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(American Journal of Pathology. 2000;156:29-35.)
© 2000 American Society for Investigative Pathology

Short Communications

Estrogen Receptor ß Is Coexpressed with ER and PR and Associated with Nodal Status, Grade, and Proliferation Rate in Breast Cancer

Tero A. H. Järvinen^*, Markku Pelto-Huikko, Kaija Holli and Jorma Isola^*

From the Laboratory of Cancer Biology,^*
Institute of Medical Technology, Tampere University and University Hospital, Tampere; the Medical School,
University of Tampere, Tampere; and the Department of Oncology,
Tampere University Hospital, Tampere, Finland

	Abstract

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The role of estrogen (ER) and progesterone receptors (PR) in breast cancer is well established. Identification of the second human estrogen receptor, the estrogen receptor ß (ERß), prompted us to evaluate its role in breast cancer. We studied the expression of ERß by immunohistochemistry and mRNA in situ hybridization in 92 primary breast cancers and studied its association with ER, PR, and various other clinicopathological factors. Sixty percent of tumors were defined as ERß-positive (nuclear staining in >20% of the cancer cells). Normal ductal epithelium and 5 of 7 intraductal cancers were also found to express ERß. Three-fourths of the ER- and PR-positive tumors were positive for ERß, whereas ER and PR were positive in 87% and 67% of ERß-positive tumors, respectively. ERß was associated with negative axillary node status (P < 0.0001), low grade (P = 0.0003), low S-phase fraction (P = 0.0003), and premenopausal status (P = 0.04). In conclusion, the coexpression of ERß with ER and PR as well as its association with the other indicators of low biological aggressiveness of breast cancer suggest that ERß-positive tumors are likely to respond to hormonal therapy. The independent predictive value of ERß remains to be established.

	Introduction

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It is well known that up to 30 to 40% of breast tumors with positive hormone receptor status do not respond to endocrine therapy.¹ Reasons for the lack of response have remained poorly understood, although steroid-independent growth factor signaling (eg, via HER-2/neu),⁴ functionally deficient splicing variants of the ER gene,² and heterogeneity of ER expression⁵ may partly explain poor therapy outcome of ER-positive tumors. However, these mechanisms explain only a fraction of the hormone receptor-positive tumors that do not respond to endocrine therapy. Therefore, the search for alternative explanations continues.

The recent discovery of a second estrogen receptor, termed ERß,^6,7 indicates that the mechanism of action of estrogens is far more complex than anticipated. Due to its recent discovery, relatively little is known about the ERß at the moment.⁷ Human ERß has a structure highly homologous to the previously known human ER, now termed ER.^8,9 Estrogens are known to bind ERß with affinity similar to ER⁷ and the transcriptional activation via the estrogen response element (ERE) is identical for both receptor forms.^6,8,10 ER and ERß can also form biologically functional receptor heterodimers in the tissues in which they are coexpressed.^11-13 So far, only limited data are available on the activity and expression of ERß in human neoplasms. Pilot studies have indicated that ERß is expressed in breast cancer as its mRNA has been detected in breast carcinoma samples by reverse transcriptase-polymerase chain reaction (RT-PCR).^14-17 However, due to the small numbers of tumors studied, the role of ERß has remained obscure.^14-17 Here we studied the expression of ERß by immunohistochemistry and mRNA in situ hybridization in a set of unselected breast tumors. Expression of ERß was correlated with ER, PR, and known clinicopathological indicators of malignant potential to clarify the role of ERß in the pathobiology of breast cancer.

	Materials and Methods

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Patients and Tumors

We studied surgical biopsy specimens from a set of 92 female breast cancer patients whose tumor samples were sent for hormone receptor analysis to the Laboratory of Cancer Biology at Tampere University Hospital. The tumor material consisted of 79 invasive ductal carcinomas, 6 lobular, and 7 intraductal carcinomas, according to the WHO tumor classification. The median age of the patients was 58 years (range, 35–88). Patients were operated with segmental resection or mastectomy and had not received any preoperative chemo- or endocrine therapy. Tumor samples were snap-frozen in OCT tissue embedding medium (Tissue-Tek, Miles Inc., Naperville, IL) within 20 minutes of removal during surgery. Cryostat sections (5–7 µm) were cut for intraoperative diagnosis, hormone receptor analysis, and DNA flow cytometry. Extra sections were stored air-tight at -70°C until used in immunohistochemistry and mRNA in situ hybridization of ERß. All histopathological diagnoses were re-evaluated and histopathological grading was performed according to the Bloom and Richardson system.¹⁸

