's activity in OVCAR5 cells, confirming baicalein's activity is mediated through the GR. In vivo, baicalein administered intraperitoneally to female mice twice a week for 4weeks at a dose of 25mg/kg induced the GR target gene GILZ in the reproductive tract, which was blocked by RU486. In summary, baicalein has PR antagonist and GR agonist activity in vitro and demonstrates GR agonist activity in the uterus in vivo.

Effect-Directed Analysis of Progestogens and Glucocorticoids at Trace Concentrations in River Water.

Effect-based monitoring is increasingly applied to detect and-in conjunction with chemical analysis-to identify endocrine-disrupting compounds (EDCs) in the environment. Although this approach of effect-directed analysis has been successfully demonstrated for estrogenicity and androgenicity, data on progestogens and glucocorticoids driving endocrine disruption are quite limited. We investigated progestogenic and glucocorticoid activities in Danube River water receiving untreated wastewater from Novi Sad, Serbia. After a 2-step fractionation, all fractions were tested with reporter gene bioassays for agonistic and antagonistic hormonal responses at progestogenic and glucocorticoid hormone receptors as well as with target and nontarget analytical screening of active fractions by liquid chromatography-high-resolution mass spectrometry. Due to masking by cytotoxic mixture components, the effects could not be detected in the raw water extract but were unraveled only after fractionation. Target chemical screening of the fraction that was active in the progesterone receptor (PR) assay revealed that progesterone and megestrol acetate were predominant drivers of PR-mediated activity along with medroxyprogesterone, dihydrotestosterone, androsterone, and epiandrosterone. Hydrocortisone was detected at sub-ng/L concentration in the active fraction in the glucocorticoid receptor (GR) assay but could not explain a significant fraction of the observed GR activity. The present study indicates that effect-based monitoring is a powerful tool to detect EDCs in the aquatic environment but that fractionation may be required to avoid masking effects of mixture components. Future effect-directed analysis studies are required to better understand the occurrence of EDCs and masking compounds in different lipophilicity windows, to finally reduce fractionation requirements for monitoring to a smart clean-up. Environ Toxicol Chem 2019;39:189-199. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

PH-responsive allochroic nanoparticles for the multicolor detection of breast cancer biomarkers

Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) are the three crucial biomarkers for the clinical diagnosis of breast cancer. Sensitive and precise detection of ER, PR, and HER2 is of great significance for the diagnosis of breast cancer. Herein, through a simple solvent-induced self-assembly process, the self-carried allochroic nanoparticles were prepared by using some hydrophobic pH indicator molecules for allochroic NPs-linked immunosorbent assay (named ALISA) of ER, PR, and HER2, respectively. Meanwhile, the introduction of bovine serum albumin (BSA) and antibody (Ab) enhanced the dispersity of the self-assembled nanoparticles as signal tags. Since the ultra-high loading and high-efficiency release of pH indicators, the ALISA exhibitssatisfactory selectivity and sensitivity, which demonstrated the great potential in the early diagnosis and postoperative monitoring of breast cancers. Furthermore, the smartphone was introduced to combine with the ALISA for point-of-care testing, indicating the high feasibility in practical applications.

A Statistical Model with Non-Linear Effects and Non-Proportional Hazards for Breast Cancer Survival Analysis

The Cox proportional hazard model is being used extensively in oncology in studying the relationship between survival times and prognostic factors. The main question that needs to be addressed with respect to the applicability of the Cox PH model is whether the proportional hazard assumption is met. Failure to justify the subject assumption will lead to misleading results. In addition, identifying the correct functional form of the continuous covariates is an important aspect in the development of a Cox proportional hazard model. The purpose of this study is to develop an extended Cox regression model for breast cancer survival data which takes non-proportional hazards and non-linear effects that exist in prognostic factors into consideration. Non-proportional hazards and non-linear effects are detected using methods based on residuals. An extended Cox model with non-linear effects and time-varying effects is proposed to adjust the Cox proportional hazard model. Age and tumor size were found to have nonlinear effects. Progesterone receptor assay status and age violated the proportional hazard assumption in the Cox model. Quadratic effect of age and progesterone receptor assay status had hazard ratio that changes with time. We have introduced a statistical model to overcome the presence of the proportional hazard assumption violation for the Cox proportional hazard model for breast cancer data. The proposed extended model considers the time varying nature of the hazard ratio and non-linear effects of the covariates. Our improved Cox model gives a better insight on the hazard rates associated with the breast cancer risk factors.

