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Evaluation of hormone receptor expression for use in predicting survival of female dogs with malignant mammary gland tumors Chao-Chin Chang, dvm, phd; Min-Hsuan Tsai, dvm, mvs; Jiunn-Wang Liao, dvm, phd; Jacky Peng-Weng Chan, dvm, phd; Min-Liang Wong, phd; Shih-Chieh Chang, dvm, phd
Objective—To evaluate the prognostic potential of expression of hormone receptors in malignant mammary gland tumors of dogs. Design—Cohort study. Animals—89 female dogs with malignant mammary gland tumors and 24 female dogs with benign mammary gland tumors. Procedures—Female dogs with malignant (n = 89 dogs) and benign (24) mammary gland tumors were evaluated to determine the prognostic value of the expression of estrogen receptor (ER)a or the progesterone receptor (PR), as determined by use of immunohistochemical methods. Results—In this study, 68 (60.2%) and 88 (77.9%) of the 113 dogs with mammary gland tumors had expression of ERa and PR, respectively. Expression of ERa and PR was detected proportionately more frequently in benign tumors (23/24 [95.8%] and 24/24 [100%], respectively) than in malignant tumors (45/89 [50.6%] and 64/89 [71.9%]). Percentage of tumors with positive results for ERa and PR was significantly higher in tumors < 5 cm in diameter; as clinical stage I, II, or III; and without metastasis to lymph nodes or distant metastasis. However, only PR expression in tumor cells was significantly associated with 1-year survival after surgical removal of the tumor. Moreover, dogs with malignant tumors expressing ERa and PR had a significantly higher survival rate, compared with the rate for dogs with malignant tumors expressing ERa but not PR. Conclusions and Clinical Relevance—These findings strongly suggested that expression of PR could be used as a prognostic factor for survival, especially in female dogs with malignant mammary gland tumors with ERa expression. (J Am Vet Med Assoc 2009;235:391–396)
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any tumor markers have been investigated as prognostic indicators in humans with breast cancer. Among these markers, the ER, PR, and HER-2 have been suggested as useful biomarkers for humans with breast cancer.1 In dogs, both benign and malignant mammary gland tumors express ERs.2,3 However, ERa and ERb expression were found to be higher in benign tumors than in malignant tumors.4,5 Lack of PRs was associated with a high histologic grade, tumor invasion, lymph node involvement, or metastases.6 Therefore, the reason for failure of ERM treatment in human breast cancer patients with ERs could be attributed to a lack of PRs. Clinical responses after ERM are significantly better in human patients whose tumors express ERs and PRs than in patients whose tumors express ERs but not PRs.7,8 Moreover, lack of expression of PRs has been significantly associated with disease progression, high tumor invasion, tumor recurrence, and death.9,10 It is of interest to investigate whether PRs are an independent prognostic factor in dogs with malignant mammary gland tumors. From the Graduate Institute of Microbiology and Public Health (CC Chang), Department of Veterinary Medicine (Tsai, Chan, Wong, SC Chang), Graduate Institute of Veterinary Pathology (Liao), and Veterinary Medical Teaching Hospital (SC Chang), College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan. Address correspondence to Dr. Shih-Chieh Chang. JAVMA, Vol 235, No. 4, August 15, 2009
Abbreviations ER ERM HER-2 OHE PR PS TNM
Estrogen receptor Estrogen receptor modulator c-erbB-2 receptor Ovariohysterectomy Progesterone receptor Proportion score Tumor, lymph nodes, and metastasis
Biologically, estrogens and progesterones play major roles in normal development of the mammary glands,11,12 but these hormones have also been implicated in tumor development.13,14 In dogs, OHE performed early in a dog’s life significantly reduces the risk of developing mammary gland tumors.13 However, OHE performed in dogs of an advanced age may influence the protective effect against mammary gland tumors or affect survival time after surgical removal of the tumor.13–18 Only a few studies4,6 have been conducted to evaluate the role of hormone receptors in survival of dogs with mammary gland tumors after surgical removal of the tumor. In another study19 conducted by our laboratory group, dogs with malignant mammary gland tumors with overexpression of HER-2 generally had a higher survival rate for 2 years after surgical removal of the Scientific Reports
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tumor, compared with the survival rate for dogs with mammary gland tumors with typical expression of HER2. Because this result differed from findings in human patients with breast cancer, it indicated that additional investigation of possible features of endocrine biomarkers in mammary gland tumors of dogs was needed. The objectives of the study reported here were to evaluate the expressions of ERa and PR in mammary gland tumors of dogs by use of immunohistochemical methods, to investigate the relationship of these hormone receptors and clinicopathologic manifestations, and to analyze the prognostic potential of hormone receptors in dogs with malignant mammary gland tumors. Materials and Methods Animals—Dogs with mammary gland tumors (n = 113 dogs, including 89 with malignant tumors and 24 with benign tumors) were identified for use in the study. Dogs had been examined at National Chung Hsing University Veterinary Medical Teaching Hospital between January 2000 and March 2006. Dogs were eligible for inclusion when a diagnosis of a mammary gland tumor had been confirmed by histologic examination and adjuvant chemotherapy was not administered after surgical removal of the tumor. Surgical procedures performed on dogs included lumpectomy, simple mastectomy, modified radical mastectomy (regional mastectomy), or radical mastectomy (complete unilateral or bilateral mastectomy). The extent of the surgical excision was determined by the size, location, and number of tumors and the status of regional lymph nodes.