Immunohistochemistry

The frozen sections were fixed with Zamboni’s fluid for 15 minutes. Nonspecific antibody binding was blocked with Tris-buffered saline containing 1.0% bovine serum albumin and 1.0% nonfat milk powder for 10 minutes at room temperature. ERß was detected with a rabbit polyclonal antibody (PAI-313, Affinity Bioreagents, Golden, CO; dilution 5 µg/ml). The antigen used for immunization is a KLH-conjugated synthetic peptide corresponding to the C-terminal amino acid residues 467 to 485 of human ERß. According to the manufacturer, the antibody reacts with human ERß and displays no cross-reactivity with human ER expressed in a baculovirus system. The primary antibody was incubated overnight at 4°C using Shandon Sequenza immunostaining coverplates (Shandon, Pittsburgh, PA). A streptavidin-biotin-peroxidase complex technique was used for visualization with diaminobenzidine as a chromogen (Histostain Plus kit, Zymed Inc., South San Francisco, CA). Sections were counterstained with hematoxylin. Immunostainings were evaluated by light microscopy using a 25x objective by a researcher unaware of immunohistochemical or clinical data. The immunohistochemical controls included omission of primary and secondary antibodies, and a pre-absorption experiment, where the antibody was incubated with the concentration of 10 times excess of the peptide immunogen (PAI-313p, Affinity Bioreagents) for 1 hour at room temperature before applying to slides. Adjacent sections from the same tumors were immunostained normally for comparison.

ER and PR were immunostained on adjacent Zamboni-fixed frozen sections using the ER-ICA and PR-ICA kits (Abbott Laboratories, Naperville, IL). Overexpression of c-erbB2 oncoprotein was detected by the monoclonal antibody CB-11 (Novocastra Laboratories, Newcastle, UK). Details of the ER, PR, and c-erbB2 staining method have been shown in our previous studies.¹⁹ DNA flow cytometry was performed using adjacent frozen sections 200 µm thick as starting materials, as previously described.²⁰

mRNA in Situ Hybridization

mRNA in situ hybridization was carried out as previously described.^6,19 Four different synthetic antisense oligonucleotide probes directed against ERß mRNA (nucleotides 542–589, 1089–1136, 1326–1373, and 1384–1431) were labeled to specific activity of 1 x 10⁹ cpm/mg at the 3' end with ³³P-dATP (DuPont-New England Nuclear Research Products, Boston, MA) using terminal deoxynucleotidyl transferase (Amersham, Buckinghamshire, UK). A cocktail of similarly labeled irrelevant oligonucleotides was used as control. The hybridization was carried out by incubating unfixed and air-dried frozen sections in humidified boxes at 42°C for 18 hours with 5 ng/ml of the labeled probe in the hybridization mixture. The sections were then washed four times (15 minutes each) in 1x SSC at 55°C. In the final rinse, the sections were left to cool to room temperature (approximately 1 hour). The sections were dipped in Kodak NTB2 nuclear track emulsion and exposed for 90 days at 4°C. The sections were stained with cresyl violet and analyzed under bright-field and epipolarization conditions in a Nikon Microphot-FX microscope. Alternatively, autoradiograph films (Amersham ß-max; Amersham) were overlaid on slides, exposed for 30 to 60 days, and developed using LX24 developer and AL4 fixative (Kodak, Rochester, NY). Irrelevant control probes of the same length, with similar GC content and specific activity, were used to ascertain the specificity of the hybridizations. Addition of 100 times excess of the unlabeled probe abolished all hybridization signals (data not shown).

	Results

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Expression of ERß in Ductal Epithelium and in Breast Cancer

Immunohistochemical staining using the polyclonal ERß antibody showed strong nuclear immunoreaction and weak cytoplasmic and extracellular background staining (Figure 1) . Positive immunostaining was confined to the nuclei of carcinoma cells, whereas the stromal and inflammatory cells in the tumor stained always stained negative. When 20% of positively stained carcinoma cells was used as a cutoff point to classify tumors as ERß-positive, 55 of 92 (59.8%) tumors were defined as ERß-positive. The specificity of ERß immunohistochemistry was confirmed by mRNA in situ hybridization (Figure 1) . Positive autoradiographic signals indicating presence of ERß mRNA were obtained from immunohistochemically ERß-positive tumors (Figure 2) . ERß mRNA and immunoreactivity were found also in the normal ductal epithelium and immunoreactivity in intraductal carcinoma (Figures 1 and 2) . Immunostaining of ERß was confirmed by pre-absorbing ERß antibody with immunogen peptide (Figure 1) . Incubation of the ERß antibody with the peptide abolished the nuclear immunoreaction completely from adjacent sections.