Evaluation of three commercial progesterone receptor assays in a single tamoxifen-treated breast cancer cohort

Estrogen receptor and progesterone receptor status are routinely assessed using immunohistochemistry assays to assist in patient prognosis and clinical management. Three commonly utilized autostainer vendors—Dako, Leica and Ventana—provide ready-to-use progesterone receptor assays; however, they have never been directly compared in a single breast cancer cohort. We looked at three immunohistochemical progesterone receptor assays, in addition to original ligand-binding assay results, in a single retrospective, tamoxifen-treated breast cancer cohort to investigate inter- and intra-observer agreement, concordance, prognostic ability and measures of test performance. All immunohistochemical assays utilized the manufacturer's specified protocols. Five-year disease-free survival was the endpoint of interest, and multivariate models were adjusted for lymph node status, tumor grade, tumor size and human epidermal growth factor 2 status. All assays showed substantial to almost perfect agreement between the three observers (Dako κ=0.69–0.90; Leica κ=0.70–0.89; and Ventana κ=0.78–0.94) and concordance (Dako/Leica κ=0.81; Dako/Ventana κ=0.78; and Leica/Ventana κ=0.82). Univariate survival analyses showed that only the ligand-binding assay, Dako and Ventana assays achieved statistical significance. No statistically significant results were seen in multivariate models, although a strong trend was seen with the Ventana progesterone receptor assay. All assays performed similarly with regards to measures of test performance with ligand-binding assay set as the reference, and all immunohistochemical assays outperformed the ligand-binding assay in regards to 5-year disease-free survival. Despite similar agreement and concordance with the progesterone receptor assays, clear differences were noted with regards to 5-year disease-free survival. Additional survival analyses suggest that clinical utility of estrogen receptor assays vary when investigated in combination with progesterone receptor.

Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water

In vitro bioassays have shown promise as water quality monitoring tools. In this study, four commercially available in vitro bioassays (GeneBLAzer® androgen receptor (AR), estrogen receptor-alpha (ER), glucocorticoid receptor (GR) and progesterone receptor (PR) assays) were adapted to screen for endocrine active chemicals in samples from two recycled water plants. The standardized protocols were used in an interlaboratory comparison exercise to evaluate the reproducibility of in vitro bioassay results. Key performance criteria were successfully achieved, including low background response, standardized calibration parameters and high intra-laboratory precision. Only two datasets were excluded due to poor calibration performance. Good interlaboratory reproducibility was observed for GR bioassay, with 16–26% variability among the laboratories. ER and PR bioactivity was measured near the bioassay limit of detection and showed more variability (21–54%), although interlaboratory agreement remained comparable to that of conventional analytical methods. AR bioassay showed no activity for any of the samples analyzed. Our results indicate that ER, GR and PR, were capable of screening for different water quality, i.e., the highest bioactivity was observed in the plant influent, which also contained the highest concentrations of endocrine active chemicals measured by LC–MS/MS. After advanced treatment (e.g., reverse osmosis), bioactivity and target chemical concentrations were both below limits of detection. Comparison of bioassay and chemical equivalent concentrations revealed that targeted chemicals accounted for ≤5% of bioassay activity, suggesting that detection limits by LC–MS/MS for some chemicals were insufficient and/or other bioactive compounds were present in these samples. Our study demonstrated that in vitro bioassays responses were reproducible, and can provide information to complement conventional analytical methods for a more comprehensive water quality assessment.