kitsc,d were used with the ERa monoclonal antibody, and another commercial secondary antibody kite was used with the PR monoclonal antibody. Positive control samples consisted of normal tissues obtained from the ovary, uterus, and mammary glands. Normal ovarian and uterine tissues were obtained from healthy dogs undergoing OHE at our veterinary hospital. Normal mammary gland tissues were obtained from a location far from the neoplastic tissues in dogs with mammary gland tumors. Negative control samples consisted of tissues incubated with PBS solution in place of the primary antibody. Tissue sections were cut at a thickness of 4 mm, mounted onto silanized slides,f and placed in an oven at 45oC for 1 hour. Slides were deparaffinizated by 2 immersions in xylene (5 min/immersion) and then immersed 2 times (3 min/immersion) in 100% ethyl alcohol followed by 2 times in 95% ethyl alcohol. Antigen epitope retrieval was achieved by incubation in epitope retrieval solution (Tris-EDTA [pH, 9.0]) for 40 minutes at 95o to 99oC. Endogenous peroxidase activ-
Data collection—Historical information was obtained from the medical records and used for data analysis. Information collected included breed, age, sex, body weight, size of tumor, number of tumors, location of tumor or affected mammary glands, OHE status, reason for OHE, interval between identification of tumor and surgical removal, clinical stage as determined by use of the TNM system, surgical procedure, tumor type, histologic grade as determined on the basis of the World Health Organization–Armed Forces Institute of Pathology classification system, score for a histologic grading system (ie, tubule formation, hyperchromatism and mitoses, and irregular size and shape of nuclei) of canine mammary gland carcinomas,20,21 and overall survival time. For all dogs, follow-up examinations, including thoracic and abdominal radiography, were performed every 2 to 3 months during the first year after surgical removal of the tumor. Thereafter, owners were interviewed by telephone every 6 months. Formalin-fixed, paraffin-embedded tissue specimens from 89 malignant and 24 benign mammary gland tumors were evaluated by use of immunohistochemical analysis for the identification of hormone receptors. Immunohistochemical analysis for identification of hormone receptors—Primary antibodies used in the study have been reported elsewhere6 and included mouse anti-human ERa monoclonal antibodya (diluted 1:35) and mouse anti-human PR monoclonal antibodyb (diluted 1:100). Two commercial secondary antibody 392
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Figure 1—Photomicrograph of tissue sections from malignant mammary gland tumors of dogs. A—A representative simple carcinoma in which most of the neoplastic epithelial cells have a strong nuclear immunoreactivity with the ERa antibody. B—A moderate number of cells in this simple carcinoma have strong immunoreactivity with the PR antibody. Immunohistochemical stain with 3,39 diaminobenzidine substrate and counterstain with hematoxylin. Bar = 25 mm. JAVMA, Vol 235, No. 4, August 15, 2009
Table 1—Hormone receptor expression in 24 benign mammary gland tumors and 89 malignant mammary gland tumors of dogs.
ERα+
Tumor type
No. of tumors
No.
10 2 12 24 42 35 12 89
10 2 11 23 28 17 0 45
Benign Adenoma* Fibroadenoma Benign mixed tumor Total Malignant Complex carcinoma Simple carcinoma Sarcoma† Total
PR+
%
No.
