comparisonof immunocytochemicaland steroid ... · an estrogen receptor immunocytochemical assay...

[CANCER RESEARCH 45, 293-304, January 1985]

Comparisonof Immunocytochemicaland Steroid-bindingAssaysfor EstrogenReceptorin HumanBreastTumors1

WilliamJ. King,2EugeneR.DeSombre,ElwoodV. Jensen,andGeoffreyL.Greene3

The Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637

ABSTRACT

An estrogen receptor immunocytochemical assay which usesmonoclonal antibodies to the estrogen receptor protein [Nature(Lond.), 307: 745-747, 1984] was applied to several humantissues, including human breast tumors, and the results werecompared to those of steroid-binding assays performed on cy-

tosol extracts of the same tissues. Specific immunoperoxidasestaining in fixed, frozen sections was confined to the nucleus ofselected cell populations within each tissue examined. In 117human breast cancers, the presence or absence of nuclearstaining was significantly associated with the concentration ofcytosolic estrogen receptor. Thirty-eight estrogen receptor immunocytochemical assay-positive tumors were further assessedfor several quantifiable features of the staining, including intensity, cellularity, and the proportion of tumor cells stained. Ofthese, epithelial cellularity showed the highest degree of correlation with the results of steroid-binding assays.

INTRODUCTION

Correlations of patient responses to endocrine therapies withthe ER4 content of human breast cancers have clearly estab

lished the clinical usefulness of biochemical ER assays (7, 19,30). While only 25 to 30% of unselected breast cancer patientsobtain objective remission of metastatic disease following various endocrine therapies, more than one-half of patients whose

cancers are found to contain ER benefit (7, 8, 35). Furthermore,there is evidence that the patients whose cancers have moresubstantial quantities of ER, designated as receptor rich, have abettsr probability to benefit from such treatments (8), as dopatients whose cancers also contain progestin receptor (4, 30).Moreover, patients whose primary cancers are ER positive arelikely to have a longer disease-free interval (1,4,43) and possibly

longer survival than those whose lesions lack the receptor.Despite this usefulness, current assay methods for ER are

problematic in that they depend on the association of radiola-

beled estrogen with its very labile receptor protein. Attempts toprovide quality control for this assay among various laboratorieshave been difficult. Furthermore, current assays do not allowassessment of the possible heterogeneity of ER among cells or

1Supported by research grants from the American Cancer Society (BC-86),Abbott Laboratories, the National Cancer Institute (CA-02897. CA-14599, CA-27476), and by the Women's Board of the University of Chicago Cancer Research

Foundation. This work was presented in preliminary form at the 64th AnnualMeeting of the Endocrine Society, San Francisco, CA, June 1982 (26).

2 Supported by a postdoctoral training grant (NIH/NCI09183). Present address:

Abbott Laboratories, North Chicago, IL 60064.3To whom requests for reprints should be addressed.4The abbreviations used are: ER, estrogen receptor; ER-ICA, estrogen receptor

immunocytochemical assay; PBS, phosphate-buffered saline containing 10 mwsodium phosphate, pH 7.4, and 150 mm sodium chloride; DAB, 3,3'-diaminobenz-

idine.Received February 6,1984; accepted September 24,1984.

regions within a lesion. Such information may be of specialimportance in tumors containing mixed histological profiles, especially when such a mixture includes benign lesions, generallyfound to contain low levels of ER but unlikely to be a source offuture mÃ©tastases(47). It is also of value when analyzing specimens which have metastasized to tissues, such as the ovary,which may contain their own complement of ER. In addition, incertain cases, the lesion may be too small to permit definitiveER assays by conventional methods, or the only material available may be from a needle biopsy.

Several methods for detecting ER with fluorescent estrogensor conjugated anti-estradiol antibodies in frozen or paraffin-

imbedded tumor sections have been developed to fill this needfor histochemical detection of ER (28, 33, 37, 42), but seriousquestions remain concerning the ability of such assays to detectreceptor and to discriminate between ER and other estrogen-

binding proteins (2, 32, 36, 48). Recently, Raam ef al. (41)reported that polyclonal antisera raised against a high-molecular-weight, estrogen-binding fraction of ER-containing human breastcancers could be used to identify receptor-positive cells in frozen

sections of such tumors; however, little is known regarding thecross-reactivity of these antibodies with respect to nonreceptorcomponents of ER-containing cells.

Over the last several years, we have purified ER from MCF-7human breast cancer cells (10, 14, 16) and calf uterus (6, 11-

14) and subsequently used these receptor preparations to generate both polyclonal and monoclonal antibodies against ER (11-

14). We have since established a library of monoclonal antibodiesthat recognize several unique receptor determinants and whichshow negligible cross-reactivity with other cellular proteins.These antibodies have been used to develop both immunoradi-

ometric and immunocolorimetric assays for ER (15). In the present study, we report the characteristics of an ER-ICA for detect

ing receptor in estrogen target tissues and the correlation of ERICA staining with biochemical ER determinations performed ona large sampling of human breast tumors. Preliminary reports ofportions of these data have appeared elsewhere (25, 26).

MATERIALS AND METHODS

Reagents. [2,4,6,7-3H]Estradiol (108 Ci/mmol) was obtained from

New England Nuclear, Boston, MA. DAB was obtained as the tetrachlo-

ride (Grade II) from Sigma Chemical Co., St. Louis, MO.Preparation of Immunological Reagents. Details concerning the

preparation of purified En, immunization of Lewis rats, and the preparation of monoclonal antibody have been described elsewhere (14,16).Briefly, confluent MCF-7 human breast cancer cells were used to prepare

cytosol from which receptor was partially purified by steroid affinitychromatography (10,14,16). This material was used to immunize maleLewis rats whose spleen cells were fused with mouse myeloma cells toobtain hybridoma lines making antibody to the receptor protein (16).Goat antiserum against Lewis rat igG was obtained as described previ-