Abstract P5-02-12: Concordance between semiquantitative immunohistochemical assay and oncotype DX RT-PCR assay for estrogen and progesterone receptors

Abstract Introduction: Estrogen receptor (ER) and progesterone receptor (PR) expression are generally determined by semiquantitative immunohistochemistry (IHC) as recommended in the clinical practice guidelines of the American Society of Clinical Oncologists. This method may be subject to variability because of differences in fixation, ER antibody clones, as well as to immunostain interpretation and use of arbitrary cut-off points. Materials and methods: We identified 72 breast cancer cases at Basurto University Hospital from 1 September 2012 to 31 May 2014 that have been analyzed by Oncotype DX with reporting on ER/PR expression levels by RT-PCR (Genomic Health Inc, Redwood City, CA). According to the company’s report, a tumor is considered as ER positive with expression units of >=6.5 and PR positive with expression units of >=5.5. Oncotype DX is a commercial assay that predicts tumor recurrence in node-negative ER-positive breast cancers. It is a reverse transcription-polymerase chain reaction (RT-PCR) based assay that analyzes the expression of 21 genes (16 cancer-related and 5 control genes) to provide a distant disease recurrence score ranging from 0 to 100. At our institution, hormone receptors by IHC were analyzed on corresponding surgery breast specimens at the time of initial diagnosis. They were evaluated by IHC on formalin-fixed paraffin-embedded tissue, between 8-72 hours. ER was assessed using Roche antibody clone SP1 and PR was assessed using antibody clone 1E2. Percentage of positive nuclei was determined by visual microscopic estimation: <1 negative and ≥1 positive. We compared our semiquantitative method of reporting ER and PR status based on IHC to the RT-PCR hormone receptor results from Genomic Health. Results: There was 100% concordance between IHC and the RT-PCR assay for estrogen receptor status. PR findings between IHC and Oncotype Dx revealed a lower concordance (82%) than for ER. RT-PCR was negative in 10 (14%) cases in which IHC was positive. Pearson correlation coefficient for PR was 0.313. Although our results demonstrated high concordance between IHQ and Oncotype Dx for ER, our data showed poor concordance for PR. Citation Format: Patricia Novas, Elena Galve, Sara Fernández, Maria Ángeles Sala, Alberto Arévalo, Juan Arango, Borja López de San Vicente, Seila Fernández, Ane Zumárraga, Purificación Martínez del Prado. Concordance between semiquantitative immunohistochemical assay and oncotype DX RT-PCR assay for estrogen and progesterone receptors [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 P5-02-12.

Considerations when assessing antagonism in vitro: Why standardizing the agonist concentration matters

There is increasing recognition of the importance of assessing both agonism and antagonism in parallel for environmental samples. Cell-based in vitro assays have the advantage over receptor binding assays as they are able to differentiate between agonist and antagonist activity, but at present there is no standardized approach to assess antagonism in vitro, and in particular the competing agonist concentration can vary in the literature anywhere from half maximal to maximal effect concentrations. In this study, we investigated the influence of changing agonist concentrations in the estrogen receptor alpha (ERα), progesterone receptor (PR) and glucocorticoid receptor (GR) assays run in antagonist mode. The antagonistic effect varied by over two orders of magnitude when using the range of agonist concentrations applied in the literature, clearly indicating the need for standardization. By comparing antagonist EC50 values with different background agonist concentrations, an EC80 background agonist concentration is recommended when assessing antagonism in vitro to optimise both assay sensitivity and reproducibility.

Abstract P3-05-06: A better clinical cutpoint for progesterone receptor expression in tamoxifen treated breast cancer

