cytoplasmic estrogen receptor in breast cancer

Cytoplasmic Estrogen Receptor in breast cancer

Allison W. Welsh1, Donald R. Lannin2, Gregory S. Young3, Mark E. Sherman4, Jonine D.Figueroa4, N. Lynn Henry5, Lisa Ryden6, Chungyeul Kim7, Richard R. Love8, RachelSchiff9,10, and David L. Rimm1

1Department of Pathology, Yale University School of Medicine2Department of Surgery and Oncology, Yale University School of Medicine3Center for Biostatistics, Ohio State University4Division of Cancer Epidemiology and Genetics, National Cancer Institute5Department of Internal Medicine, University of Michigan Medical School6Department of Surgery, Clinical Sciences, Lund, Lund University, Sweden7Division of Pathology, National Surgical Adjuvant Breast and Bowel Project Foundation8International Breast Cancer Foundation, Madison, WI9Departments of Medicine and of Molecular and Cellular Biology, Baylor College of Medicine10Lester and Sue Smith Breast Center, Baylor College of Medicine

AbstractPurpose—In addition to genomic signaling, it is accepted that ERα has non-nuclear signalingfunctions, which correlate with tamoxifen resistance in preclinical models. However, evidence forcytoplasmic ER localization in human breast tumors is less established. We sought to determinethe presence and implications of non-nuclear ER in clinical specimens.

Experimental Design—A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, 1D5) werevalidated by western blot and quantitative immunofluorescent (QIF) analysis of cell lines andpatient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed forcytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, 128) and four others (NCIYTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort)from other sites around the world.

Results—Four of the antibodies specifically recognized ER by western and QIF, showed linearincreases in amounts of ER in cell line series with progressively increasing ER, and the antibodieswere reproducible on YTMA 49 with pearson’s correlations (r2 values)ranging from 0.87-0.94.One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence forspecific cytoplasmic staining with the other 4 antibodies across eight cohorts. The averageincidence was 1.5%, ranging from 0 to 3.2%.

Conclusions—Our data shows ERα present in the cytoplasm in a number of cases usingmultiple antibodies, while reinforcing the importance of antibody validation. In nearly 3,200 cases,cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be ofroutine clinical value.

Address all correspondence to: David L Rimm, Dept of Pathology, BML 116, Yale University School of Medicine, 310 Cedar St,New Haven, CT 06520-8023, Phone: 203-737-4204, Fax: 203-737-5089, [email protected].

NIH Public AccessAuthor ManuscriptClin Cancer Res. Author manuscript; available in PMC 2013 January 1.

Published in final edited form as:Clin Cancer Res. 2012 January 1; 18(1): 118–126. doi:10.1158/1078-0432.CCR-11-1236.

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Keywordsnon-nuclear; Estrogen Receptor; cytoplasmic; breast cancer

IntroductionThe Estrogen Receptor (ER) is the oldest and most successful biomarker that exists in breastcancer today (1-3); however, roughly 50% of ER-positive patients still exhibit de novo oracquired resistance to tamoxifen, suggesting that more complex mechanisms are operatingin these patients (4). In addition to the classical view of the Estrogen Receptor (ER) as anuclear hormone receptor, in the past ten years, it has become accepted that ER-alpha hasnon-nuclear signaling functions, referred to as non-genomic signaling. In the case of breastcancer, this non-genomic signaling can involve full-length receptor or other isoforms (5-9),as well as cross-talk with other growth-factor receptors (GFRs) (4, 10-12) or cytoplasmickinases such as Src (13-15). In preclinical models, non-genomic signaling has been shown tounderlie tamoxifen resistance (13, 16-20).