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ER IMMUNOCYTOCHEMISTRY

ously (14) and was found to have little or no cross-reactivity with human

IgG.Immunocytochcmical Assay for ER. Surgical specimens sufficiently

large to permit both biochemical and immunocytochemical analysis weretrimmed of obvious fat and necrotic tissue. Care was taken to selectportions of the tumor for immunocytochemical analysis which by grossinspection were representative of that portion to be analyzed biochemically. If not already frozen, the specimens were then quick-frozen in

liquid nitrogen and stored for no more than 1 week before furtherprocessing. Immunocytochemical staining for ER was performed asfollows. Tumor samples were sectioned at 8-^m thickness across theirwidest diameter at -20Â°, and the sections were thaw mounted onto

acetone-cleaned, glass microscope slides and were immediately placed

without drying into an appropriate fixative. In order to permit meaningfulcomparisons, several sections of each tumor were fixed by successiveimmersions for 1 to 2 min each in 70% ethanokPBS at 4Â°,followed by

95% ethanol at room temperature. Slides were then transferred to PBSfor 5 to 30 min before proceeding. Sections fixed in this manner werevery stable with respect to immunocytochemical detection of ER andcould be stored for a period of several months at -20Â° in 50% glyc-

erol:PBS with no apparent loss of staining intensity. In most cases, ERwas localized immediately and was therefore evaluated several daysbefore the results of steroid-binding assays were available.

All of the following incubations were performed at room temperaturein a humidified chamber. Sections were first incubated for 15 min with a10% (w/v) solution of ovalbumin (Sigma) in PBS in order to reducenonspecific binding of subsequent reagents, all of which were added inPBS alone. After gentle rinsing in PBS, the sections were incubatedsuccessively with monoclonal anti-ER, goat anti-Lewis rat IgG serum(1:50 dilution), and rat horseradish peroxidase:anti-horseradish peroxi-dase complexes (1:80 dilution; Sternberger-Meyer, Jarrettsville, MD). Alltumors were incubated with the same monoclonal anti-ER, designatedH226Sp27,5 at 10 to 20 ^g/ml, a concentration which gives maximum

staining intensity under the incubation conditions used. However, specificstaining of ER-positive breast tumors is seen with as little as 0.3 Â¿Â¡gof

this antibody per ml. Each antibody incubation was carried out for 30min and was followed by a 5- to 15-min wash in PBS. After the final

wash, sections were incubated for 15 min with the chromogen (1.7 HIMDAB and 0.06% hydrogen hydroxide in PBS). Sections were then rinsedin running tap water and dehydrated through graded ethanols and xylenebefore mounting in Pro-Texx (American Scientific Products, McGaw Park,

IL). Duplicate sections were lightly stained with hematoxylin after the tapwater rinse to facilitate the identification of cellular elements within thetumor. Negative controls were run with every specimen and consistedof adjacent sections treated with a control monoclonal rat IgG againstbovine choline acetyltransferase or torpedo acetylcholine receptor (generously provided by Dr. Bruce Wainer and Dr. David Richman, respectively) or with normal Lewis rat IgG. Positive controls consisted ofsections of human proliferative-phase or postmenopausal endometriumand biochemically confirmed, ER-rich breast tumors.

Biochemical Determination of ER. ER was measured in humanbreast tumor cytosols by sucrose density gradient centrifugation asdescribed previously (22). Under these conditions, ER-rich tumors are

defined as having greater than 750 fmol ER/g, wet weight, for specimensfrom postmenopausal patients or greater than 300 fmol/g for premeno-

pausal specimens and represent that group of tumors with a frequencyof remission that in response to endocrine-ablative therapy approaches

70% (21).Semiquantitative Evaluation of Immunocytochemical Staining.

Thirty-eight breast tumors, containing immunocytochemically positive

cells and representing a wide range of cytosol receptor values (41 to2833 fmol/g), were further examined for staining features that might

relate to the tumor ER content.6 Following the decision to perform these

analyses, all tumors containing ER-ICA-positive cells were examined.

The intensity of nuclear staining was subjectively given an integer scoreof 1, 2, or 3, with 3 representing the most intense staining. Since somevariability in intensity is often noted within various regions of the sametumor section, the assigned value was chosen to reflect the averagedegree of staining seen in that population of cells which were ER-ICApositive. With the aid of a microscope grid, we also estimated theproportion of each tumor cross-section which was occupied by epithelial

cells, i.e., tumor cellularity, ranging from 0.05 to 0.95. Finally, becausemost ER-ICA-positive tumors contained subpopulations of ER-ICA-neg-

ative epithelial cells, the proportion of ER-ICA-positive epithelial cells,

which ranged from 0.05 to 1.00, was evaluated in each tumor byassessing at least 200 cells from each tumor. No more than 50 cellswere assessed in any single high-power (x250) field. Each of these

semiquantitative measures was correlated with the cytosolic ER contentdetermined by sucrose density gradient analysis.

The possible influence of sampling error in making the above evaluations was assessed by sectioning 4 tumors throughout their thickness;a section from each quartile was used for immunocytochemical evaluation. The ranges of values for intensity, positive tumor fraction, andcellularity for 4 sections of Tumor A (3 fmol/g) were 1 to 2, 0.15 to 0.54,and 0.02 to 0.06, respectively. For Tumor B (1813 fmol/g), the corresponding values were 1 to 2, 0.70 to 1.00, and 0.36 to 0.56; for TumorC (1789 fmol/g), they were 1 to 2, 0.60 to 0.95, and 0.30 to 0.50; andfor Tumor D (2100 fmol/g), they were 2, 0.78 to 0.97, and 0.23 to 0.32.

Statistics. Simple and multiple regression analyses were performedusing the MINITAB (copyright, Pennsylvania State University) statisticalpackage. The ER value obtained by assay of each tumor cytosol wasregressed on the immunocytochemical variables taken singly and in allpossible combinations. A f value was calculated by the program for theregression coefficient of each immunocytochemical variable by dividingthis coefficient by its S.D. ("f ratio"). In the case of multivariate testing,

the immunocytochemical values were entered in all possible orders, andthe t value given for each variable tests the significance of that variablewhen entering the equation last. The significance was evaluated byreferral to a table of the t distribution, testing the null hypothesis of zerovalue for the regression coefficient of each variable. When appropriate,TWOSAMPLE (a MINITAB program to compare means without assumingequal population variances) or x2 analysis was also used.