Abstract INTRODUCTION: Hormone receptors are routinely measured by immunohistochemistry (IHC) to classify breast cancers (BC) and guide treatment decisions. Both estrogen receptor (ER) and progesterone receptor (PR) are measured yet “the precise role of PR in patient management has not been strongly established” (ASCO/CAP; JCO 2010 28(16)). PR expression is under the transcriptional control of ER, and its been suggested that BC with high PR expression are more estrogen-dependent and thus more sensitive to endocrine therapy. We hypothesized that using a higher PR threshold would be prognostic in hormone therapy (HT) treated ER+/HER2- BC patients compared to the current PR cutpoint (Allred Score≥3). METHODS: We analyzed PR expression using the Calgary Tamoxifen BC Cohort (Cal-TBCC), a retrospective database that has a clinically annotated tissue microarray (TMA) series of 532 BC patients treated with HT. The NCI Stage I BC TMA set (N = 590) was obtained for validation. ER was visualized by staining with Dako ER pharmDx, and HER2 with Dako HercepTest. We visualized PR expression using ready-to-use PR assays from 3 commercial IHC vendors (Dako, Leica, Ventana) for the Cal-TBCC. The NCI validation set was stained only using the Dako assay. Allred scores were generated, capturing intensity and percentage coverage of PR expression. Only patients receiving HT that were ER+/HER2- by IHC were included for study. RESULTS: We found that ER+/HER2- patients from the Cal-TBCC stratified using a newly defined PR Allred cutpoint to capture only PR-High BC had significantly better disease free survival (DFS) compared to the current cutpoint (PR+≥3), regardless of platform used (Table 1). Multivariate analysis - adjusting for tumor grade, size and lymph node status - confirmed the improved prognostic significance of the new PR cutpoint (Table 2A). We next validated the prognostic power of the newly defined cutpoint using the NCI series. This set also has a group of ER+/HER2- BC patients who were not treated with HT, allowing us to evaluate the ability of ER+/HER2-/PR-High to predict DFS in patients treated with or without HT. We found that in patients who weren't treated with HT, PR was not prognostic using either cutpoint (Logrank p = 0.952; p = 0.611); but in the HT group, the new cutpoint was predictive of treatment response (p = 0.343; p = 0.047) (Cox Table 2B). Table 1 Current CutpointNew CutpointDako PRp = 0.024p = 0.0002Leica PRp = 0.102p = 0.0002Ventana PRp = 0.058p = 0.0001 CONCLUSIONS: We have identified a new cutpoint for PR expression in BC that is prognostic within a tamoxifen treated BC cohort, and that maintained significance across 3 commercial PR assays. Moreover, this new cutpoint seems to be predictive of HT, as the untreated NCI controls did worse than the HT treated group. We intend to continue to evaluate the clinical role for the new PR cutpoint by re-analyzing PR data from completed clinical trials. Table 2 Current Cutpoint New Cutpoint 2A: Cal-TBCCHR95% CIp-valueHR95% CIp-valueDako PR0.7020.295-1.6700.420.6390.369-1.1060.11Leica PR0.7590.301-1.9130.560.5020.283-0.8880.018Ventana PR0.4480.201-0.9980.0490.4620.253-0.8440.0122B: NCI No HT0.9690.349-2.6910.9520.8530.461-1.5770.61HT0.5980.204-1.7510.3490.4340.185-1.0150.054 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-06.

Semi-quantitative immunohistochemical assay versus oncotype DX® qRT-PCR assay for estrogen and progesterone receptors: an independent quality assurance study

Semi-quantitative immunohistochemical assay versus oncotype DX® qRT-PCR assay for estrogen and progesterone receptors: an independent quality assurance study

Abstract P3-07-06: Initial treatment and survival among elderly breast cancer patients with positive estrogen receptor status by progesterone receptor status and stage: an analysis of US national registry data 2000–2009

