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Immunohistochemical Expression of Estrogen Receptor β in Normal and Tumoral Canine Mammary Glands J. Martín De Las Mulas, J. Ordás, M. Y. Millán, F. Chacón, M. De Lara, A. Espinosa De Los Monteros, C. Reymundo and A. Jover Vet Pathol 2004 41: 269 DOI: 10.1354/vp.41-3-269 The online version of this article can be found at: http://vet.sagepub.com/content/41/3/269
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Brief Communications and Case Reports
Vet Pathol 41:3, 2004
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Immunohistochemical Expression of Estrogen Receptor  in Normal and Tumoral Canine Mammary Glands J. MART´ıN DE LAS MULAS, J. ORDA´S, M. Y. MILLA´N, F. CHACO´N M. DE LARA, A. ESPINOSA DE LOS MONTEROS, C. REYMUNDO, AND A. JOVER Abstract. To date, two isoforms of estrogen receptors (ER) have been identified, cloned, and characterized from several species, estrogen receptor–␣ (ER␣) and estrogen receptor– (ER). Although the presence of ER␣ has been demonstrated in normal and tumoral canine mammary tissues, the issue of ER expression has not been addressed in the dog. In this study, we have analyzed the expression of ER in formalin-fixed, paraffinembedded tissue samples of nonaltered mammary gland, 30 malignant (six complex carcinoma, 12 simple carcinoma, three carcinosarcoma, and nine carcinoma or sarcoma in benign tumor), and five benign (one fibroadenoma, one complex papilloma, one complex adenoma, and two benign mixed tumors) mammary tumors of the dog by using a polyclonal ER antibody and the avidin–biotin–peroxidase complex immunohistochemical technique. Our results show that high numbers of normal ductal and acinar epithelium and approximately one third of canine mammary tumors express ER. This expression was higher in benign than in malignant tumors. Furthermore, expression was higher in complex and mixed histologic subtypes of malignant tumors when compared with simple subtypes. Key words:
Dogs; estrogen receptor ; mammary gland; tumor.
The presence of estrogen receptors (ER) in variable proportions of benign and malignant tumors of the mammary gland of the dog has been demonstrated by both biochemical and immunohistochemical (IHC) methods.4,5,12–14 Its presence has been associated with a favorable prognosis in malignant tumors.12,13 After the cloning of a second ER in rats and humans, designated ER,9,11 it became known that currently reported data correlated with the isoform ␣ of ER (ER␣). In humans, messenger ribonucleic acid, western blot, and IHC analyses have shown that ER is expressed in a wide variety of tissues including the mammary gland and breast cancer cell lines and tumors.2,3,8,15,16 In this study, we have analyzed the expression of ER by immunohistochemistry in formalin-fixed, paraffin-embedded tissue samples of normal mammary gland, benign tumors, and malignant tumors of the dog. Proliferative mammary lesions were collected from 28 female dogs aged 4–16 years (mean 9.3 years). Nonaltered mammary tissues were from glands without tumoral lesions included in regional or block mastectomy pieces. The 35 excised mammary lesions (four dogs had multiple tumors) were fixed in neutral-buffered formalin and embedded in paraffin wax. The histologic types of tumors, determined on hematoxylin and eosin–stained tissue sections,10 were as follows: 30 malignant tumors (six complex carcinoma, 12 simple carcinoma, three carcinosarcoma, and nine carcinoma or sarcoma in benign tumor) and five benign tumors (one fibroadenoma, one complex papilloma, one complex adenoma, and two benign mixed tumors). Enlarged regional lymph nodes free of metastases were observed in seven dogs. The polyclonal rabbit anti-ER antibody developed against the N-terminal region of the human ER sequence (Upstate Biotechnology, Lake Placid, NY) diluted 1 : 50 was used, using the avidin–biotin–peroxidase complex Vectastain kit for polyclonals (Vector, Burlingame, CA). According to the manufacturer, the antibody recognizes ER and does not detect