RESULTS

Staining Specificity. As described previously (25, 26), incubation of ethanol-fixed, frozen sections of ER-rich human breasttumors with monoclonal anti-ER results in a pattern of nuclear

localization with little or no cytoplasmic staining (Fig. 1). Thisintracellular distribution was noted regardless of the menstrualstatus of the patient at the time of surgery and was limited totumor epithelial cells, although intermittent cells in adjacent "normal" or hyperplastic epithelium also stained (Fig. 2). A similar

pattern of intermittent staining was seen in epithelial cells ofnormal breast tissue obtained as a result of reductive mammo-plasty (not shown).

Optimal staining was critically dependent upon proper handlingand fixation of the tissue sections. Sections were fixed immediately after thaw mounting on glass slides. Drying the sectionsfor as little as 5 min prior to or following fixation can result indrastic decreases in staining intensity. Although the optimalduration for fixation differed somewhat for the various fixatives

5Monoclonal antibodies designated H222Sp2-xand H226Sp2-r were first isolated and characterized by Dr. Larry Miller of Abbott Laboratories. All of the otheranti-ERs used Â¡nthis study were developed in our laboratory.

"These consisted of 15 tumors from premenopausal women with a mean

cytosolic ER content of 657 Â±613.1 (S.D.) fmol/g, and 23 tumors from postmenopausal women with a mean cytosolic ER content of 1197 Â±804.3 fmol/g.


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tested (Table I), we generally found that relatively brief (1 to 2min for alcohols, up to 10 min for aldehydes) treatments weremost effective. Fixations extending beyond this period resultedin progressive loss of nuclear staining with no correspondingincrease in staining in other subcellular compartments.

At subsaturating antibody concentrations, the nuclear stainingintensity was proportional to the concentration of the monoclonalanti-ER. However, even at saturating antibody concentrations(10 Â¿ig/mlfor H226Sp27), the intensity among various tumorsranged from a punctate distribution of oxidized DAB evenlydistributed throughout the nucleus to an intense, solid nuclearcoloration. Corresponding biochemical assays suggest that thisintertumor variation in staining intensity reflects cellular receptorcontent, although this relationship may be obscured by variationsin tumor cellularity and heterogeneity which may also contributeto biochemical ER values (see below and Refs. 18, 24, 38, 39,and 47). With appropriate fixation, 5 different monoclonal anti-

ERs, known to recognize 5 different receptor determinants (10),showed a predominantly nuclear localization, although at equivalent concentrations, the intensity of this staining varied amongthe antibodies with H222Sp27, H226Sp27, and D58P3M affording the most intense staining (Fig. 3). Conversely, no nuclearstaining occurred when normal rat IgG or any one of a numberof control monoclonal rat IgG's was substituted for the mono

clonal anti-ERs (Figs. 16 and 3/4). In addition, the nuclear stainingwhich is associated with anti-ER is effectively inhibited by prein-

cubating the antibodies with highly purified receptor, as shownpreviously (25).

Nuclear staining was also seen in selected cell populations ofboth human uterus (40) and fallopian tube (Fig. 4) as well as inthe receptor-containing MCF-7 human breast cancer line (25). In

uterus and fallopian tube, staining was seen in cells of both theglandular epithelium and surrounding stroma and in smoothmuscle cells of the myometrium. Connective tissue cells and

Table 1Relativenuclear staining intensity observed in frozen sections ot humanbreast

tumors following various fixation proceduresFrozensections obtained from unfixed receptor-richtumors were thaw-mounted

onto glass slides.They were then immediatelyimmersedin fixative for the indicatedperiod of time. After rinsing for 10 to 15 min in PBS, immunocytochemicalstainingwas performed as described in "Materials and Methods," using antibody H226Sp->

to localize receptor. An adjacent section of each tumor was stained using a controlmonoclonal IgG (see text).

FixativeEthanolGraded

ethanols (30-100%)"Gradedethanols +xylenes"10%

bufferedformalin1%glutaraldehydeAcetone

(-20Â°or roomtemperature)AcroteinBouin'sPicric

acid:paraformaldehyde(Zamboni'sfPeriodic

acid:lysine:paraformaldehydeDuration5

min60min50

min90min10min16

hr10min10min10min10

min10min10minNuclear

staining+++++Â±++++++â€”+++++++++

inflammatory cells within all of the above target tissues did notstain, and ER-negative breast tumors contained either no or only

focal staining (Fig. 5; also see below). Human colon epitheliumwas Â¡mmunocytochemically negative (not shown).

Correlation of Nuclear Staining with Biochemical Assay.One hundred seventeen tumors of mammary origin were assayed by ER-ICA during a 14-month period, and the results were

compared to those of sucrose density gradient analyses. Approximately 77% of these specimens were diagnosed as beingpredominantly infiltrating ductal carcinoma with or without accompanying intraductal carcinoma. Seven % were infiltratinglobular carcinoma, 12% metastatic breast adenocarcinoma, 1%intraductal carcinoma alone, and 3% benign disease. Tumorswere classified as ER-ICA positive if they contained any cells

showing nuclear staining, regardless of the staining intensity, thecellularity of the tumor, or the proportion of epithelial cells showing such staining.

A clear and significant (p < 0.001) association was notedbetween the presence or absence of nuclear staining and thelevel of cytosolic receptor measured in tumor extracts by steroid-binding assay (Table 2). As seen in Chart 1, all ER-rich tumorsfrom postmenopausal patients (n = 32) were also ER-ICA posi

tive and were further characterized by their high degree ofcellularity and high percentage of tumor cells (usually 60 to 100%)which showed immunocytochemical staining (Figs. ^A and 3, Bto F). Similarly, all ER-ICA-negative tumors (n = 23) had insignif

icant levels of cytosolic ER. On the other hand, 18 tumors foundto have cytosolic ER levels between 0 and 636 fmol/g, andtherefore presumed to be largely hormone unresponsive, contained ER-ICA-positive cells. However, as cytosol ER values

decreased within this group, the cellularity and the percentageof stained tumor cells also decreased (Figs. 6 and 7). Specimensthat contained less than 300 fmol/g of cytosolic ER showed onlyfocal nuclear staining (Fig. 8) and were easily distinguished fromreceptor-rich tumors.

Tumors of premenopausal patients showed a pattern of localization similar to those of postmenopausal patients: all ER-ICA-negative tumors were receptor poor; and all receptor-rich tumorswere ER-ICA positive (Chart 2). However, in contrast to the

situation described for postmenopausal specimens, 5 of the 7ER-poor, ER-ICA-positive tumors were indistinguishable fromreceptor-rich tumors containing 300 to 500 fmol cytosolic ER

per g, despite the presumed differences in prognostic outlookfor these 2 groups.