%
100 100 91.7 95.8
10 2 12 24
100 100 100 100
66.7 48.6 0 50.6
34 24 6 64
to 1 in 10, 3 = PS from 1 in 10 to 1 in 3, 4 = PS from 1 in 3 to 2 in 3, and 5 = PS > 2 in 3) and an intensity score for estimating staining intensity of positive-staining tumor cells (0 = negative, 1 = weakly positive, 2 = mildly positive, and 3 = strongly positive). A total score was then calculated by adding the PS and intensity score. For both ER and PR, a total score ≥ 3 was considered a positive result. Statistical analysis—The Kaplan-Meier method was used to construct survival curves for dogs with mammary gland tumors with differences in expression of hormone receptors. The log-rank test was used to identify factors and expression of hormone receptors associated with survival 1 year after surgical removal of the tumor. The Pearson c2 test was used to detect associations between the characteristics of dogs or tumors and hormone receptors. For all analyses, values of P ≤ 0.05 were considered significant. All analyses were performed with commercial software.g Results
81.0 68.6 50.0 71.9
Dogs—Age of the 113 female dogs at the time of tumor removal ranged from 2 to 15 years (mean ± SD, 9.8 ± 2.7 years; median, 10 years). There were 21 breeds represented in these 113 dogs, with mixed-breed dog (n = 31 [27.4%] dogs), Maltese (23 [20.4%]), Pomeranian (10 [8.8%]), and Toy Poodle (7 [6.2%]) the 4 breeds most commonly represented. The majority of the dogs
*Includes simple and complex adenomas. †Includes carcinosarcoma, osteosarcoma, and fibrosarcoma. ERα+ = Expression of ERα. PR+ = Expression of PR.
Table 2—Relationship between hormone receptor expression and features of malignant mammary gland tumors of dogs. Variable
ERα+ No. of tumors
No.
%
ERα– No.
%
PR+ No.
PR– %
No.
%
Tumor size 5 cm 49 28 57.1 21 42.9 43 87.8‡ 6 12.2 5 cm 40 17 42.5 23 57.5 21 52.5 19 47.5 Clinical stage* I, II, or III 52 31 59.6‡ 21 40.4 43 82.7‡ 9 17.3 IV or V 37 14 37.8 23 62.2 21 56.8 16 43.2 Metastasis to lymph nodes Yes 36 13 36.1‡ 23 63.9 20 55.6‡ 16 44.4 No 53 32 60.4 21 39.6 44 83.0 9 17.0 Distant metastasis Yes 21 6 28.6‡ 15 71.4 12 57.1 9 42.9 No 68 39 57.4 29 42.6 52 76.5 16 23.5 Clinical stage I 24 17 70.8 7 29.2 21 87.5 3 12.5 II or III 53 28 52.8 25 47.2 37 69.8 16 30.2 Tubule formation† Score 1 34 23 67.6‡ 11 32.4 27 79.4‡ 7 20.6 Score 2 26 16 61.5 10 38.5 22 84.6 4 15.4 Score 3 17 6 35.3 11 64.7 9 52.9 8 47.1 Mitotic index† Score 1 29 18 62.1 11 37.9 23 79.3‡ 6 20.7 Score 2 40 25 62.5 15 37.5 32 80.0 8 20.0 Score 3 8 2 25.0 6 75.0 3 37.5 5 62.5 20,21 *Score based on the TNM system. †Score based on a histologic grading system. ‡Significant (P 0.05) association between the investigated variable and expression of the specific hormone receptor. ERα– = No expression of ERα. PR– = No expression of PR. See Table 1 for remainder of key. JAVMA, Vol 235, No. 4, August 15, 2009
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ity was reduced by addition of peroxidase-blocking reagent. Subsequent immunochemical procedures were performed in accordance with the instruction manual of the manufacturer. Criteria for determination of staining proportion and staining intensity were based on a scoring system for immunohistochemical staining of ER and PR established in another study.7 The scoring system included a PS as an estimation of the proportion of positive-staining tumor cells (1 = PS < 1 in 100; 2 = PS from 1 in 100