Abstract BACKGROUND: There are few recent studies of initial treatment and survival among elderly, newly diagnosed breast cancer (BC) patients with positive estrogen receptor (ER) status, by progesterone receptor (PR) status and cancer stage at diagnosis. METHODS: The linked Surveillance and Epidemiology End Results-Medicare (SEER-Medicare) database was used for this analysis. SEER is an epidemiologic surveillance system consisting of population-based tumor registries designed to track cancer incidence and survival in geographically defined areas that represent approximately 25% of the US population. The SEER registry file (available through 2007) is linked to Medicare claims and enrollment data available through 2009. We identified female patients newly diagnosed with ER positive (ER assay reported to SEER as positive/elevated) Stage I, II, III, or IV breast cancer in a SEER registry between January 2002 and December 2007. Study patients were required to be aged 66+ years with no prior history of any other (non-breast) cancer. Patients were followed from the date of breast cancer diagnosis through death or December 31, 2009. PR status was collected in the breast cancer site-specific factor variable, with patients identified as PR+ (PR−) if the PR assay was reported to SEER as positive/elevated (negative/normal). Demographics, initial treatment (surgery or radiation within 4 months of diagnosis), and survival (proportion of patients who died during the study period, survival at 96 months and median survival) were evaluated, by PR status and cancer stage. Kaplan-Meier (KM) survival curves were estimated by PR status and stage. RESULTS: 57,571 female ER+ BC patients (47,385 PR+, and 10,186 PR−) were identified. Age at diagnosis was similar for PR+ and PR- BC patients (mean [SD] 75–77 [7]; median 75–77 years) across all stages. Compared to PR- patients, those with PR+ status were more likely to be diagnosed at Stage I (56.6% vs. 50.7%), and slightly less likely to be diagnosed at other stages (Stage II: 31.7% PR+ vs. 33.2% PR−; Stage III: 7.7% vs. 10.4%; Stage IV: 4.0% vs. 5.7%). Most patients had surgery in the first 4 months following diagnosis (96% PR+; 94% PR−), with Stage IV patients least likely (42% for both PR+ and PR−). Similar proportions of PR+ and PR- patients received initial radiation (49% vs. 46%), with minor variation across stages. Overall, fewer PR+ patients died during the evaluation period (22% vs. 28% PR−). KM survival analyses suggest that survival rates were higher for PR+ than PR- patients overall (survival at 96 months: 64% vs. 59%), and, with the exception of Stage I (73% for both), by stage (Stage II: 61% vs. 55%; Stage III: 40% [median survival 76 months] vs. 31% [median 58 months]; Stage IV: 14% [median 25 months] vs. 9% [median 19 months]). CONCLUSIONS: Among elderly, newly diagnosed ER+ BC patients, those with PR+ status were diagnosed at earlier stages and had better survival outcomes. Further studies of treatment patterns and outcomes in the ER+ BC population are warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-07-06.

Comparison of the oestrogen and progesterone receptor status in primary breast carcinomas as evaluated by immunohistochemistry and immunocytochemistry: a consecutive series of 267 patients

The use of cytological specimens to evaluate tumour biomarkers in metastatic breast cancer lesions has attracted increased interest because of the considerable number of reports that have shown discordance between the primary tumour and metastatic lesion. Oestrogen receptor (ER) and progesterone receptor (PgR) assays are crucial for the management of patients with breast cancer, in both adjuvant and palliative settings. The aim of this study was to compare the ER and PgR immunocytochemical analysis of fine needle aspiration (FNA) samples with the immunohistochemistry (IHC) of surgical specimens and core biopsies from primary breast cancers. The FNA specimens were prepared as cell blocks (n = 25) or ThinPreps (n = 258) for the immunocytochemistry (IC) ER and PgR analyses. Sixteen patients were excluded because of lack of follow-up (n = 1), neoadjuvant therapy (n = 3) or cell counts in their fine needle aspirates that were too low (n = 12). The results of IC on 25 cell blocks and 242 ThinPreps were compared with IHC on the corresponding core needle biopsies (n = 16) or excised tumours (n = 251). The ER and PgR status was defined as negative (when less than 10% of the nuclei were stained) or positive (when equal or more than 10% of the nuclei were stained). Kappa statistics were used to evaluate the concordance. The ER concordance was 98% with ThinPrep (κ = 0.93) and 92% with cell block (κ = 0.82). The corresponding values for PgR were 96% (κ = 0.91) and 96% (κ = 0.92). Our results confirm that, in cases in which biopsies or surgical specimens are not available, IC (with either cell block or ThinPrep techniques) is a reliable method for the determination of the ER and PgR status performed under strict conditions using primary breast carcinomas, and is therefore potentially useful in metastatic settings.