Morphometric Correlates of Steroid-binding Assay Results.

Our initial observations concerning the degree and distributionof immunocytochemical staining suggested that several of thestaining features reflected the results of the corresponding biochemical receptor determinations. Thus, the intensity of nuclearstaining, the proportion of the cross-sectional area occupied by

Table 2

Comparisonof immunocytochemicalresults with quantitativeER valuesfor 117humanbreast cancers

"Fixation consisted of successive 10-min immersions in 30, 50, 75, 90, and

100% ethano!.All dilutions were made with PBS (41).Fixation was in graded ethanols as above (41), followed by 10 min in xylene.

Sections were then rehydrated by reimmersionin each ethanol solution in reverseorder for 3 min each.

0As in Ret. 46."As in Ref. 31.

Receptor levels(fmol/g)'<300

>300ER-ICA

stainingNegative

Positive48

161 52

' Determinedby sucrose density gradient ultracentrifugationwith [3H]estradiol

(22). Three hundred fmol of receptor per g of tissue approaches the limit ofsensitivity of this assay.


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Chart 1. Comparison of immunocytochemical and sucrose density gradientassays for ER in breast tumors from postmenopausal women. Tumor sectionswere stained with monoclonal antibody H226Sp2 (20 pg/ml) as described inâ€¢Materialsand Methods," and the staining results were compared to those of

sucrose density gradient assays of cytosol prepared from another portion of thesame tumor. Gradient assay values are corrected for nonspecific binding. Tumorswere classified as ER-ICA positive if they contained any cells which showed nuclearstaining, regardless of the staining intensity or the number of cells stained.

epithelial cells (cellularity), and the proportion of the epitheliumthat was stained appeared in various specimens to indicate notonly the presence of receptor, but also to some degree itsabundance.

Among the 38 ER-ICA-positive tumors subsequently assessed

semiquantitatively for these features, nuclear staining intensityshowed a low, but marginally significant correlation (r = 0.382,p < 0.05) with tumor cytosol ER content. As seen in Chart 3,tumors scored as having light (Grade 1) nuclear staining had amedian cytosolic receptor content of 275 fmol/g of tissue, whilethe median values for tumors with Grades 2 and 3 staining were1056 and 1103 fmol/g, respectively. A wide range of biochemicalassay values was associated with each intensity grade due inpart to the overwhelming contributions of cellularity and heterogeneity to these values in each tumor (see below). ER-ICA-

positive tumors removed from premenopausal patients tendedto stain less intensely [1.5 Â±0.19 (S.E.), n = 15] than thoseobtained from postmenopausal patients [2.2 Â±0.16, n = 23].

The proportion of stained epithelial cells also correlated positively with tumor receptor content (r = 0.565, p < 0.01); the

distinguishing feature of this relationship was the consistentlyhigh proportion of ER-ICA-positive tumor cells in ER-rich tumors

(Chart 4). In contrast, tumors with low levels of cytosolic receptorexhibited a wide range of proportions of stained cells, againreflecting the influence of the other morphometric parameters,particularly tumor cellularity. Thus, classification of a tumor asER poor may apparently arise from either a lack of tumor cells(even if such cells are largely ER positive) or, in the case of more

ERICA Nuclear Staining

Chart 2. Comparison of immunocytochemical and sucrose density gradientassays for ER in breast tumors from premenopausal women. Same procedure asin Chart 1.

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STAINING INTENSITYCharts. ER concentration in cytosols of 38 ER-ICA-positive human breast

tumors plotted against the intensity of staining. Sucrose density gradient assays,immunocytochemical staining, and subjective scoring of the intensity of nuclearstaining were performed as described in "Materials and Methods." The menstrual

status of the patient at the time of surgery is indicated for premenopausal patients(O) and postmenopausal patients (â€¢).

highly cellular specimens, from a low percentage of ER-positivecells.

Specimens from postmenopausal patients contained a higherpercentage of immunocytochemically stained epithelial cells thandid tumors from premenopausal patients (0.75 Â±0.050 versus0.430 Â±0.075, respectively; p < 0.05). While some correlation(r = 0.405) was noted between the positive tumor fraction and

staining intensity, we often observed specimens which containeda high percentage of lightly stained cells or other tumors in which


296




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POSITIVE TUMOR FRACTION

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Chart 4. ER concentration in cytosols of 38 ER-ICA-positive human breasttumors plotted against the proportion of tumor cells graded ER-ICA positive.Sucrose density gradient assays, immunocytochemical staining, and scoring of thepositive tumor cell fraction were performed as described in "Materials and Methods.' The menstrual status of the patient at the time of surgery is indicated for

pnemenopausal women (O) and postmenopausal women (â€¢).

Table3Correlationsbetweensemiquantitativefeaturesof ER-ICAstaining and the ER

content of 38 ER-ICA-positivebreast tumors

Correlation coefficients (r) were obtained from simple and multiple linear regression analyses. Analyses were performed using MINITAB as outlined in "Materialsand Methods.' ER content was determined by sucrose density gradient analyses

(22).

ER content vs. t r

IntensityCellularityPositive cell proportion

Intensity + cellularity

Intensity + positive cell proportion

Cellularity + positive cell proportion

Intensity + cellularity + positive cell proportion

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Charts. ER concentration in cytosols of 38 ER-ICA-positive human breasttumors plotted against the proportion of the cross-sectional area of the tumoroccupied by tumor cells (cellularity). Sucrose density gradient assays, immunocytochemical staining, and scoring of the cellularity were performed as described in"Materials and Methods." The menstrual status of the patient at the time of surgery

is indicated for premenopausal patients (O) and postmenopausal women (â€¢).

the staining was intense but focal, suggesting that these featurescan vary independently.

As implied above, the strongest single correlate with tumorreceptor levels was found to be tumor cellularity (r = 0.675; p <0.01). ER-ICA-positive specimens containing little tumor epithe

lium tended to have lower levels of cytosolic receptor than didhighly cellular specimens (Chart 5) with no significant differencenoted in the degree of cellularity of tumors obtained from pre- or

postmenopausal women. The prediction of biochemical assayresults by the regression equation was consistently improvedupon by also considering either staining intensity or the proportion of positive cells with the highest degree of correlation (r =

0.764) noted when all 3 parameters were considered in theregression analyses (Table 3).