The current guidelines for measuring ER in a clinical setting, however, assess only nuclearstaining using immunohistochemistry (IHC) (21). The presence of cytoplasmic ormembranous immunoreactivity is ignored or assumed to be non-specific, and whileindividual pathologists may observe it from time to time, there is no available record of theincidence of such staining. The few reports in literature are all in cell line models, and nonehave shown concrete evidence to date of any cytoplasmic ER in actual breast cancer cases.Furthermore, some of these studies have used antibodies with less rigorous validation. Wehave previously found antibody validation to play a critical role in evaluation of proteinlocalization (22), and have thus developed extensive antibody validation protocols (23). Wealso have established the use of quantitative immunofluorescence (QIF), commercialized asAQUA technology (HistoRx Inc, New Haven, Connecticut), to assess expression andlocalization of a wide range of biomarkers on tissue microarrays (TMAs) (24-27). Inaddition to the benefit of quantification on a continuous scale, QIF allows more accurateassessment of protein localization, since each slide is stained with DAPI as well ascytokeratin, and a protein of interest can be assessed for co-localization with each.

In this study, we first sought to validate a panel of ER antibodies in order to determine ifnon-nuclear ER existed in clinical specimens using QIF on a number of retrospectivecohorts. We then reviewed the localization of expression in a series of eight cohortsdisplayed on TMAs. We hypothesized that patients with high levels of cytoplasmic ERwould show less benefit from endocrine therapies than those with low levels.

Materials and MethodsCell Culture

A panel of ATCC breast cancer cell lines was chosen to span a range of ER expression.Additionally MCF-7 cells engineered with doxycyclin-inducible ER overexpression wereused, which were a gift from Elaine Alarid (see Fowler et al (28)). All cells were maintainedat 37°C and 5% CO2, and grown either in suggested media, or in RPMI 1640 culturemedium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gemini BioProducts),100units/mL penicillin G and 100μg/mL streptomycin (Gibco), 1mM sodium pyruvate(Gibco), and 2mM L-glutamine (Gibco).

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AntibodiesAntibodies were selected for validation based on previous use in the literature. The fiveantibodies selected were F10 (Santa Cruz) used at 1:1000 overnight at 4deg, SP1 (Thermo)used at 1:500 overnight at 4deg, 60c (Upstate) used at 1:2000 overnight at 4deg, 1D5 (Dako)at 1:500 overnight at 4deg, and MC20 (Santa Cruz) at 1:500 for 1hr at RT. β-tubulin (CellSignaling Technology, 2146), diluted 1:4000, was used as a loading control for westernblots. Rabbit and mouse cytokeratin antibodies (Dako) were used at 1:100 forimmunostaining.

Western BlottingWhole-cell lysates were prepared in buffer containing 1% Nonidet P-40, 20nM TrisHClpH8.0, 137mM NaCl, 10% glycerol, 2mM EDTA, 1mM DTT, 1mM NaVO3, and completemini EDTA-free protease inhibitor cocktail (Roche) in dH20. 25μg of each lysate wasresolved by SDS-PAGE on a 4-12% Bis-Tris gel (NuPage), using NuPage MOPS SDSRunning Buffer at 45mA. Resolved protein was transferred using NuPage Transfer Buffer at50V for 2h. Western blotting was performed according to standard procedures, using the ERantibodies above.

Index TMAWhole cell pellets (fixed in formalin and paraffin-embedded) were created from the cell linepanel (for a detailed protocol, see Dolled-Filhart et al and McCabe et al (29, 30)). Thesepellets were cored and placed on a tissue microarray along with a panel of 40 breast cancerpatient controls (spanning the range of ER expression). This TMA (referred to as the IndexTMA) was used as a control array during antibody validation and immunofluorescentstaining.

Patient CohortsIn total, eight different cohorts of archival breast cancer cases, present on formalin-fixedparaffin-embedded TMAs, were examined for evidence of cytoplasmic ER byimmunofluorescence. One of the cohorts (YTMA 49) was also examined by traditionalimmunohistochemistry. Four of the TMAs were constructed at Yale according to previouslypublished guidelines (31): YTMA 49 (diagnosed 1962-1982, n = 619, stained with 1D5, 60c,SP1, and F10), YTMA 107 (collaboration with University of Michigan as part of aTamoxifen pharmacogenetics clinical trial (32), n = 179, stained with 1D5), YTMA 130(diagnosed 1976-2005, n = 526, stained with 1D5 and SP1), and YTMA 128 (diagnosed2003-2006, n = 257, stained with 1D5 and SP1). Four other cohorts came from outsideinstitutions: NCI YTMA 99 (a Polish multi-institutional trial, n = 732 with triplicate coresfor each case, stained with 1D5), NSABP B14 (a multi-institutional trial (33), n = 956,stained with SP1), South Swedish Breast Cancer Group SBII (34, 35) (n = 556, stained with1D5), and a Vietnamese Oophorectomy cohort (premenopausal women with operable breastcancer, see Love et al (36) for more information, n = 156, stained with SP1).