Semi-quantitative immunohistochemical assay versus oncotype DX® qRT-PCR assay for estrogen and progesterone receptors: an independent quality assurance study

AbstractEstrogen receptor (ER) status is a strong predictor of response to hormonal therapy in breast cancer patients. Presence of ER and level of expression have been shown to correlate with time to recurrence in patients undergoing therapy with tamoxifen or aromatase inhibitors. Risk reduction is also known to occur in ER-negative, progesterone receptor (PR)-positive patients treated with hormonal therapy. Since the 1990s, immunohistochemistry has been the primary method for assessing hormone receptor status. Recently, as a component of its oncotype DX® assay, Genomic Health began reporting quantitative estrogen and PR results determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). As part of an ongoing quality assurance program at our institution, we reviewed 464 breast cancer cases evaluated by both immunohistochemistry and oncotype DX® assay for estrogen and PR. We found good correlation for ER status between both assays (98.9% concordance), with immunohistochemistry being slightly more sensitive. Concordance for PR was 94.2% between immunohistochemistry and qRT-PCR with immunohistochemistry again more sensitive than RT-PCR. The results also showed linear correlation between immunohistochemistry H-scores and qRT-PCR expression values for ER (correlation coefficient of 0.579), and PR (correlation coefficient of 0.685). Due to the higher sensitivity of hormone receptor immunohistochemistry and additional advantages (ie preservation of morphology, less expensive, faster, more convenient), we conclude immunohistochemistry is preferable to qRT-PCR for determination of estrogen and PR expression.

P2-14-01: Race, Response to Chemotherapy, and Outcome within Clinical Breast Cancer Subtypes.

Abstract Introduction: Racial disparity in breast cancer (BC) outcome has been attributed to access to care, socioeconomic factors, and biologic factors. In particular, the prevalence of triple negative BC is significantly higher among African-American (AA) women than white women (non-AA). However a recent population-based study suggests that racial disparities persist even if stratified by subtype, although it is possible that bias in stage at presentation may have influenced these results. In this study, we examined 464 women of uniform clinical stage II-III breast cancer, all treated with neoadjuvant chemotherapy, and examined pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS) to see if racial disparity exists within a more homogeneous stage and treatment cohort. Methods: The UNC Neoadjuvant Breast Cancer Database is a prospectively maintained cohort with clinical and outcome annotation. We identified women diagnosed between 1991–2011 and assigned BC subtype by clinical assay for estrogen receptor and progesterone receptor (hormone receptors, HR) and HER2. Fisher's Exact and Wilcoxon Rank sum tests compared characteristics between AA and non-AA. The Kaplan-Meier method and Log Rank test compared OS and RFS curves. Results: 142 (31%) of 464 women were AA, 294 (63%) were white, and 28 (6%) were other races (for analysis white and “other” were categorized as non-AA). 191 (41%) were clinical stage II and 273 (59%) stage III at diagnosis. Stage at diagnosis did not differ between AA and non-AA (p=0.07). AA were slightly older (median age 49 v 45, p=0.01) and were more likely to be HR-/Her2- (44% v 25%, p=0.003). All patients received preoperative chemotherapy. AA received less biologic therapy such as trastuzumab or on-protocol bevacizumab (10% v 18%, p=0.03) and received significantly less endocrine therapy (46% v 59%, p=0.01) than non-AA patients. 102 (22%) of patients had a pCR to neoadjuvant chemotherapy; pCR rates did not differ significantly by race (p=0.7) or within subtype by race (all p>0.14). At a median follow-up of 4.5 years, 121 (26%) of patients have died. Among HR-/HER2− (n=114) and HR-/HER2+(n=47) patients, OS and RFS did not differ between AA and non-AA (all p>0.5). Conversely, among HR+/HER2− patients (n=159) AA had worse OS (p=0.01) and RFS (p= 0.03) and among HR+/HER2+ patients (n=53), AA had worse OS (p=0.0004) and RFS (p=0.005). Conclusion: In this cohort of clinical stage II-III BC patients, AA women were over 75% more likely to have triple negative BC than non-AA patients. Pathologic reponse to chemotherapy did not differ between AA and non-AA patients. Examining the entire cohort, AA women had worse RFS and OS, however there was no difference among ER-negative subtypes. The difference in RFS and OS was driven by worse outcome among women with ER or PR+ tumors (regardless of HER2). It is possible that this racial disparity in outcome among hormone receptor-positive BC patients reflects unknown biologic differences by race, or differences in access to or receipt of endocrine therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-14-01.