DISCUSSION

The need for a reliable histochemical method to localize ER inhuman breast tumors is evident from the proliferation of suchmethods during the past several years (28, 33, 37, 41, 42).However, most of these procedures have been criticized forlacking adequate sensitivity or specificity to distinguish receptorfrom other more abundant intracellular binding proteins (32, 48).We showed previously that monoclonal antibodies raised againsthuman breast cancer ER localize receptor within cell nuclei offixed, frozen sections of both human and animal tissues and thatthis staining is eliminated by preincubating the antibody witheither receptor-rich cytosol or with receptor-depleted (by affinity

absorption) cytosol to which highly purified ER has been added(25). Additional evidence of specificity is provided by (a) theabsence of staining when control monoclonal or polyclonal antibodies are substituted for anti-ER; (fa) the identical pattern ofstaining observed with 5 different monoclonal anti-ERs, each of

which recognizes different receptor determinants; (c) the tissuespecificity of such staining; and (d) the correlation betweennuclear staining and the results of biochemical assays for cytosolic receptor.

Whether this procedure presents a true picture of intracellularreceptor distribution is a difficult question to address by animmunocytochemical approach alone. We cannot, for example,dismiss the possibility that unoccupied receptor is associatedwith cytoplasmic elements in such a way that the determinantsare unavailable to antibody, although it seems unlikely that thedeterminants for all 5 of the antibodies tested would be similarlyobscured. Given the consistent predominance of nuclear stainingunder a variety of conditions of tissue fixation and processing, itseems improbable that the absence of cytoplasmic staining canbe attributed to experimental artifacts leading to cytoplasmicreceptor solubilization or translocation during the freeze/thaw orfixation processes. We have on occasion treated fresh tissuebiopsies and intact MCF-7 cells with precipitating and cross-linking fixatives at temperatures of 4-37Â° prior to freeze-section-


297




ing or vibrotomy and have consistently found that, as long asthe duration of fixation is relatively brief, the specific staining isnuclear. The effectiveness of such fixations was confirmed byelectron microscopic inspection of these specimens.7 The nu

clear predominance persists following more extended fixations,but the staining intensity decreases with length of fixation withno evidence of a corresponding increase in the cytoplasm. Furthermore, as discussed previously (25), cytosolic ER covalentlylabeled with the antiestrogen derivative, tamoxifen aziridine,precipitates quantitatively in buffers containing as little as 35%ethanol and cannot be resolubilized by PBS.

That the estrogen receptor may be a nuclear protein even inthe absence of hormone was suggested previously by othersand us (25, 29, 44, 50). According to this model, unoccupied,nonactivated receptor is loosely associated with the nucleus sothat, upon homogenization, it is easily extracted into low-salt

buffers. Estradiol binding leads to a tighter association of thereceptor with nuclear components, requiring the use of high-salt

buffers for receptor extraction. Thus, as originally proposed,interaction of estrogens with receptor protein induces a temperature- and steroid-dependent transformation to a more nucleo-

tropic hormone:receptor complex (9, 20, 23). However, translocation would be reinterpreted as an altered association withintranuclear elements rather than movement of the receptorprotein from cytoplasm to nucleus. It should be noted in thisregard that other hormone receptors, most notably those forvitamin D and thyroid hormone, are also thought to reside withinthe nucleus even under basal conditions (5, 27, 34, 45, 49)and that a reexamination of the autoradiographic localization of[3H]estradiol in rat uteri at low temperature suggested a similar

residence for ER (29). Finally, Welshons ef al. (50) have observedrecently that cytochalasin-induced enucleation of rat pituitary

(GH3) cells leads to partitioning of ER almost exclusively into thenucleoplast fraction, although the anuclear cytoplasts retain asignificant portion of the total cellular protein (50).

The heterogeneous pattern of nuclear staining observed heremay reflect the polyclonal origins of such tumors (39) and isconsistent with previous reports of the disparity in receptorvalues which can occur when sampling different portions of thesame specimen, even when the cellularity of these samples isapparently equivalent (38, 47). It is tempting to speculate thatthe 30 to 40% failure rate for endocrine therapy in patients withER-rich tumors (7, 8, 21, 22) results from the survival of asubpopulation of ER-poor cells the existence of which is sug

gested by the present immunocytochemical data. However, suchpatterns may also be related to experimental artifact (3) or mayrepresent transient alterations in receptor levels related to normalcell physiology. For example, in actively dividing MCF-7 cultures,

cells which are obviously in M phase show no specific stainingat the light microscopic level.8 To what extent this phenomenon

extends into late G2 or early GÃ¬phases, where the relativedispersal of the chromatin and the integrity of the nuclear membrane make phase discrimination more difficult, has yet to bedetermined.

It is unlikely that the heterogeneity observed in many humanbreast tumors is attributable to suboptimal antibody concentrations (3). When picric acid:paraformaldehyde (46) is substitutedfor ethanol as the fixative, the sensitivity of the assay increases

7 M. F. Press, personal communication.â€¢W. J. King, E. R. DeSombre, and G. L. Greene, unpublished observations.

by at least 10-fold (as judged by the minimum concentration of

monoclonal antibody which gives detectable specific nuclearstaining), yet the degree of staining heterogeneity in such tumorsis not significantly altered (not shown).

Finally, the correlation between the degree of heterogeneityand the levels of receptor assayed biochemically also suggeststhat the staining pattern indeed reflects the distribution of receptor-containing cells. Tumors having a low percentage of ER-ICA-

positive cells invariably had low levels of cytosolic receptor, evenin moderately to highly cellular tumors in which this staining wasintense (Grade 3). On the other hand, if the percentage of positivetumor cells was high, the contributions of the number of suchcells and the intensity of staining (sites per cell?) became moreapparent in determining overall tumor receptor content. Whetherthis pattern truly reflects heterogeneity in the clonal origins ofsuch tumors or alternatively in the expression of the receptorphenotype may only be resolved when tumor cells from suchpatients can be efficiently cloned in a manner in which thisexpression is reliably preserved.