Immunofluorescent (IF) StainingSlides were deparaffinized by melting at 60°C for 20min, followed by soaking twice for20min in xylene (JT Baker). Rehydration was performed twice in 100% EtOH for 1min,followed by 70% EtOH for 1min, and tap water for 5min. Antigen retrieval was performedin citrate buffer (3.84g sodium citrate dihydrate in 2L ddH20, brought to pH 6.0 with 1Mcitric acid) using the PT module from LabVision. Endogenous peroxidases were blocked by30 min incubation in 2.5% hydrogen peroxide in methanol at room temperature (RT). Afterwashing, non-specific antigens were blocked by incubation in 0.3% bovine serum albuminin TBST for 30min at RT in humidity chamber. Rabbit Cytokeratin (Dako), diluted 1:100 in

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block (BSA in TBST above), and was incubated overnight at 4°C. ER was stained usingvarious antibodies (noted under Patient Cohorts), which included 1D5 (Dako, 1:50 in block,incubated 1h at RT), SP1 (Thermo, 1:1000 in block, incubated overnight at 4°C), F10 (SantaCruz, 1:5000 in block overnight at 4), 60c (Upstate, 1:5000 in block overnight at 4deg),MC20 (1:100 in block overnight at 4deg). Primary antibodies were followed by Alexa 546-conjugated Goat anti-Rabbit or anti-Mouse secondary antibody (Molecular Probes) diluted1:100 in mouse or rabbit EnVision reagent (Dako) for 1h at RT. Signal was amplified usingCyanine 5 (Cy5)-tyramide (Perkin-Elmer) at a dilution of 1:50 for 10min at RT. Nuclei werestained using 10μg/mL DAPI (Molecular Probes) in block for 20min at RT, and coverslipsmounted with Prolong mounting medium (ProLong Gold, Molecular Probes).

AQUA analysisER immunofluorescence was quantified using AQUA. Briefly, a series of high-resolutionmonochromatic images were captured by the PM-2000 microscope (HistoRx) usingAQUAsition 2.2 software (HistoRx). Images were collected for each histospot after auto-focus and auto-exposure. Fluorophores included DAPI (to create nuclear compartment), Cy3(Alexa 546-cytokeratin to distinguish tumor from stroma and create cytoplasmiccompartment), and Cy5 for the target (ER). Image analysis was performed usingAQUAnalysis 2.2 software (HistoRx), which binarizes the cytokeratin stain (each pixelbeing “on” or “off”) to create an epithelial tumor “mask”. It uses a clustering algorithm toassign each pixel, with 95% confidence, to either a nuclear or cytoplasmic compartment.The AQUA score of ER in each subcellular compartment (nuclear, cytoplasmic, and wholetumor mask) is calculated by dividing the ER pixel intensities by the area of thecompartment within which they were measured. AQUA scores are normalized to theexposure time, bit depth, and lamp hours at which the images were captured, allowing scorescollected at different exposure times to be directly comparable.

Immunohistochemical StainingIHC staining was performed on YTMA 49 in a CLIA-certified laboratory, using either the1D5 (Dako) or SP1 (Ventana) staining system for ER in accordance with manufacturer’sinstructions. Slides were digitally scanned using the BioImagene slide scanner and visuallyassessed using ImageViewer software (BioImagene).