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(72 [63.7%] dogs) had a body weight < 10 kg (< 22 lb). In 18 of 113 (15.9%) dogs, OHE was performed prior to surgical removal of mammary gland tumors; reasons for performing OHE were routine spay in 8 dogs and pyometra in 10 dogs. In addition, OHE was performed at the time of tumor removal in 35 dogs; OHE was performed in 13 of the 35 (37.1%) dogs because of pyometra. Of the 113 dogs, 89 had malignant mammary gland tumors, including 42 (47.2%) complex carcinomas, 35 (39.3%) simple carcinomas, and 12 (13.5%) sarcomas (9 carcinosarcomas, 2 osteosarcomas, and 1 fibrosarcoma). For the 77 carcinomas (complex and simple carcinomas), 24 (31.2%) were classified as grade I, 40 (51.9%) were classified as grade II, and 13 (16.9%) were classified as grade III by use of histologic grading. Expression of hormone receptors in normal and neoplastic mammary glands—Analysis was performed to detect hormone receptor expression in malignant mammary gland tumors (Figure 1). Normal ovarian, uterine, and mammary gland tissues were used as positive control samples. Expressions of ERa were in the nuclei of mucosal epithelial cells, stromal fibrocytes, and smooth muscle cells of the uterine horns; granulosa cells and interstitial glandular cells of the ovary; and glandular epithelial cells of normal mammary glands. In neoplastic tissues, staining for ERa expression was identified in the nuclei of glandular epithelial cells but not in the cartilage- or osteoid-like area. Moreover, in several tumors, stromal cells surrounding the tumor lesion had moderate to strong staining. Compared with neoplastic cells that had positive staining of various intensities, there was homogenous immunoreactivity of ERa in the normal mammary glands. Characteristics of PR expression in positive control tissues were similar to those for ERa expression, except that the stromal cells did not have positive staining results for PR. The staining intensity for PR varied in neoplastic tissues. Relationship between hormone receptors and characteristics of mammary gland tumors—Detection of ERa and PR expression in benign and malignant mammary gland tumors was summarized (Table 1). Except for some benign mixed tumors, most of the benign mammary gland tumors had positive results when tested for ERa and PR expression. However, percentages of expression of ERas and PRs varied among types of malignant tumors. Accordingly, 68 (60.2%) and 88 (77.9%) mammary gland tumors of the 113 dogs tested, respectively, had positive results when tested to detect expression of ERa and PR. Expression of ERa and PR was detected proportionately more frequently in benign tumors (24/24 [100%] and 23/24 [95.8%], respectively) than in malignant tumors (45/89 [50.6%] and 64/89 [71.9%], respectively). Univariate analysis by use of a c2 test revealed that hormone receptor expression was not associated with age, breed, OHE prior to tumor removal, history of pyometra, and number and location of affected mammary glands (data not shown). However, among the 11 dogs that had malignant mammary gland tumors with OHE prior to tumor removal, 3 had ERa expression in tumors; the proportion was lower than in malignant mammary gland tumors of dogs (42/78 [53.8%]) with394
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out OHE prior to tumor removal. Similar results were also detected with regard to PR expression in dogs that had malignant mammary gland tumors with (6/11) and without (58/78) OHE prior to tumor removal. To further evaluate the relationship between hormone receptor expression and characteristics of malignant tumors, univariate analysis by use of a c2 test was used (Table 2). Dogs with malignant tumors with a low clinical stage (I, II, or III) without metastasis to lymph nodes or distant metastasis were significantly more likely to have a higher percentage of ERa expression. With regard to PR expression, the percentage was significantly higher in malignant tumors < 5 cm in diameter, with a low clinical stage (I, II, or III), without metastasis to lymph nodes, and with a low histologic score for tubule formation or mitotic index.