Pregnancy outcome in patients with stage 1a endometrial adenocarcinoma, who conservatively treated with megestrol acetate

The most effective treatment of well-differentiated endometrial carcinoma is surgery. The aim of this study is the evaluation of megestrol acetate on young patients with well-differentiated endometrial cancer who wish to preserve their fertility, with regard to the receptors. 16 patients were treated initially with 160 mg/d of megestrol acetate and continued with 320 mg/d for non-responsive cases. All patients followed with FD&C and hysteroscopy. The responsive patients were referred to IVF group. Response rate to hormonal therapy was 10/16 (62.5%). The mean time of responding was 7.5 months. Other six (37.5%) patients underwent total abdominal hysterectomy (TAH). Of 10 patients who responded to hormonal therapy, one exited of the study because of her husband's infertility. Two patients are under IVF. Three patients did not get pregnant and four patients became pregnant and finally underwent TAH. All patients had progesterone receptors. Only one patient lacked estrogen receptors; who also responded to treatment. Progestins treatment of these patients who want to have child may be useful, but close long-term follow-up is necessary. The evaluation of estrogen and progesterone receptors assay may be useful in predicting response to the treatment.

Abstract P4-07-05: Correlation of a Proliferation Index Combined with Progesterone Receptor to Oncotype DX® in Early Stage ER-Positive Breast Cancer

Abstract Background: Different gene expression sets have been reported to reproducibly assess the metastatic potential of early stage operable breast tumors. Knowledge of the biology-based components of amalgamated expression scores may provide insight into the relative contributions of the cellular processes involved. We sought to compare a composite metastasis score (cMS) consisting of a previously reported Metastasis Score (MS), that serves essentially as a proliferation index, and the progesterone receptor (PR) with the Oncotype DX® Recurrence Score (RS) in a regional cancer center setting. Methods: We examined sections from 152 blocks collected in the Blumenthal Cancer Center that had Oncotype DX® scores (a commercially available service performed by Genomic Health Inc. (GHI)). The study protocol was approved by the Institutional Review Board of the Blumenthal Cancer Center. ER and PR were initially scored by IHC; HER2 was determined by FISH. RT-PCR multiplex TaqMan assays of a 14-gene Metastasis Score (Tutt et al. (2008)) and a multiplex assay for ER, PR and HER2 (Iverson et al. (2009)) were carried out. A cMS was generated with MS and PR using previously constructed weighting. Risk classification of cMS was based on a single pre-established cut point; GHI recommended cut points were used for categorized Oncotype DX® risk. Correlations were estimated by Pearson's product-moment correlation coefficient; a goodness-of-fit test was used to determine differences in the categorical proportions. Results: Eighty-nine (59%), 51 (34%), and 12 (8%) of the 152 tumors tested had low, intermediate and high Oncotype DX® Recurrence Scores, respectively. The continuous scores for cMS and RS were correlated (r=0.71). Of the 89 tumors categorized as low risk by RS, 78 (88%) and 11 (12%) were at low and high risk by cMS, respectively; all (12) of the tumors categorized as high risk by RS were high risk by cMS; and of the 51 tumors with intermediate RS, 27 (53%) were categorized as low risk and 24 (47%) as high risk by cMS. Correlations of RT-PCR PR levels with overall RS and cMS scores were 0.70 and 0.71, respectively. The MS had correlations of 0.50 and 0.80 with RS and cMS, respectively. The categorized risk proportions of this patient population differed from those reported by Paik et al. (2004) (p=1.47x10-7). Tumors with HER2 gene amplification by FISH [3/141 (2%)] were under-represented (p=0.016) relative to Paik et al. (2004) [55/715 (8%)]; the proportion of PR negative tumors for Paik et al. [126/668 (19%)] and this study [14/149 (9%)], were also dissimilar (p=0.006). Conclusions: Despite sharing few genes, the continuous scores for cMS and RS show good correlation. The correlation of PR with these two scores suggests the level of this hormone receptor makes a similar significant contribution to both scores in this patient population. MS was more highly correlated to cMS than RS. Distinguishable features of cMS relative to the amalgamated RS include binary categorization of a continuous score and availability and transparency of discrete and combined scores for the endocrine and proliferation components. Under-representation of HER2- amplified tumors suggests exclusion of this patient group from testing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-05.