Assessment of Cytoplasmic ERIn order for a case to be considered positive for cytoplasmic ER, the following conditionshad to be met: 1) immunoreactivity was observed using one of four valid monoclonalantibodies (1D5, SP1, 60c, F10), 2) immunoreactivity co-localized with cytokeratin but didnot co-localize with DAPI, 3) immunoreactivity was robust (at least 25% the intensity ofnuclear staining, or greater than nuclear staining), 4) immunoreactivity was not due to out-of-focus tissue or bleed-through from any other channel, 5) immunoreactivity was observedon the same slide as a positive (nuclear staining with no background) and negative (nostaining) control cases. For each cytoplasmic case, conditions 1-5 were confirmed by twoseparate individuals (AWW and DLR), including a certified pathologist (DLR).

Statistical AnalysisAll statistical analyses (bivariate regressions) were performed using StatView analysissoftware (SAS Institute Inc., Cary, NC).

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ResultsAntibodies to multiple epitopes of ER are highly reproducible

In order to see assess the existence of cytoplasmic ER in breast cancer cases, we firstvalidated a panel of five antibodies raised against different epitopes of ER. We chose fiveantibodies previously reported in the literature to be specific and/or to detect cytoplasmic ER(see Figure 1A). These included 1D5 (an N-terminal mouse monoclonal from Dako, theclinical standard), SP1 (a C-terminal rabbit monoclonal, reported to be more sensitive than1D5 and less sensitive to signal degradation due to delays in fixation (37-40)), 60c (an N-terminal rabbit monoclonal from Upstate), F10 (a C-terminal mouse monoclonal we hadpreviously validated in the lab), and MC20 (a rabbit polyclonal reported to showcytoplasmic ER staining(41-48)). First, a panel of cell lines with known ER status wasexamined by western blot. SP1, 60c, and F10 showed specific detection of the expected66kD band for full-length ER (Figure 1B) in the four lines known to be ER-positive (BT474,MCF7, T47D, ZR751). These four antibodies (1D5, F10, SP1, 60c) were then used to stain alarge retrospective cohort of breast cancer cases from Yale (YTMA 49, diagnosed1962-1982) by QIF, and levels of nuclear ER were quantified using AQUA. All antibodiesshowed strong reproducibility on these 619 cases (Figure 1C-H), with pearson’s r2 valuesranging from 0.87 to 0.94 (r-values ranging from 0.93-0.97).

The next step in our antibody validation protocol is titration and analysis of specificity andreproducibility by immunofluorescence. Each cell line in the panel was formalin-fixed,paraffin-embedded and cored as tissue blocks to create the Index TMA, which alsocontained a panel of 40 breast cancer control cases in duplicate (spanning the range of ERexpression). After titration on breast test TMAs to optimize their dilution, each antibody wasindividually used to stain the Index TMA by QIF. Specific nuclear staining was observed inthe four ER-positive cell lines using 1D5, SP1, 60c and F10. Furthermore, progressivelyincreased staining was seen in a cell line series induced to gradually overexpress amounts ofER (data only shown with SP1 as a representative case, see Figure 2F, top panels),confirming the specificity of these antibodies by IF. Duplicate control cases also showedstrong reproducibility core-to-core.

We also sought to test the MC20 antibody since it is commonly cited in publisheddescriptions of cytoplasmic ER(41-48). We found that, at low exposure, the MC20 antibodyappeared to detect a specific 66kD band on a western blot of cell line controls (Figure 2A,top panel). However, when the blot was left for longer exposure, and compared to SP1,MC20 showed non-specific immunoreactivity in a series of ER-negative cell lines (Figure2A), as well as multiple immunoreactive bands. Similar non-specific immunoreactivity wasobserved when MC20 was used to quantify ER by IF on the cell line panel (Figure 2B), incontrast to SP1 (Figure 2C). On the panel of patient controls present on the same IndexTMA, QIF analysis of ER expression using MC20 again did not correlate with SP1 (Figure2D), and showed strong cytoplasmic staining in cases that were strictly nuclear with SP1(Figure 2E). Lastly, in a panel of cell lines engineered to overexpress increasing amounts ofERα, MC20 showed constitutively high levels of cytoplasmic immunoreactivity, in contrastto increasing nuclear immunoreactivity seen with SP1 (Figure 2F). To confirm this data wasnot due to a poor antibody lot, we repeated each experiment with a second lot of MC20, andfound the same results. Thus MC20 was not included in the panel of valid antibodies.