View larger version (110K): [in this window] [in a new window]   Figure 1. Immunohistochemical demonstration of ERß in human breast cancer by immunohistochemistry (A) and mRNA in situ hybridization (B). ERß is expressed also in intraductal carcinoma (C), and in normal ductal epithelium (D). The expression of ERß in normal ducts was confirmed by mRNA in situ hybridization (E). F and H demonstrate the specificity control of the immunostaining. Adjacent tumor sections were immunostained with or without pre-absorption of the ERß antibody by the immunogen peptide. The nuclear immunoreaction is completely abolished after pre-absorption.

View larger version (90K): [in this window] [in a new window]   Figure 2. Localization of ERß mRNA by in situ hybiridization using a sensitive X-ray film autoradiography detection. A demonstrates hybridization of an immunohistochemically ERß-positive tumor with a cocktail of five ERß antisense oligonucleotides. The tumor area and a normal duct (upper left corner) are labeled, whereas no specific labeling can be seen with a cocktail of irrelevant oligonucleotides (B). Bar, 0.4 mm.

Association of ERß with ER and PR

Three-fourths of the ER-positive tumors (76%, 48/63) were positive for ERß, whereas 7 of 29 (24%) ER-negative tumors expressed ERß (Table 1) . A similar strong association was identified between ERß and PR status (Table 1) . Seventy-six percent of the PR-positive tumors were ERß-positive (37/49), whereas 42% of the PR-negative breast tumors were ERß-positive (18/43). When ER and PR status were combined, 77% of ER-positive/PR-positive tumors were found to be ERß-positive, while almost as high precentage of the ERß-positivity was identified in ER+/PR- tumors (75%, Table 1 ). Although a majority of ER-positive/PR-negative tumors (12/16) were positive for ERß, only 22% of the tumors that were negative for both ER and PR were positive for ERß (Table 1) . Patterns of ER and ERß coexpression are illustrated in Figure 3 .

View this table: [in this window] [in a new window]   Table 1. Association of ERß with ER, PR, and Receptor Status in 92 Breast Cancers

View larger version (137K): [in this window] [in a new window]   Figure 3. Patterns of ER and ERß coexpression in breast cancer. A and B demonstrate a tumor expressing both ER and ERß. A tumor expressing ER but not ERß is shown in C and D, and a tumor expressing ERß but not ER in E and F, respectively. All stainings were done from adjacent frozen sections. Counterstained with hematoxylin.

Expression of ERß was significantly associated with several clinicopathological features of breast cancer. Positive ERß status was more common in axillary node-negative than in node-positive tumors (P < 0.0001, Table 2 ), but no correlation was found with the size of the primary tumor (Table 2) . Expression of ERß was more common in pre- and perimenopausal than postmenopausal patients (P = 0.04). There was no association between the histological type of the tumor and the ERß expression, in that 46/79 invasive ductal carcinomas, 4/6 invasive lobular, and 5/7 intraductal carcinomas showed positive ERß immunostaining. ERß had strong association with histological grade (P = 0.0003), and ERß-positive tumors were also characterized by diploid DNA content and lower S-phase fractions than ERß-negative tumors (P = 0.03 and P = 0.002, respectively; Table 2 ). A nearly significant association was found between negative ERß status and overexpression of ErbB-2 oncoprotein (P < 0.08, Table 2 ). For comparison, association of ER with clinicopathological features was also determined. Similarly to ERß, there was a correlation between the ER and histological grade, DNA ploidy, S-phase fraction, ErbB-2 oncoprotein overexpression, and tumor size (Table 2) . No significant association was found between ER and menopausal and nodal status of the tumor.

	Discussion

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Our study revealed that both ER receptors, and ß, are expressed in morphologically normal ductal epithelium, indicating that ERß is likely to have a function in the normal mammary gland. More importantly, coexpression of ER and ERß was retained in a majority of breast cancers, suggesting that ERß may be an equal target with ER for hormone therapy. In this context, it is known that ERß is equivalent to ER in its binding affinity for natural estrogens as well for anti-estrogens.⁷ ER and -ß can both activate gene transcription by binding either to the classical estrogen response elements (EREs) or the AP1 enhancer elements.^7,10,22 Anti-estrogens prevent gene transactivation via ER through both EREs and AP1 elements.¹⁰ Unlike ER, the anti-estrogen-ERß -complex inhibits gene transcription when bound to ERE, but works as an agonist when bound to AP1 elements.¹⁰ It is, therefore, possible that anti-estrogens could have also agonistic effects in ERß-positive breast tumors, which could decrease the effect of the hormone therapy. An alternative possibility is that ER and -ß are expressed in the form of the heterodimers.^11-13,22 As ER and ERß were coexpressed in most breast tumors, the heterodimers may also have a significant role in breast cancer. However, the presence and significance of ER and ERß heterodimers in breast cancer remains to be established.

