Original Article

Modern Pathology (2004) 17, 1372–1377, advance online publication, 2 July 2004; doi:10.1038/modpathol.3800201

Estrogen receptor bold italic beta expression in vascular neoplasia: an analysis of 53 benign and malignant cases

Andrea T Deyrup1, Maria Tretiakova2, Andrey Khramtsov2 and Anthony G Montag2

  1. 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
  2. 2Department of Pathology, The University of Chicago, Chicago, IL, USA

Correspondence: Dr AG Montag, MD, Department of Pathology, MC6101, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA. E-mail: amontag@mcis.bsd.uchicago.edu

Received 22 January 2004; Revised 6 April 2004; Accepted 6 April 2004; Published online 2 July 2004.

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Abstract

The importance of estrogen in vascular neoplasia is suggested by a predilection for women and a tendency for rapid growth during pregnancy. Although early experiments using radioligand assays demonstrated estrogen receptor (ER) expression, these findings were not confirmed by subsequent immunohistochemical studies which were performed with antibodies raised against ERalpha. A newly discovered estrogen receptor subtype, ERbeta, has not been previously characterized in vascular lesions. In order to verify the expression of estrogen receptors in vascular neoplasms as well as to clarify the inconsistency between radioligand and early immunohistochemical studies, we examined a series of 53 benign and malignant vascular neoplasms for ERbeta expression. All of the subtypes of vascular neoplasia examined had nuclear expression of ERbeta. The majority of cases (94%) displayed 2+ to 3+ staining. The discrepancy between radioligand studies and previous immunohistochemical studies is attributable to the use of antibodies raised against ERalpha, which is not expressed in vascular lesions, and not ERbeta, which is broadly expressed in both benign and malignant vascular neoplasms. Although ERbeta may be of limited diagnostic use in vascular neoplasia due to its broad expression, the potential exists for a therapeutic approach using ER agonists.

Keywords:

vascular, estrogen receptor, radioligand, immunohistochemical, estrogen receptor beta

The role of estrogen in vascular neoplasia has long been suspected due to the observation that vascular neoplasms tend to grow rapidly during pregnancy and in adolescence.1 These clinical observations were initially substantiated by early experiments in the 1980s using radioligand assays, which confirmed the presence of the estrogen receptor (ER) in vascular tumors;2, 3 however, with the advent of immunohistochemical techniques, subsequent studies performed in the 1990s with antibodies raised against the ER refuted these earlier data and it appeared that the newer technology had exposed the limitations of the older biochemical assays.4, 5, 6, 7 Recently, an additional ER subtype, estrogen receptor beta (ERbeta),8 was identified and the previous ER against which the original antibodies were raised was renamed estrogen receptor alpha (ERalpha). Although ERalpha has been assessed immunohistochemically in vascular neoplasms and found to be absent, ERbeta has not been previously characterized in these entities. In order to verify the expression of ER in vascular tumors as well as to clarify the inconsistency between radioligand and early immunohistochemical studies, we examined a series of 53 benign and malignant vascular neoplasms for ERbeta expression.

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Materials and methods

In all, 22 cases of angiosarcoma, three cases of Kaposi sarcoma, one case of epithelioid hemangio-endothelioma, two cases of spindle-cell heman-gioma, four cases of infantile hemangio-endothelioma, 13 cases of hemangioma, seven cases of lymphangioma and one case of papillary endothelial hyperplasia were identified and retrieved from the paraffin archives of the University of Chicago. Formalin-fixed paraffin-embedded specimens were cut into 4-mum sections and mounted on positively charged slides. Sections were deparaffinized, rehydrated, then washed in Tris-buffered saline (TBS) and subjected to heat epitope retrieval in a microwave. Slides were then incubated in 1% hydrogen peroxide in methanol for 5 min to block endogenous peroxidase activity, followed by incubation for 20 min in a protein-blocking solution to reduce nonspecific antibody binding. The primary anti-human polyclonal antibody (Biogenex, San Ramon, CA, USA, 1:50) was applied for 1 h at room temperature. Slides were then incubated for 30 min at room temperature with anti-mouse or anti-rabbit IgG conjugated to a horseradish peroxidase (HRP)-labeled polymer (DAKO Envision™+System, DAKO Corp., Carpinteria, CA, USA), treated for 5 minutes with 3-3'-diaminobenzidine (DAB) chromogen, counterstained with hematoxylin, and coverslipped. Negative controls received a nonimmune polyclonal rabbit antiserum or monoclonal mouse antibody as appropriate.