Detection of cytoplasmic ER by multiple antibodies and methodsOnce specific and reproducible detection of nuclear ER was confirmed using multipleantibodies, we examined a series of large retrospective cohorts of breast cancer cases onTMAs to determine the frequency of non-nuclear expression of ER. We were able to find

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cases with strong cytoplasmic immunoreactivity in epithelial cells (co-localized withcytokeratin, not co-localized with DAPI, Figure 3A). In all cases, the staining appeared to bemore cytoplasmic than membranous, and while a few cases were strictly cytoplasmicwithout any nuclear reactivity (Figure 3A, top panel), in a majority of instances thecytoplasmic staining was observed along with nuclear staining (Figure 3A, bottom panel).When subsequent cuts were available (this was the case in our Yale cohorts), we were ableto assess expression using multiple ER antibodies and confirm that the cytoplasmicreactivity was observed using more than one antibody (Figure 3B), suggesting it was not anepitope-specific artifact, and was potentially full-length ER or an isoform containing anintact N and C terminus. Furthermore, the cytoplasmic immunoreactivity was observedusing traditional IHC methods as well (Figure 3C), with both N- (1D5) and C-terminal (SP1)antibodies.

Analysis of multiple retrospective cohorts suggests incidence of cytoplasmic ER is lowWe have assessed cytoplasmic ER by QIF on nearly 3,200 individual cases from eightdifferent retrospective breast cancer cohorts, four from Yale (YTMA 49, 107, 128, and 130)and four from outside sources, including two multi-institutional trials (NSABP B14 andSouth Swedish Breast Cancer Group SBII) as well as two others (NCI YTMA 99 and aVietnamese Oophorectomy cohort). One of these (YTMA 49) was assessed by QIF induplicate (a second core from each patient) as well as by traditional IHC. In order for a caseto be considered “positive” for cytoplasmic ER, five conditions had to be met, as describedin the methods section.

Overall the incidence of cytoplasmic staining only averaged 1.49%, ranging from 0% to3.2% at best (Table I), an incidence that was too low to discover any prognostic orpredictive significance, even with relatively large cohorts. Since many of the cytoplasmiccases were observed on cohorts from outside institutions, we did not have broad access tothe original tissue to perform any follow-up analysis on the individual cases. We attemptedto extract RNA from formalin-fixed, paraffin-embedded (FFPE) cores of the cases found inYTMA 49 and 130 (in Table I), however since most of the cases are older then 10 years, theRNA was insufficiently preserved for successful RT-PCR analysis with ER-specific primers.

DiscussionWe have found that four antibodies to ER-alpha (1D5, SP1, F10 and 60c) are highlyreproducible and specific by both western blot as well as QIF, in cell lines and in FFPEcases. Using each of these valid antibodies, we have found evidence for cytoplasmic ER inbreast cancer specimens, with both IF and traditional IHC. However, across nearly 3,200cases, spanning eight different retrospective cohorts, we found cytoplasmic ER is present atvery low incidence. Thus, its measurement by these methods is unlikely to be of prognosticor predictive value in untreated cases.

Confirmation of immunoreactivity by multiple antibodies suggests that the cytoplasmiclocalization is not an epitope-specific artifact. However, there are other artifacts that cannotbe ruled out, including the possibility that the cytoplasmic localization is a function offixation method, tissue age, processing, or some other ill-defined pre-analytic variable. Thespecimens assessed in this study came from a variety of institutions and in many cases wehave no record of how the tissue was handled. Follow-up studies on many of the cases wasnot possible since we did not have access to tissue. Nevertheless, studies are ongoing todetermine the identity of the cytoplasmic immunoreactivity we have observed. To date, wehave seen no evidence of higher incidence on tissue with longer time-to-fixation, or otherpre-analytic variables. We also have no evidence for correlation between cytoplasmic ERimmunoreactivity and expression of other biologically-relevant markers, including HER2,

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PR and stem-cell markers (ALDH and CD44 co-expression). However, we have observedsome cytoplasmic staining with ER antibodies on melanoma tissue, and are in the process ofdetermining the identity of this immunoreactivity.