It is perhaps less surprising that a relationship exists betweentumor cellularity and receptor concentrations, particularly whenthe biochemical data are expressed in terms of tumor weight asthey are here. This correlation is also evident when receptorvalues are expressed relative to cytosol protein content (notshown). The connection between cellularity and tumor ER levelshas also been noted by other investigators (24, 38) and impliesthat certain tumors are found to be receptor poor largely as aresult of extensive fibrosis, despite the fact that the epitheliumin such specimens is ER positive (e.g., Fig. 6). Although classification of these tumors with respect to endocrine responsivenesswill have to await the outcome of prospective clinical studies,their frequency appears to exceed the 5% expected rate ofresponsiveness in ER-poor specimens (21,22) and may therefore

comprise a tumor subgroup of particular interest for understanding the basis of treatment failures.

The several differences observed between tissue obtainedfrom pre- and postmenopausal patients also bear further inves

tigation. Among the premenopausal tumors, several highly cellular, ER-ICA-positive specimens with very poor (less than 300

fmol/g) cytosolic receptor levels were observed, while all postmenopausal tumors having a similar receptor content displayedvery poor cellularity. The former may have arisen if a significantportion of the receptor was occupied by endogenous estradiol,converting the receptor to the activated, high-salt extractable

form (9, 20). Total tumor receptor may thus be underestimatedby conventional cytosol assays which use extraction buffers oflow ionic strength (18, 24, 38, 39, 47). On the other hand, theimmunocytochemical method, which uses antibodies which recognize both cytosolic and more highly nucleotropic forms of thereceptor (10, 14, 16), might be expected to reveal receptorregardless of the strength of its association.

The less intense staining generally observed in tumors frompremenopausal women (regardless of their cellularity) could bepartly explained if the total (low- plus high-salt extractable)

receptor content were less than that of postmenopausal materials. This would assume that many premenopausal specimensdisplay low cytosolic steroid-binding activity not only because of

an increased portioning of receptor to the nucleus, but becauseof other hormonal influences, e.g., progestational, which maydecrease total cellular ER levels (17). Clearly, more basic work


298




is needed, perhaps using a number of model systems, to clarifythe influence which these factors may have on the regulation oftumor receptor content and dynamics and the consequences ofsuch changes for immunocytochemical detection of the receptorprotein.

Finally, while correlations may provide some insight into general features of receptor distribution which contribute to the ERcontent of tumor extracts, they do not imply usefulness forpredicting the outcome of biochemical assays in individual tumors except in a most general way. The sampling error inherentin analyzing one or even a few sections from a clinical specimen(see "Materials and Methods") as well as the subjective nature

of semiquantitative evaluations of their staining may both contribute significantly to disparities between receptor values obtained by steroid-binding assays and those predicted fromregression equations obtained as a result of immunocytochemical procedures. On the other hand, while the clinical usefulnessof steroid-binding assays for ER is well established, investigatorshave long been concerned by the loss of information concerningregional variations in receptor content which occur during ho-

mogenization. Since current receptor assays fail to preciselyidentify which patients will actually respond to endocrine therapyin a significant number of cases, more information which couldbe relevant to the hormone sensitivity of the cancers could behelpful. However, until more is known about the relationshipbetween various patterns of immunocytochemical staining andclinical outcome, it would be prudent to consider the immunocytochemical procedure not as a replacement for, but rather anadjunct to, biochemical assays of receptor. The present data,which contain evidence of a relationship between ER-ICA resultsand those of steroid-binding assays, support a hope that theimmunocytochemical method will ultimately prove to be at leastas valuable as biochemical procedures for predicting patientresponsiveness and prognosis. Observations concerning theheterogeneity of staining may enable us to further improve theaccuracy of such predictions by identifying patients whose ER-

positive tumors may contain significant subpopulations of hormone-unresponsive cells.

ACKNOWLEDGMENTS

We thank Richard Blough of the BiomÃ©dicalComputation Facilities, Universityof Chicago, for his advice and assistance in the statistical analysis of the data.

REFERENCES

1. Allegra, J. C., Lippman, M. E., Simon, R., Thompson, E. B., Barlock, A., Green,L, Huff, K. K., Do, H. M. T., Aitken, S. C., and Warren, R. Association betweensteroid hormone receptor status and disease-free interval in breast cancer.Cancer Treat Rep., 63:1271-1277,1979.

2. Chamness, G. C., Mercer, W. D., and McGuire, W. L. Are histochemicalmethods for estrogen receptor valid? J. Histochem. Cytochem., 28: 792-798,1980.

3. Ciocca, D. R., Adams, D. J., Bjercke, R. J., Sledge, G. W., Jr., Edwards, D.P., Chamness, G. C., and McGuire, W. L. Monoclonal antibody storageconditions, and concentration effects on immunohistochemical specificity. J.Histochem. Cytochem., 31: 691-696,1983.

4. Clark, G. M., McGuire, W. L., Hubay, C. A., Pearson, O. H., and Marshall, J.S. Progesterone receptors as a prognostic factor in Stage II breast cancer. N.Engl. J. Med., 309:1343-1347,1983.

5. OeGroot, L. J., and Strausser, J. L. Binding of T3 in rat liver nuclei. Endocrinology, 95: 74-83,1974.

6. DeSombre, E. R., and Gorell, T. A. Purification of estrogen receptors. MethodsEnzymol., 36. 349-365,1975.

7. DeSombre, E. R., Greene, G. L., and Jensen, E. V. Estrogen receptors andthe hormone dependence of breast cancer. In: M. J. Brennan, C. M. McGrath,

and M. A. Rich (eds.), Breast Cancer: New Concepts in Etiology and Control,pp. 69-87. New York: Academic Press, Inc., 1980.

8. DeSombre, E. R., and Jensen, E. V. Estrophilin assays in breast cancer:quantitative features and application to the mastectomy specimen. Cancer(Phila.), 46: 2783-2790,1980.

9. Gorski, J., and Gannon, F. Current models of steroid hormone action: a critique.Annu. Rev. Physiol., 38: 425-450,1976.

10. Greene, G. L. Application of immunochemical techniques to the analysis ofestrogen receptor structure and function. In: G. Litwack (ed.), BiochemicalActions of Hormones, Vol. 11, pp. 207-239. New York: Academic Press, Inc.,

1984.11. Greene, G. L., Closs, L. E., DeSombre, E. R., and Jensen, E. V. Antibodies to

estrophilin: comparison between rabbit and goat antisera. J. Steroid Biochem.,11: 333-341,1979.