Comparison of the ER and ERß expression with PR status may also shed light on the roles of ER and ERß in breast cancer. Transcription of the PR gene is enhanced and maintained by estrogens; thus, a positive PR status has long been regarded as a marker of a functional ER pathway. PR was positive in a majority of ER-positive/ERß-negative tumors (11/15, 73%), similarly to the situation in the ER-positive/ERß-positive tumors (36/48, 75%). The semiquantitative PR histoscores were not different in these groups (data not shown). Thus, ERß does not seem to be an important factor defining the expression of PR in the breast cancer. This may indicate indirectly that ERß has a smaller role in defining the responsiveness to hormonal therapy in breast tumors. From the therapeutic point of view, the most interesting receptor combination explaining the lack of response to hormone therapy in hormone-positive breast tumors is ER-positive/ERß-negative/PR-positive. In other words, it will be important to know whether lack of ERß in ER-positive/PR-positive tumors may lower the likelihood for response to anti-estrogen therapy. In our material ERß was negative in 23% of the ER-positive/PR-positive tumors, which is close to the proportion of ER-positive/PR-positive tumors that are known to respond poorly to tamoxifen. The predictive value of ERß remains to established in forthcoming studies.

The correlation of ERß with various clinicopathological factors revealed that ERß is expressed predominantly in the well-differentiated, diploid, and slowly proliferating breast cancers. The correlations were similar to those obtained for ER. These results indicate that the expression of both ER and -ß are lost in an identical manner during dedifferentiation of the tumor cells. However, two interesting differences were found in the clinicopathological associations of ER and ERß. First, ERß was tightly associated with axillary lymph node status, whereas a less strong association was identified for ER. This suggests that the loss of ERß expression might be an indicator of a tumor phenotype with high metastatic potential. From the endocrinological point of view, it is worth noting that expression of ERß was significantly more common in pre- and perimenopausal than in postmenopausal patients. This association usually goes in the opposite direction for ER; in other words, ER status is more often positive in postmenopausal patients. It is therefore possible that circulating estrogens favor ERß as their primary target at the expense of ER in premenopausal patients. After menopause the situation may then change. Obviously the endocrinological aspects of ER/ERß expression in breast cancer need to be studied with larger arrays of patient material, taking into account the use of oral contraceptives, hormone replacement therapy, and the possible anti-estrogen therapy (for previous breast cancer).

In conclusion, we have shown that normal ductal epithelium and a majority of breast cancers express the second human receptor for estrogens, the ERß. The ERß-positive breast cancers tumors are predominantly ER- and PR-positive, node-negative, well differentiated and slowly proliferating. The coexpression of ERß with ER and PR as well as its association with indicators of low biological aggressiveness suggest that ERß-positive tumors are likely to respond to hormonal therapy. The independent predictive value of ERß remains to be established.

	Acknowledgements

 
We thank Mrs. Sari Toivola and Mrs. Anne Luuri for skillful technical assistance.

	Footnotes

 
Address reprint requests to Prof. Jorma Isola, University of Tampere, Institute of Medical Technology, P.O. Box 607, FIN-33101 Tampere, Finland. E-mail: bljois{at}uta.fi

Supported by the Tampere University Hospital Research Foundation, Pirkanmaa and Finnish Cultural Foundations, Emil Aaltonen Foundation, Farmos Research Foundation, and the Finnish Cancer Society (to T. J. and J. I.).

Accepted for publication September 16, 1999.

	References

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				S. D. K. Berry, P. M. Jobst, S. E. Ellis, R. D. Howard, A. V. Capuco, and R. M. Akers Mammary Epithelial Proliferation and Estrogen Receptor {alpha} Expression in Prepubertal Heifers: Effects of Ovariectomy and Growth Hormone J Dairy Sci, June 1, 2003; 86(6): 2098 - 2105. [Abstract] [Full Text] [PDF]

				S. A. W. Fuqua, R. Schiff, I. Parra, J. T. Moore, S. K. Mohsin, C. K. Osborne, G. M. Clark, and D. C. Allred Estrogen Receptor {beta} Protein in Human Breast Cancer: Correlation with Clinical Tumor Parameters Cancer Res., May 15, 2003; 63(10): 2434 - 2439. [Abstract] [Full Text] [PDF]

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				H. Buteau-Lozano, M. Ancelin, B. Lardeux, J. Milanini, and M. Perrot-Applanat Transcriptional Regulation of Vascular Endothelial Growth Factor by Estradiol and Tamoxifen in Breast Cancer Cells: A Complex Interplay between Estrogen Receptors {alpha} and {beta} Cancer Res., September 1, 2002; 62(17): 4977 - 4984. [Abstract] [Full Text] [PDF]

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