Figure 2—Kaplan-Meier survival curves in dogs with malignant mammary gland tumors grouped on the basis of expression of ERa (A) and PR (B). Analysis revealed that expression of ERa (ERa+) was not significantly associated with survival 1 year after surgical removal of the tumor (log-rank statistic = 1.66; P = 0.198), whereas expression of PR (PR+) was significantly associated with survival 1 year after surgical removal of the tumor (log-rank statistic = 3.93; P = 0.047). Survival time indicates the interval after surgical removal of the tumor. ERa– = No expression of ERa. PR– = No expression of PR. JAVMA, Vol 235, No. 4, August 15, 2009
Comparison of survival in dogs with malignant mammary gland tumors on the basis of hormone receptor expression—Kaplan-Meier survival analysis and log-rank tests were used to investigate the relationship between hormone receptor expression and 1-year survival rate. Results revealed that only PR expression was significantly associated with 1-year survival in dogs with malignant mammary gland tumors (Figure 2). A higher proportion of dogs survived for 1 year when the mammary gland tumor had positive results for PR expression (52/63 [82.5%]), compared with the proportion that survived for 1 year when the mammary gland tumor had negative results for PR expression (15/24 [62.5%]). Additional comparisons were made of the survival curves with regard to the interaction of various hormone receptor expressions (Figure 3). Dogs with tumors that expressed both ERa and PR were more likely to survive for 1 year after surgical removal of the tumor (35/41 [85.4%]), compared with the likelihood of survival for 1 year in dogs with tumors that expressed ERa but not PR (1/3 [33.3%]); survival did not differ significantly between dogs with tumors that expressed both ERa and PR, compared with dogs with tumors that expressed PR but not ERa (17/22 [77.3%]) or expressed neither ERa or PR (14/21 [66.7%]). Discussion The study reported here was conducted to evaluate the relationship of hormone receptors (ERa and PR) and 1-year survival in dogs after surgical removal of mammary gland tumors. Analysis of the results indicated that expression of ERa or PR was significantly associated with tumor size, clinical stage, and metastasis to lymph nodes or distant metastasis, which was similar to results in another report.6 Furthermore, hormone receptor expression also was associated with the histologic scores of tubule formation and mitotic index. In other studies,4,6–10,22,23 it has been suggested that hormone receptor expression plays an important role in JAVMA, Vol 235, No. 4, August 15, 2009
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Figure 3—Kaplan-Meier survival curves in dogs with malignant mammary gland tumors with various expressions of ERa and PR. Curves differed significantly (P = 0.004) between dogs with tumors that had expression of both ERa and PR (ERa+PR+) and dogs that had tumors with expression of ERa but not PR (ERa+PR–). Curves did not differ significantly between dogs that had tumors with ERa+PR+, dogs that had tumors with expression of PR but not ERa (ERa–PR+ [P = 0.302]), and dogs that had tumors without expression of ERa or PR (ERa–PR– [P = 0.063]).
predicting the biological behavior of breast cancer in humans and mammary gland neoplasia in dogs. However, in the study reported here, analysis of survival curves revealed that only PR expression was significantly associated with postoperative survival in dogs with malignant mammary gland tumors. Furthermore, dogs with malignant mammary gland tumors that had expression of both ERa and PR had a significantly higher survival rate, compared with that for dogs with malignant mammary gland tumors that had expression of ERa but not PR. Results of this study were similar to results reported for humans with breast cancer7–10,24 in which a lack of PR expression was associated with secondary breast cancer and PR expression was a better outcome predictor than ER status alone. These observations strongly suggested that expression of PR could be used as a prognostic factor, especially in dogs with malignant gland tumors with ERa expression. In dogs with malignant mammary gland tumors, the proportion of tumors with PR expression (64/89 [71.9%]) was higher than the proportion of tumors with ERa expression (45/89 [50.6%]); these results were similar to those in another study6 in dogs (76% vs 31% for PR and ERa, respectively). However, our findings differed from those in reports25,26 for humans with breast cancer, which had a higher percentage (68.8% to 81%) of ERa than that of PR (54% to 70%). Furthermore, lower percentages of ERa expression were