Slides were evaluated for strong nuclear staining and scored as 0, 1+ (<25%), 2+ (25–50%), or 3+ (>50%) by two pathologists (ATD and AGM). Ovarian follicles or granulosa cell tumors served as positive controls for ERbeta.

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Results

All of the subtypes of vascular neoplasia examined displayed nuclear expression of ERbeta (Table 1, Figure 1). The majority of cases (93%) displayed 2+ to 3+ nuclear staining intensity. There was no significant difference between benign and malignant lesions. Specifically, staining intensity, percentage of tumor stained and pattern of expression were similar in both sets of tumors. Only one case of angiosarcoma was negative, with all others having 2+ to 3+ staining intensity. Normal endothelium (Figure 2) and the proliferating endothelium of papillary endothelial hyperplasia were also strongly positive for ERbeta.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

ER beta expression in (a, b) capillary hemangioma; (c, d) Kaposi sarcoma; (e, f) angiosarcoma.

Full figure and legend (1,186K)

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

ER beta expression in normal capillaries.

Full figure and legend (147K)


The cases included approximately equal numbers of male subjects and (24 M:29 F) and there was no significant difference in the presence or degree of ERbeta expression across genders (Table 2).


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Discussion

The ER and other steroid hormone receptors are members of the nuclear receptor superfamily of ligand-activated transcription factors.9 Following ligand binding, the ER undergoes dimerization, complexes with coregulatory molecules and binds to the promoter area of targeted genes.10 Regulation of ER function depends on the relative agonistic and antagonistic properties of the ligands and the combination of repressing and stimulating co-regulatory molecules present in the cell. In the 1970s, antibodies to the ER were produced with subsequent development of paraffin-stable monoclonal antibodies in the 1990s. In 1996, a second ER protein was cloned from rat prostate and ovary8 and was consequently identified in humans.11 The originally defined ER has since been designated ERalpha, while the more recently discovered receptor has been designated ERbeta. ERbeta displays significant homology with ERalpha in the transcription activating and ligand binding domains, although the domains involved in the binding of coactivators and corepressors show substantially less homology. ERbeta has a number of isoforms as a result of differential splicing, which appear to have some tissue specificity.12, 13

ERalpha and ERbeta have different tissue distributions, although with considerable overlap in breast and other organs. ERbeta is present in most CNS cells, prostate, ovary, connective tissues of many organs and lymphocytes,14, 15 decreasing its diagnostic utility. Modulation of estrogen binding depends on the variable distribution of receptor types and subtypes, their varying avidity for ligands, and a constellation of coactivators and corepressors. For example, U2OS osteosarcoma cell lines transfected with inducible ERalpha or ERbeta and studied with an Affymetrix GeneChiptm array were found to have fewer than a quarter of their estraidiol-, raloxifene- and tamoxifen-regulated genes in common.16 The differential function of the two ER genes is illustrated in the placenta, where ERbeta is expressed in syncytiotrophoblast, ERalpha in cytotrophoblast and where they appear to promote proliferation and differentiation, respectively.17