12. Greene, G. L., Closs, L. E., DeSombre, E. R., and Jensen, E. V. Estrophilin:pro anti. J. Steroid Biochem., 72:159-167,1980.

13. Greene, G. L., Closs, L. E., Fleming, H., DeSombre, E. R., and Jensen, E. V.Antibodies to estrogen receptor: immunochemical similarity of estrophilin fromvarious mammalian species. Proc. Nati. Acad. Sci. USA, 74:3681-3685,1977.

14. Greene, G. L., Fitch, F. W., and Jensen, E. V. Monoclonal antibodies toestrophilin: probes for the study of estrogen receptors. Proc. Nati. Acad. Sci.USA, 77:157-161,1980.

15. Greene, G. L., and Jensen, E. V. Monoclonal antibodies as probes for estrogenreceptor detection and characterization. J. Steroid Biochem., 76: 353-359,

1982.16. Greene, G. L., Nolan, C., Engler, J. P., and Jensen, E. V. Monoclonal antibodies

to human estrogen receptor. Proc. Nati. Acad. Sci. USA, 77: 5115-5119,1980.

17. Hsueh, A. J. W., Peck, E. J., Jr., and Clark, J. H. Control of uterine estrogenreceptor levels by progesterone. Endocrinology, 98: 438-444,1976.

18. Hull, D. F., Clark, G. M., Osbome, C. K., Chamness, G. C., Knight, W. A., andMcGuire, W. L. Multiple estrogen receptor assays in human breast cancer.Cancer Res., 43: 413-416, 1983.

19. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., and DeSombre, E. R. Estrogenreceptors and breast cancer response to adrenalectomy Nati. Cancer Inst.Monogr., 34: 55-70,1971.

20. Jensen, E. V., and DeSombre, E. R. Estrogen-receptor interaction. Science(Wash. DC), 782:126-134,1973.

21. Jensen, E. V., Greene, G. L., Closs, L. E., DeSombre, E. R., and Nadji, M.Receptors reconsidered: a 20-year perspective. Recent Prog. Horm. Res., 38:1-40,1982.

22. Jensen, E. V., Smith, S., and DeSombre, E. R. Hormone dependency in breastcancer. J. Steroid Biochem., 7: 911-917,1976.

23. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W., andDeSombre, E. R. A two-step mechanism for the interaction of estradici withrat uterus. Proc. Nati. Acad. Sci. USA, 59: 632-638,1968.

24. Kiang, D. T., and Kennedy, B. J. Factors affecting estrogen receptors in breastcancer. Cancer (Phila.), 40:1571-1576,1977.

25. King, W. J., and Greene, G. L. Monoclonal antibodies localize oestrogenreceptor in the nuclei of target cells. Nature (Lond.), 307: 745-747,1984.

26. King, W. J., Jensen, E. V., Miller, L., and Greene, G. L. Immunocytochemicaldetection of estrogen receptor in frozen sections of human breast tumors withmonoclonal anti-receptor antibodies. Endocrinology, 770(Suppl.): 258,1982.

27. Lawson, D. E. M., and Wilson, P. W. Intranuclear localization and receptorproteins for 1,25-dihydroxycholecalciferol in chick intestine. Biochem. J., 144:573-583,1974.

28. Lee, S. H. Cytochemical study of estrogen receptor in human mammary cancer.Am. J. Clin. Pathol., 70:197-203, 1978.

29. Martin, P. M., and Sheridan, P. J. Toward a new model for the mechanism ofaction of steroids. J. Steroid Biochem., 76: 215-229,1982.

30. McGuire, W. L. Steroid receptors and breast cancer. In: H. Aldercrentz, R. D.Bulbrook, H. J. Van der Molen, A. Vermeulen, and F. Sciana (eds.), Researchon Steroids, Vol. 9, pp. 11-17. Amsterdam: Exerpta Medica, 1981.

31. McLean, I. W., and Nakane, P. K. Periodate-lysine-paraformaldehyde fixative.

A new fixative for immunoelectron microscopy. J. Histochem. Cytochem., 22:1077-1083,1974.

32. Mercer, W. D., Edwards, D. P., Chamness, G. C., and McGuire, W. L. Failureof estradici Â¡mmunofluorescence in MCF-7 breast cancer cells to detectestrogen receptors. Cancer Res., 47: 4644-4652,1981.

33. Nenci, I., Beccati, M. D., Piffanelli, A., and Lanza, G. Detection and dynamiclocalization of estradiol-receptor complexes in intact target cells by immunoflu-orescence techniques. J. Steroid Biochem., 7: 505-510,1976.

34. Oppenheimer, J. H., Schwartz, H. L., Surko, M. I., Koemer, D., and Dillman,W. H. Nuclear receptors and the initiation of thyroid hormone action. RecentProg. Horm. Res., 32:529-565,1976.

35. Osbome, C. K., Yochmowitz, M. G., Knight, W. A., and McGuire, W. L. Thevalue of estrogen and progesterone receptors in the treatment of breastcancer. Cancer (Phila.), 46: 2884-2888,1980.

36. Pari, F. F., Wetherall, N. T., Halter, S., Schuffman, S., and Mitchell, W. M.Comparison of histochemical and biochemical assays for estrogen receptor inhuman breast cancer cell lines. Cancer Res., 44: 415-421,1984.

37. Pertschuk, L. P., Gaetjens, E., Carter, A. C., Brigati, D. J., Kim, D. S., and


299




Fealey, T. E. An improved histochemfcal method for detection of estrogenreceptors in mammary cancer. Am. J. Clin. Pathol., 77; 504-508, 1979.

38. Poulsen, H. S. Oestrogen receptor assay-limitation of the method. Eur. J.Cancer. 77:495-501,1981.

39. Poulsen, H. S., Jensen, J., and Hermansen,C. Human breast cancer: heterogeneity of estrogen binding sites. Cancer (Phila.).48:1791 -1793,1981.

40. Press, M. F., and Greene, G. L. An immunocytochemicalmethod for demonstrating estrogen receptor in human uterus using monoclonal antibodies tohuman estrophilin. Lab. Invest., 50: 480-486,1984.