The low incidence overall which we have observed is surprising, given the extent of existingdata on functional non-genomic ER. One explanation for this is the discrepancies betweendifferent antibodies. Our data suggests that extensive antibody validation, as we havepreviously described (22, 23) is critical in assessment of ER localization. Additionally,essentially all of the evidence for cytoplasmic ER is shown in preclinical models, most ofthem cell lines where rapid localization of the receptor was observed. A patient tissuesample, obtained and fixed at a single moment in time, may not be able to detect such ashort-lived event. Additionally, the majority of cell lines where cytoplasmic ER wasobserved in a more permanent nature were treated with tamoxifen. It is difficult to testpatient tissue after treatment with tamoxifen, since most cohorts are made from primarybreast tumors prior to any sort of treatment. In the future, neoadjuvant endocrine therapycohorts may be able to resolve this issue.

Another explanation for our observations could be the use of TMAs instead of wholesections. If localization of ER is heterogeneous within tumors, it may be seen in wholesections, but missed in low sampling (one-fold redundant) TMA cohorts. While wholesections were not available for any of these cohorts, the use of TMAs also allowed us tomaximize the number of specimens included in this study. Furthermore, in smaller serieswhere we assessed ER on whole sections, cytoplasmic ER was not observed (49). We did,however, have triplicate cores for each case in the NCI YTMA 99 cohort, and in cases withevidence for cytoplasmic ER, we were able to observe it in multiple cores. Additionally, thelargest Yale cohort (YTMA 49) has been produced at 12-fold redundancy. We found that thetwo cases initially positive for cytoplasmic ER were also positive in other cores from thesame patient. Furthermore, examination of other cores did not reveal any new cytoplasmiccases when examining other blocks. Finally, it is possible that low levels of cytoplasmic ERexist in many clinical specimens, but the levels of expression are below the threshold ofdetection using the QIF or IHC assays. We have not yet determined a way to isolate andquantify ER that is present at very low levels in human tissue specimens.

Our initial goal was to develop an assay that measured cytoplasmic ER in order to predictpatients who would show less benefit from endocrine therapies. However, after extensiveantibody validation and examination of a large number of cases, we cannot find evidence forcytoplasmic ER on more than 4% of cases. Although these cohorts are reasonably large, thelow event frequency results in insufficient statistical power to look for meaningfulassociations with outcome.

AcknowledgmentsThis study was supported by additional grants from the Intramural Research Program of the National CancerInstitute to Mark Sherman and Jonine Figueroa, and an American Society of Clinical Oncology Young Investigatoraward to Lynn Henry.

Support for this research comes from NIH R33 CA 106709 (to DLR) and a US Army CDMRP pre-doctoralfellowship (AWW).

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Statement of Translational Relevance

Clinicians today rely on measurement of nuclear Estrogen Receptor (ER) as a hallmarkof breast cancer diagnosis and treatment. However, for over 10 years it has been widelyaccepted that ER exists and functions outside the nucleus, contributing to growth andsurvival as well as resistance to endocrine therapies in cellular models, suggesting it’smeasurement could be of prognostic or predictive value, and could help address theproblem of tamoxifen resistance. However, in the first study to thoroughly examine theincidence of cytoplasmic ER in human cases, we find an incidence of only 1.5%,suggesting cytoplasmic ER may only be a transient occurrence or an artifact ofimmunostaining, and is therefore unlikely to be of predictive value in the current clinicalsetting.