detected in malignant tumors involving metastasis to lymph nodes or distant metastasis. Higher percentages of PR expression were detected in tumors < 5 cm in diameter and tumors with histologic tubule formation and a low mitotic index. These findings may imply that expression of ERa and PR is an indicator of differentiation of neoplastic cells. Similarly, lack of ERa and PR expression has frequently been reported in tumor cells with poor differentiation.27,28 It has also been suggested that neoplastic cells with or without ERa and PR expression could originate from different stem cells.29,30 On the basis of results in other studies,11,31 cells with PR expression should have ERa expression because PR expression is modulated by ERa and ERa expression is an essential factor for inducing PRs. In the study reported here, the reason 23 malignant tumors had expression of PR but not of ERa is unclear. However, such a discrepancy could have been caused by the detection limit of the anti-ERa antibody for ERa or as a result of defective ligand binding for biologically functional ERa variants.24,27 Expression of ERas and tumor cell growth can be suppressed after an ERM (ie, tamoxifen) binds to ERas.32 Therefore, this technique has been used in treatments for humans with breast cancer. In women with breast cancers, 51.5% of patients whose tumors had expression of both ER and PR responded to tamoxifen treatment; however, only 16.7% of patients whose tumors had expression of ER but not PR responded to the same treatment.8 Similarly, it was also reported10 that patients receiving adjuvant endocrine treatment had a significant decrease in the rate of relapse (53%) and death (38%) when the tumor had expression of both ER and PR, compared with the results when the tumor had expression of ER but not PR. Therefore, from the aspect of clinical treatment,
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PR expression may play an important role for use in predicting better clinical outcome after clinical treatments in human patients whose tumors have expression of ER. Currently, tamoxifen is not recommended as an adjuvant treatment in dogs because of adverse effects such as pyometra, urogenital infection, and vulvar edema.33,34 On the basis of our findings, OHE may be used as an adjuvant treatment because the removal of ovaries that produce most of the estrogen may have benefits similar to those of an ERM in humans. Expression of ERa and PR was detected significantly more often in benign tumors than in malignant tumors. In malignant tumors, expression of ERa and PR was higher in complex carcinomas than in simple carcinomas and sarcomas. Expression of ERa was also associated with other recognized prognostic factors, such as clinical stage, tumor size, and metastasis status. Furthermore, expression of PR was associated with clinical stage, tumor size, metastasis status, tubule formation, and mitotic index. Of the hormone receptors evaluated, only PR was significantly associated with postoperative survival in dogs with malignant mammary gland tumors. These observations strongly suggested that expression of PR could be used as an important prognostic factor, especially in dogs with malignant mammary gland tumors that have ERa expression. a. b. c. d. e. f. g.
Clone 1D5, DakoCytomation, Glostrup, Denmark. Clone PR10A9, Immunotech, Marseille, France. EnVision+Dual Link system, DakoCytomation, Glostrup, Denmark. HRP(DAB+), DakoCytomation, Glostrup, Denmark. IHC Select immunoperoxidase secondary detection system, Chemicon (Millipore), Temecula, Calif. Muto Pure Chemicals Co, Tokyo, Japan. SPSS, version 15.0, SPSS Inc, Chicago, Ill.
References 1.
2. 3. 4.
5. 6.
7. 8. 9.
396
Bast RC Jr, Ravdin P, Hayes DF, et al. American Society of Clinical Oncology Tumor Markers Expert Panel. 2000 update of recommendations for the use of tumor markers in breast and colorectal cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001;19:1865–1878. MacEwen EG, Patnaik AK, Harvey HJ, et al. Estrogen receptors in canine mammary tumors. Cancer Res 1982;42:2255–2259. Donnay I, Rauïs J, Devleeschouwer N, et al. Comparison of estrogen and progesterone receptor expression in normal and tumor mammary tissues from dogs. Am J Vet Res 1995;56:1188–1194. Nieto A, Peña L, Pérez-Alenza MD, et al. Immunohistologic detection of estrogen receptor alpha in canine mammary tumors: clinical and pathologic associations and prognostic significance. Vet Pathol 2000;37:239–247. Martín de las Mulas J, Ordás J, Millán MY, et al. Immunohistochemical expression of estrogen receptor β in normal and tumoral canine mammary glands. Vet Pathol 2004;41:269–272. Martin de las Mulas J, Millán Y, Dios R. A prospective analysis of immunohistochemically determined estrogen receptor a and progesterone expression and host and tumor factors as predictors of disease-free period in mammary tumors of the dog. Vet Pathol 2005;42:200–212. Allred DC, Harvey JM, Berardo M, et al. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11:155–168. Yamashita H, Yando Y, Nishio M, et al. Immunohistochemical evaluation of hormone receptor status for predicting response to endocrine therapy in metastatic breast cancer. Breast Cancer 2006;13:74–83. Balleine RL, Earl MJ, Greenberg ML, et al. Absence of progesterone receptor associated with secondary breast cancer in postmenopausal women. Br J Cancer 1999;79:1564–1571. Scientific Reports
10. Bardou VJ, Arpino G, Elledge RM, et al. Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. J Clin Oncol 2003;21:1973–1979. 11. Arafah BM, Finegan HM, Roe J, et al. Hormone dependency in N-nitrosomethylurea-induced rat mammary tumors. Endocrinology 1982;111:584–588. 12. Rutteman GR. Contraceptive steroids and the mammary gland: is there a hazard?—Insights from animal studies. Breast Cancer Res Treat 1992;23:29–41. 13. Schneider R, Dorn CR, Taylor DO. Factors influencing canine mammary cancer development and postsurgical survival. J Natl Cancer Inst 1969;43:1249–1261. 14. Selman PJ, Mol JA, Rutteman GR, et al. Progestin-induced growth hormone excess in the dog originates in the mammary gland. Endocrinology 1994;134:287–292. 15. Yamagami T, Kobayashi T, Takahashi K, et al. Influence of ovariectomy at the time of mastectomy on the prognosis for canine malignant mammary tumours. J Small Anim Pract 1996;37:462–464. 16. Sorenmo KU, Shofer FS, Goldschmidt MH. Effect of spaying and timing of spaying on survival of dogs with mammary carcinoma. J Vet Intern Med 2000;14:266–270. 17. Philibert JC, Snyder PW, Glickman N, et al. Influence of host factors on survival in dogs with malignant mammary gland tumors. J Vet Intern Med 2003;17:102–106. 18. Chang SC, Chang CC, Chang TJ, et al. Prognostic factors associated with survival two years after surgery in dogs with malignant mammary tumors: 79 cases (1998–2002). J Am Vet Med Assoc 2005;227:1625–1629. 19. Hsu WL, Huang HM, Liao JW, et al. Increased survival in dogs with malignant mammary tumours overexpressing HER-2 protein and detection of a silent single nucleotide polymorphism in the canine HER-2 gene. Vet J 2009;180:116–123. 20. Misdorp W. Tumors of the mammary gland. In: Meuten DJ, ed. Tumors in domestic animals. 4th ed. Ames, Iowa: Iowa State Press, 2002;575–606. 21. Misdorp W, Else R, Hellmen E, et al. Histological classification of mammary tumors of the dog and the cat. World Health Organization international histological classification of tumors of domestic animals. 2nd series. Vol 7. Washington, DC: Armed Forces Institute of Pathology, 1999;1–59. 22. Walker B, Figgs LW, Zahm SH. Differences in cancer incidence, mortality, and survival between African Americans and whites. Environ Health Perspect 1995;8:275–281. 23. Kommoss F, Pfisterer J, Idris T, et al. Steroid receptors in carcinoma of the breast. Results of immunocytochemical and biochemical determination and their effects on short-term prognosis. Anal Quant Cytol Histol 1994;16:203–210. 24. McGuire WL, Chamness GC, Fuqua SA. Estrogen receptor variants in clinical breast cancer. Mol Endocrinol 1991;5:1571–1577. 25. Wenger CR, Beardslee S, Owens MA, et al. DNA ploidy, S-phase, and steroid receptors in more than 127,000 breast cancer patients. Breast Cancer Res Treat 1993;28:9–20. 26. Pichon MF, Broet P, Magdelenat H, et al. Prognostic value of steroid receptors after long term follow-up of 2257 operable breast cancers. Br J Cancer 1996;73:1545–1551. 27. Fuqua SA, Fitzgerald SD, Chamness GC, et al. Variant human breast tumor estrogen receptor with constitutive transcriptional activity. Cancer Res 1991;51:105–109. 28. Locker GY. Hormonal therapy of breast cancer. Cancer Treat Rev 1998;24:221–240. 29. Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumors. Nature 2000;406:747–752. 30. Sørlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001;98:10869–10874. 31. Horwitz KB, McGuire WL. Specific progesterone receptors in human breast cancer. Steroids 1975;25:497–505. 32. Paech K, Webb P, Kuiper GG. Differential ligand activation of estrogen receptors ERa and ERb at AP1 sites. Science 1997;277:1508–1510. 33. Kitchell BE, Fidel JL. Tamoxifen as a potential therapy for canine mammary carcinoma, in Proceedings. Vet Can Soc 1992;91. 34. Morris JS, Dobson JM, Bostock DE. Use of tamoxifen in the control of mammary neoplasia. Vet Rec 1993;133:539–542. JAVMA, Vol 235, No. 4, August 15, 2009