Clinical measurement of ER protein has been a routine practice since the 1970s; however, as biomedical science has advanced, the methodology has changed over the years, with varying results. Early assays were based on ligand-binding techniques, which involved either radioligand binding or fluorescent hormone binding. In the 1980s, these methods were supplanted by monoclonal antibody-based techniques, such as immunohistochemistry and immunoenzyme assays.18, 19 Although initial comparisons of the methods in breast carcinoma found a good correlation between the two techniques, no systematic comparison was made in mesenchymal lesions.20 Since ligand-binding assays assess binding of a radiolabeled or fluorescein-labeled ligand, this technique measures both ERalpha and ERbeta. Today, virtually all ER determinations are performed with an antibody-based method, typically using a monoclonal antibody against the ERalpha protein which, due to the specificity of the antibody, only detects ERalpha. Therefore, any tissue expressing only ERbeta will result in a false negative, accounting for the historical discrepancy in the literature regarding ER in vascular tumors.

The influence of hormones in vascular neoplasia has long been suspected due to an increased incidence in women and a tendency for rapid growth during pregnancy. Ligand binding assays of steroid hormone receptors in vascular tumors gave variable results, with only two studies reporting the presence of ER. (Table 3). Subsequently, Kuma-gami21 used antibodies to steroid hormones to detect bound hormone in tissue sections and discovered evidence of bound estradiol and progesterone in 5/5, testosterone in 2/5, and dihydrotestosterone in 3/5 cases. Later, immunohistochemical studies were uniformly negative for ER, including a large series by Hwang et al22 and Liang et al.23 These studies employed commercially available anti-ERalpha anti-bodies and, consequently, would be expected to fail to detect ERbeta. The antibody against ERbeta used in the current study was raised against a peptide sequence from the carboxy terminus of ERbeta, a region of the protein that lacks homology to the corresponding area of ERalpha.24 Lack of cross reactivity with ERalpha was confirmed via Western blot (Biogenex, personal communication). The data described here clarify the earlier errors promoted through the use of antibodies raised against ERalpha, and establish that ERbeta is strongly expressed in vascular neoplasms.


Although the spectrum of mesenchymal neoplasms remains to be evaluated, ERbeta expression has been characterized in a number of adenocarcino-mas including those of the breast,25 ovary,26, 27, 28 endo-metrium,29 esophagus,30 stomach31 and colon.31, 32 Interestingly, in breast,25 ovary25, 26, 27, 28 and endometrium,29 malignancy is associated with loss of ERbeta expression while in adenocarcinoma of the esophagus30 increased expression is seen. One study has shown decreased ERbeta expression in colonic adenocarcinoma31 while another study32 suggests that, as is seen in gastric adenocarcinoma,30 there is no significant difference between normal and dysplastic mucosa. The situation is complicated by the recent discovery of additional splice variants and isoforms of ERbeta in normal tissue33, 34, 35 as well as evidence that the interaction of ERalpha and ERbeta levels may play a role in carcinogenesis.28

The demonstration of ERbeta in vascular neoplasms suggests a role for estrogen antagonists in the therapy of the most aggressive vascular tumors: angiosarcomas. Although rare, angiosarcomas have a uniformly poor prognosis, which correlates with tumor site and size.36, 37 Moreover, perhaps due to their association with the systemic vasculature, angiosarcomas metastasize widely and often present initially with multifocal disease, rendering them particularly refractory to surgical therapy. A new generation of antiestrogenic compounds and selective ER modulators (SERM) are in development, some of which preferentially target ERbeta.38, 39, 40 As yet, none of these SERM has been tested in angiosarcomas and it remains uncertain whether the effect on normal endothelial cells would preclude systemic therapy. Nonetheless, further study is warranted.

We have demonstrated that, despite previous immunohistochemical evidence to the contrary, a broad range of benign and malignant vascular neoplasms as well as normal blood vessels express ER, specifically ERbeta. Furthermore, we have showed that the previous inconsistencies in the ER status of vascular neoplasms—and perhaps other entities as well—are attributable to the unexpected existence of a second ER which remains to be fully classified. It is perhaps worth contemplating the existence of a third ER, as yet undetected by our more modern methods but fully present to the biochemists of before.

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