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Figure 1. Antibodies to multiple epitopes of ER are highly reproducibleFive antibodies binding to different epitopes of ER were chosen for validation, includingmouse monoclonals 1D5 and F10, rabbit monoclonals SP1 and 60c, and rabbit polyclonalMC20 (A). Specific detection of a 66kD band for ER was detected by western blot in a cellline panel using F10, SP1 and 60c, with β-tubulin as loading control (B). Antibodies thatpassed validation (1D5, SP1, F10, 60c) were stained using QIF on YTMA 49, and nuclearER expression levels (AQUA scores) were correlated between each antibody (C-H) showingPearson’s coefficients (r2-values) from 0.87-0.94. AF1, activation function 1; BD, bindingdomain; AF2, activation function 2.

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Figure 2. Significance of antibody validation in detection of cytoplasmic ERA panel of breast cancer cell lines was analyzed by western blot for ER (full-length 66kD)using both MC20 and SP1 antibody and developed at different exposures (A, β-tubulin wasloading control) to compare antibody specificity. Both antibodies were also stained usingQIF on the Index array, which contained the same cell line panel (AQUA scores for MC20in B, for SP1 in C), as well as a panel of patient controls. Correlation between AQUA scoresfor the patient controls is shown in D. A representative case is shown in E, with cytoplasmicimmunoreactivity observed with MC20 (top panel), where there is only nuclear staining seenwith SP1 (bottom panel). Lastly, a series of MCF-7 cells engineered with doxycyclin-inducible overexpression of ER (28) were maintained as six separate cultures, and treatedwith increasing amounts of doxycyclin for 48 hours, before pelleting and formalin-fixing forpreparation on a TMA. IF staining was then performed on these cell lines, using both SP1(F, top panels) and MC20 (F, bottom panels) in order to further validate specificity ofimmunoreactivity. Doxy, doxycyclin.

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Figure 3. Detection of cytoplasmic ER by multiple antibodies and methodsValidated antibodies (1D5, SP1, F10, 60c) were stained by QIF on eight retrospective breastcancer cohorts in order to assess presence of cytoplasmic ER according to six conditions(see Methods). Representative image of two cases are shown in A (both stained with SP1),showing cytoplasmic immunoreactivity co-localized with cytokeratin, either in the absenceof nuclear staining (top panel, A), as well as alongside or along with nuclear staining(bottom panel, B). Scale bars are 100μm, insets are higher magnification. On cases fromYTMA 49, which was stained using all four antibodies, cytoplasmic immunoreactivity wasobserved with each antibody (B, scale bars are 100μm, insets are higher magnification).Cytoplasmic immunoreactivity was also observed using traditional IHC performed in aCLIA-certified lab with both SP1 (Ventana) and 1D5 (Dako) systems (C, insets are highermagnification).

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Table I

Incidence of cytoplasmic ER on multiple retrospective breast cancer cohorts

Cohort Number of cytoplasmiccases

Number of total casesvalidated

Percent (%) of cases withcytoplasmic staining

Antibodies used

NCI YTMA 99 17 732 2.3 1D5

NSABP B14 21 655 3.2 SP1

South Swedsh BCG SBII 0 556 0 1D5

YTMA 49 2 378 0.5 1D5, SP1, 60c, F10

YTMA 130 4 316 1.3 1D5, SP1

YTMA 128 0 183 0 1D5, SP1

VIE OO Cohort 0 156 0 SP1

TMA 107 3 179 1.7 1D5

Total 47 3155 1.49

Eight retrospective cohorts were assessed for presence of cytoplasmic ER, with multiple validated antibodies, according to the six conditionsoutlined in the Methods. Incidence of cytoplasmic ER was confirmed by two separate individuals (AWW and DLR, one a board-certifiedPathologist and the other a Pathology graduate student), and reported as a percentage of the total cases with valid immunostaining (eliminated caseswith missing tissue, out of focus regions, etc). NCI, National Cancer Institute; YTMA, Yale Tissue Microarray; NSABP, National SurgicalAdjuvant Breast and Bowel Project; BCG, Breast Cancer Group; VIE, Vietnamese; OO, Oophorectomy


cytoplasmic estrogen receptor in breast cancer

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