ER␣. Dewaxed and rehydrated sections were subjected to high-temperature antigen retrieval by incubation with 0.01 M citrate buffer, pH 6.0, at 95 C for 25 minutes in a water bath. After cooling down at room temperature for 20 minutes, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 minutes at room temperature, rinsed (phosphate-buffered saline [PBS], pH 7.6, thrice for 10 minutes), treated with 10% normal goat serum in PBS, and incubated overnight at 4 C. The reaction was developed by incubation with the chromogen 3,3⬘-diaminobenzidine tetrahydrochloride (DakoCytomation, Glostrup, Denmark) for 15 minutes, rinsed in tap water, counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Normal canine tissues known to contain the antigen sought (ovary, uterus) were used as external positive controls. Tissue sections under study that were treated with normal rabbit serum, instead of the specific antibody, were run as negative controls. The number of positive cells in a given sample was determined as the percentage of positively stained cells: ⫺, when either none or less than 5% of nuclei were stained; ⫹, when positive nuclei represented between 5 and 19% of nuclei observed; ⫹⫹, when positive nuclei represented between 20 and 59% of nuclei observed; and ⫹⫹⫹, when 60% or more of the nuclei are positive. No staining was observed in the negative controls. In the canine ovary, ER expression was found in the nuclei of granulosa cells and ovarian stroma (Fig. 1a). In the canine uterus, immunoreactive products to ER antibody were found in the nuclei of endometrial (surface and glandular epithelial, and stromal fibroblasts) and myometrial (smooth muscle) cells (Fig. 1b). In the normal canine mammary gland, immunoreactive products to ER antibody were observed in the nuclei of numerous acinar and ductal glandular epithelial cells of several lobuli. The nuclei of isolated basalmyoepithelial cells were occasionally stained (Fig. 2), whereas stromal cells were unreactive. In addition to the
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Fig. 1. Ovary and uterus; dog. Fig. 1a. Ovarian granulosa cells (arrow) and ovarian stroma cells (star) show nuclear immunoreactivity with ER polyclonal antibody. In addition, a faint cytoplasmic staining is observed in both types of cells. ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 15 m. Fig. 1b. Uterine glandular epithelial (arrow) and stromal (arrowhead) cells of the endometrium and smooth muscle cells of the myometrium (star) show nuclear immunoreactivity with ER polyclonal antibody. Cytoplasmic staining is more evident in glandular epithelial cells of the endometrium. ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 25 m. Fig. 2. Mammary gland; dog. Normal lobule. Immunoreactive products to ER antibody are observed in the nuclei and, with lower intensity, in the cytoplasm of the majority of glandular epithelial cells of all acini. Both stained (arrow) and unstained (arrowhead) fusiform nuclei occupying a basal position in the acinus are also seen. Stromal cells are unreactive. ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 15 m.
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Table 1. Estrogen receptor– expression in five benign mammary tumors of the dog.
Fibroadenoma Complex papilloma Complex adenoma Benign mixed tumor Total
Total Number
ER Positive
IHC Scoring (⫹⫹⫹) (%)
1 1 1 2 5
1 0 1 1 (50%) 3 (60%)
0 — (100) (100) (67%)
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Table 2. Estrogen receptor beta expression in 30 malignant mammary tumors of the dog.
Complex carcinoma Simple carcinoma Carcinosarcoma CSBT* Total
Total Number
ER Positive
IHC Scoring (⫹⫹⫹) (%)
6 12 3 9 30
3 (50%) 2 (17%) 0 3 (33%) 8 (27%)
(67) (100) — (100) (87.5%)
* Carcinoma or sarcoma in benign tumor.