41. Raam,S., Nemeth, E., Tamura, H., O'Briain, 0. S., and Cohen,J. L. Immunc-

histochemicallocalizationof estrogen receptors in humanmammarycarcinomausing antibodiesto the receptor protein. Eur. J. Cancer Clin. Oncol., 78:1-12,1982.

42. Rao, B. R., Fry. C. G., Hunt, S.. Kuhnel, R.. and Dandliker.W. B. A fluorescentprobe for rapid detection of estrogen receptors. Cancer (Phila.),46: 2902-2906,1980.

43. Rose, C., Thorpe, S. M., Mouridsen, H. T., Andersen, J. A., Brincker, H., andAndersen, K. W. Antiestrogen retreatment of postmenopausal women withprimary high risk breast cancer. Breast Cancer Res. Treat., 3: 77-84,1983.

44. Siiteri, P. K., Schwarz, B. E., Moriyama, I., Ashby, R., Unkie, D., and Mac-Donald, P. C. Estrogen binding in the rat and human. Receptorsfor reproductive hormones.Adv. Exp. Med. Bid., 36: 97-112,1973.

45. Spindter, B. J., MacLeod, K. M., Ring, J., and Baxter, J. D. Thyroid hormonereceptors. J. Bid. Chem.,250: 4113-4119,1975.

46. Stefanini, M., DeMartino, C., and Zamboni, L. Fixation of ejaculated spermatozoa for electron microscopy. Nature (Lond.),276:173-174,1967.

47. Straus, M. J., Moran, R., Multer, R. E., and Wotiz, H. H. Estrogen receptorheterogeneityand the relationshipbetween estrogen receptor and the tritiatedthyrnidmelabeling index in human breast cancer. Oncology (Basel),39: 197-200,1982.

48. Underwood,J., Sher, E., Reed,M., Eisman,J. A., and Martin,T. J. Biochemicalassessment of histochemical methods for oestrogen receptor localization.J.Clin. Pathol., 35: 401-406,1982.

49. Walters, M. R., Hunziker, W., and Norman,A. W. Unoccupied1,25-dihydroxy-vitamin D3 receptors. J. Bid. Chem.,255: 6799-6805,1980.

50. Welshons. W. V., Ueberman, M. E., and Gorski, J. Nuclear localization ofunoccupiedoestrogen receptors:cytochalasinenucleatoti of GH3cells.Nature(Lond.),307: 747-749,1984.

Fig. 1. Immunocytochemicalstaining of an ER-richbreast tumor section. Adjacent 8->imfrozen sections of an ER-rich breast tumor were fixed in ethanol and stainedusing the immunoperoxidase technique as described in 'Materials and Methods" with either H226Sp2-y(10/ig/mi; A) or normal rat igG (10 fig/ml, B). This tumor, takenfrom a 67-year-old woman, was found to contain 1546 tmol of cytosolic ER per g of tissue by sucrose density gradient assay. Original magnification, x 400. Nocountersign.

Fig. 2. Immunocytochemicalstaining of hyperplastic breast epithelium. Frozen section (8 *im thick), fixed in ethanol and stained with H226Sp2> (10 Â»ig/ml).Note theintermittent pattern of staining. Originalmagnification, x 400. No counterstain.

Fig. 3. Similarpatterns of localizationproduced by various monoclonalantibodies to humanER. Immunoperoxidasestainingwas performed as described in "Materialsand Methods," using 10 (Â¿gof normal rat IgG (A), H226Sp2-x(8), H222Sp2-y(C),D75P3y (D),D547Spr (Â£),or D58P3^ (F) per ml. Normal rat IgM or control monoclonal

rat IgGs produce a (nonspecific)staining pattern similar to that seen with normal rat IgG. This tumor, taken from a postmenopausalwoman, was found to contain 1976fmol of cytosolic ER per g of tissue by sucrose density gradient assay. Originalmagnification, x 400. No counterstain. Picric acid:paraformaldehydefixation.

Fig.4. Immunocytochemicalstaining of human fallopian tube. Adjacent 8-jjtn-thick frozen sections of human fallopiantube removedfrom a premenopausalwoman inthe midproliferative phase of her cycle. Hysterectomy/bilateral salpingoovariectomywas performed as a consequenceof uterine fibroids. Immunocytochemicalstainingwas performed as described in "Materials and Methods," using either H226Sp2? (10 /ig/ml) (A)or normal rat IgG (B). Original magnification, x 400. No counterstain.

Picric acid:paraformaldehydefixation.Fig. 5. Lack of immunocytochemicalstaining in a receptor-poor human breast tumor. Frozen section (8 Â«mthick), fixed in ethanol and stainedwith H226Sp2->(20 tig/

ml). Tumor was removed from a 79-year-old woman and showed 113 fmol of cytosolic ER per g of tissue by sucrose density gradient assay. Original magnification,x400. No counterstain.

Fig.6. Immunocytochemicallypositive cells in a receptor-poor human breast tumor. Frozen section (8 Â«imthick), fixed in ethanol and stained with H226Sp2y (20 ng/ml). Tumor was removed from an 81-year-old woman and was found to .have 79 fmol of cytosolic ER per g of tissue by sucrose density gradient assay. Originalmagnification, x 40. No counterstain.

Fig. 7. Heterogeneous immunocytochemicalstaining in a receptor-rich human breast tumor. Frozen section (8 /umthick), fixed in ethanol and stained with H226Sp2->(20 Mg/ml).Tumor was removed from a postmenopausalwoman and contained 1724 fmol of cytosolic ER per g of tissue by sucrose density gradient assay. Originalmagnification, x 150. Section was lightly counterstained with hematoxylin to permit visualizationof immunocytochemicallynegativecells.

Fig. 8. Heterogeneousimmunocytochemicalstaining in a receptor-poor humanbreast tumor. Frozen section (8 *im thick), fixed in ethanol and stainedwith H226Sp2y(20 /ig/ml). Tumor was removed from a postmenopausal woman and showed 301 fmol of cytosolic ER per g of tissue by sucrose density gradient assay. Originalmagnification, x 400. Section was lightly counterstained with hematoxylin to permit visualizationof immunocytochemicallynegative cells.


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1985;45:293-304. Cancer Res William J. King, Eugene R. DeSombre, Elwood V. Jensen, et al. Assays for Estrogen Receptor in Human Breast TumorsComparison of Immunocytochemical and Steroid-binding

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