strong nuclear staining, faint staining was found in the cytoplasm of all types of cells (Figs. 1, 2). In the tumors, immunoreactive products to ER were found in the nuclei of glandular epithelial-type cells (Figs. 3–6). Additionally, a faint cytoplasmic staining was sometimes seen in the same type of cells (Figs. 3–6). Myoepithelial-type cells of complex (Figs. 3, 6) and mixed tumors (Fig. 4), chondrocytes of mixed tumors (Fig. 4), and sarcoma cells were unreactive. Eleven tumors expressed ER (31%) (Tables 1, 2). ER-positive cases included eight malignant tumors (26% of the group of malignant tumors) (Table 2) and three benign tumors (60% of the group of benign tumors) (Table 1). The number of positive cells was scored (⫹⫹⫹) in 87% of malignant and 67% of benign tumors. The polyclonal antibody anti-ER used in this study cross-reacted specifically with formalin-fixed and paraffinembedded canine tissues in the same way that it reacted with cells and tissues known to contain the antigen in humans,16 whereas there was no reaction in negative controls. As in human tissues, including breast carcinomas, ER immunoreactivity was found in the cell nucleus and, to a lesser extent, in the cell cytoplasm. The latter has been attributed to the production of both nuclear and cytoplasmic forms of the receptor by those cells.8,16 In the mammary gland, ER was expressed in morphologically normal acinar and ductal epithelium and in one third of the tumors analyzed. In both cases, immunoreactive cells were abundant and of the luminal- or secretory-epithelial type, whereas stromal cells and the overwhelming majority of myoepithelial cells were unreactive. In humans,
ER has been detected in normal breast epithelium and carcinoma cells by immunohistochemistry.8,16 ER-positive tumors were more frequently benign (60%) than malignant (27%). Among the latter, tumors expressing ER were more frequently of complex and mixed histologic subtypes.10 There are no data for comparison in the canine mammary tumor literature because, to our knowledge, our results are the first to address the issue of ER expression in either normal or neoplastic canine mammary gland. On the contrary, several studies have shown a similar differential expression of ER␣ in benign and malignant mammary tumors of the dog by using radioimmunoassay techniques,13 which suggests that a similar progressive loss of hormone dependence with malignant transformation occurs with respect to ER. The expression of ER in 26% of canine malignant mammary tumors is lower than the reported expression of ER␣, which varies from 37 to 70% when using radioimmunoassay techniques13 and from 10 to 87.5% when using IHC techniques.4,5,12,14 In humans, western blot and IHC studies using ER antibodies have shown ER expression in 60% of breast carcinomas,3,8 a figure similar to that of ER␣ expression in women. ER␣ expression is a favorable prognostic indicator in human and canine mammary carcinomas,12,13 and our data point to similar biological implications for ER expression. Thus, the higher expression of ER in canine malignant tumors of complex and mixed histologic subtypes suggests that the expression of ER also may be a favorable prognostic factor because the grade of malignancy of com-
← Fig. 3. Mammary gland; dog. Complex adenoma. Immunoreactive products to ER antibody are observed in the nuclei and the cytoplasm of the majority of glandular epithelial-type tumor cells (arrows). The immunoreactivity is strongest in the nucleus. The myoepithelial cell component of the neoplasm is unreactive (star). ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 15 m. Fig. 4. Mammary gland; dog. Benign mixed tumor. Immunoreactive products to ER antibody are observed in the nuclei and the cytoplasm of the majority of glandular epithelial-type tumor cells (arrows). The intensity of the reaction is similar at both levels. Stromal cells are unreactive (stars). ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 25 m. Fig. 5. Mammary gland; dog. Simple tubular carcinoma. Immunoreactive products to ER antibody are observed in the nuclei of the overwhelming majority of the neoplastic epithelial cells. A fainter reaction is also observed in the cytoplasm of these cells. Stromal cells are unreactive (arrows). ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 25 m. Fig. 6. Mammary gland; dog. Complex carcinoma. Immunoreactive products to ER antibody are observed in the nuclei and the cytoplasm of some of the glandular epithelial-type tumor cells, whereas myoepithelial cells (stars) and stromal cells (arrows) are unreactive. ABC IHC method counterstaining with Mayer’s hematoxylin. Bar ⫽ 15 m.
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plex carcinomas is lower than that of simple carcinomas.10 Older reports point to lower grade of malignancy for the recently defined ‘‘carcinoma in benign tumor’’ histologic subtype.10 However, the data with regard to ER expression in invasive tumors of the human breast and its relationship to prognosis are somewhat contradictory, with some groups reporting that the presence of this receptor is a good prognostic factor and others reporting the reverse.2,8,15 Therefore, the role played by ER in mammary tumors is not clear. However, it is interesting to note that studies in ER␣ and ER knock-out animals suggest that ER␣ is the key mediator of estradiol action in the normal mammary gland.6 Additional data suggest that ER␣ and ER play different roles in cell proliferation and carcinogenesis of breast cancer, partly by mediating the transcription of various genes via different types of deoxyribonucleic acid enhancers.1 A negative correlation between the expression of ER (but not ER␣) and the cyclin D1 gene (CCND1), a key cell cycle regulator that contains an AP1 element but not an estrogen-response element in its promoter, has been observed in human sporadic breast cancer.1 In addition, ER and ER␣ seem to have different ligand-binding specificity,9 and the results of experimental studies indicate that one role of ER is to modulate the ER␣ transcriptional activity.7 Accordingly, the relative expression level of the two isoforms may be a key determinant of cellular responses to agonists and antagonists. Therefore, ER determination may provide additional information on the responsiveness of canine mammary carcinomas to different endocrine treatments. Taken together, these data suggest that the ratio between ER␣ and ER expression in canine mammary tumors may be useful to identify subgroups of ER-positive tumors. The clinical usefulness of the subgroups remains to be explored. In conclusion, we have shown that normal ductal and acinar epithelium and approximately one third of canine mammary tumors express the second receptor for estrogens, the ER. The ER-positive tumors are more frequently benign than malignant. ER-positive malignant tumors are more frequently of the complex and mixed types than simple. The clinicopathologic associations of ER expression and the usefulness of examination of canine mammary tumors for ER expression over or in addition to ER␣ remain to be established.
Acknowledgement This study was supported by Consejeria de Educatio´n y Ciencia, Junta de Andalucı´a, Spain (Grant number CVI287).
References 1 Bieche I, Parfait B, Laurendau I, Girault I, Vidaud M, Liderau R: Quantification of estrogen receptor alpha and beta expression in sporadic breast cancer. Oncogene 20: 8109–8115, 2001 2 Dotzlaw H, Leygue E, Watson PH, Murphy LC: Estrogen receptor-beta messenger RNA expression in human breast tumor biopsies: relationship to steroid receptor status and regulation by progestins. Cancer Res 59:529– 532, 1999
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3 Fuqua SA, Schiff R, Parra I, Friedrichs WE, Su JL, McKee DD, Slentz-Kesler K, Moore LB, Willson TM, Moore JT: Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer. Cancer Res 59:5425–5428, 1999 4 Geraldes M, Ga¨rtner F, Schmitt F: Immunohistochemical study of hormonal receptors and cell proliferation in normal canine mammary glands and spontaneous mammary tumours. Vet Rec 146:403–406, 2000 5 Graham JC, O’Keefe DA, Gelberg HB: Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors. Am J Vet Res 60:627–630, 1999 6 Gustafsson J-A: Estrogen receptor —a new dimension in estrogen mechanism of action. J Endocrinol 163:379– 383, 1999 7 Hall JM, Mc Donnell: The estrogen receptor -isoform (ER) of the human estrogen receptor modulates ER transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578, 1999 8 Ja¨rvinen TAH, Pelto-Huikko M, Holli K, Isola J: Estrogen receptor beta is coexpressed with Eralpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol 156:29–35, 2000 9 Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustaffson JA: Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925– 5930, 1996 10 Misdorp W, Else RW, Hellme´n E, Lipscomb TP: Histological classification of mammary tumors of the dog and the cat, 2nd series, vol. VII, pp. 11–29. WHO International Histological Classification of Tumors of Domestic Animals. AFIP, Washington, DC, 1999 11 Mosselman S, Polman J, Dijkema R: ER-beta: identification and characterisation of a novel human estrogen receptor. FEBS Lett 392:49–53, 1996 12 Nieto A, Pen˜a L, Pe´rez-Alenza MD, Sa´nchez MA, Flores JM, Castan˜o M: Immunohistologic detection of estrogen receptor alpha in canine mammary tumors: clinical and pathologic associations and prognostic significance. Vet Pathol 37:239–247, 2000 13 Rutteman GR, Withrow SJ, MacEwen EG: Tumors of the mammary gland. In: Small Animal Clinical Oncology, ed. Withrow SJ, MacEwen BR, pp. 455–477. WB Saunders, Philadelphia, PA, 2001 14 Sobczak-Filipiak M, Malicka E: Estrogen receptors in canine mammary gland tumours. Polish J Vet Sci 5:1– 5, 2002 15 Speirs V, Parkes AT, Kerin MJ, Walton DS, Carleton PJ, Fox JN, Atkin SL: Coexpression of estrogen receptor alpha and beta: poor prognostic factor in human breast cancer? Cancer Res 59:525–528, 1999 16 Taylor Ah, Al-Azzawi F: Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol 24:145–155, 2000 Request reprints from Dr. J. Martı´n de las Mulas, Dpto. de A. y Anatomı´a Patolo´gica Comparadas, Universidad de Co´rdoba, Edificio de Sanidad Animal, Campus de Rabanales, Carretera de Madrid-Ca´diz, Km. 396, Co´rdoba 14014 (Spain). E-mail:
[email protected].
