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Many tumor markers have been investigated as prognostic indicators in humans with breast can-

cer. 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.

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 immunohisto-chemical 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 de-tected 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)

Biologically, estrogens and progesterones play major roles in normal development of the mammary glands,11,12 but these hormones have also been impli-cated in tumor development.13,14 In dogs, OHE per-formed early in a dog’s life significantly reduces the risk of developing mammary gland tumors.13 How-ever, 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 con-ducted 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

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 Vet-erinary Medical Teaching Hospital (SC Chang), College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.

Address correspondence to Dr. Shih-Chieh Chang.

Abbreviations

ER EstrogenreceptorERM EstrogenreceptormodulatorHER-2 c-erbB-2receptorOHE OvariohysterectomyPR ProgesteronereceptorPS ProportionscoreTNM Tumor,lymphnodes,andmetastasis

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tumor, compared with the survival rate for dogs with mammary gland tumors with typical expression of HER-2. Because this result differed from findings in human patients with breast cancer, it indicated that additional investigation of possible features of endocrine biomark-ers 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 tu-mors of dogs by use of immunohistochemical methods, to investigate the relationship of these hormone recep-tors and clinicopathologic manifestations, and to ana-lyze 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 Uni-versity Veterinary Medical Teaching Hospital between January 2000 and March 2006. Dogs were eligible for in-clusion when a diagnosis of a mammary gland tumor had been confirmed by histologic examination and adjuvant chemotherapy was not administered after surgical remov-al of the tumor. Surgical procedures performed on dogs included lumpectomy, simple mastectomy, modified radi-cal mastectomy (regional mastectomy), or radical mastec-tomy (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.

Data collection—Historical information was ob-tained from the medical records and used for data anal-ysis. Information collected included breed, age, sex, body weight, size of tumor, number of tumors, loca-tion of tumor or affected mammary glands, OHE status, reason for OHE, interval between identification of tu-mor 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 sur-vival 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 inter-viewed 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 identi-fication 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

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 hospi-tal. Normal mammary gland tissues were obtained from a location far from the neoplastic tissues in dogs with mammary gland tumors. Negative control samples con-sisted 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 im-mersed 2 times (3 min/immersion) in 100% ethyl al-cohol followed by 2 times in 95% ethyl alcohol. An-tigen 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-

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.

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ity was reduced by addition of peroxidase-blocking re-agent. 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 estab-lished in another study.7 The scoring system included a PS as an estimation of the proportion of positive-stain-ing tumor cells (1 = PS < 1 in 100; 2 = PS from 1 in 100

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 tu-mor 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 tu-mors and hormone receptors. For all analyses, values of P ≤ 0.05 were considered significant. All analyses were performed with commercial software.g

Results

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

Table 1—Hormone receptor expression in 24 benign mammary gland tumors and 89 malignant mammary gland tumors of dogs.

ERa+ PR+

Tumortype No.oftumors No. % No. %

Benign Adenoma* 10 10 100 10 100Fibroadenoma 2 2 100 2 100Benignmixedtumor 12 11 91.7 12 100Total 24 23 95.8 24 100 Malignant Complexcarcinoma 42 28 66.7 34 81.0Simplecarcinoma 35 17 48.6 24 68.6Sarcoma† 12 0 0 6 50.0Total 89 45 50.6 64 71.9

*Includes simple and complex adenomas. †Includes carcino-sarcoma,osteosarcoma,andfibrosarcoma.

ERa+=ExpressionofERa.PR+=ExpressionofPR.

ERa+ ERa– PR+ PR–

Variable No.oftumors No. % No. % No. % No. %

Tumorsize 5cm 49 28 57.1 21 42.9 43 87.8‡ 6 12.25cm 40 17 42.5 23 57.5 21 52.5 19 47.5 Clinicalstage* I,II,orIII 52 31 59.6‡ 21 40.4 43 82.7‡ 9 17.3IVorV 37 14 37.8 23 62.2 21 56.8 16 43.2 Metastasistolymphnodes Yes 36 13 36.1‡ 23 63.9 20 55.6‡ 16 44.4No 53 32 60.4 21 39.6 44 83.0 9 17.0 Distantmetastasis Yes 21 6 28.6‡ 15 71.4 12 57.1 9 42.9No 68 39 57.4 29 42.6 52 76.5 16 23.5 Clinicalstage I 24 17 70.8 7 29.2 21 87.5 3 12.5IIorIII 53 28 52.8 25 47.2 37 69.8 16 30.2 Tubuleformation† Score1 34 23 67.6‡ 11 32.4 27 79.4‡ 7 20.6Score2 26 16 61.5 10 38.5 22 84.6 4 15.4Score3 17 6 35.3 11 64.7 9 52.9 8 47.1 Mitoticindex† Score1 29 18 62.1 11 37.9 23 79.3‡ 6 20.7Score2 40 25 62.5 15 37.5 32 80.0 8 20.0Score3 8 2 25.0 6 75.0 3 37.5 5 62.5

*ScorebasedontheTNMsystem.†Scorebasedonahistologicgradingsystem.20,21‡Significant(P0.05)associationbetweentheinvestigatedvariableandexpressionofthespecifichormonereceptor.

ERa–=NoexpressionofERa.PR–=NoexpressionofPR.SeeTable1forremainderofkey.

Table 2—Relationship between hormone receptor expression and features of malignant mammary gland tumors of dogs.

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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 pyo-metra 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 fibrosar-coma). 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 posi-tive control samples. Expressions of ERa were in the nuclei of mucosal epithelial cells, stromal fibrocytes, and smooth muscle cells of the uterine horns; granulo-sa 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 le-sion had moderate to strong staining. Compared with neoplastic cells that had positive staining of various in-tensities, 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, ex-cept 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—Detec-tion 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 be-nign mammary gland tumors had positive results when tested for ERa and PR expression. However, percent-ages 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 affect-ed 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%]) with-

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 hor-mone receptor expression and characteristics of ma-lignant 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 expres-sion. With regard to PR expression, the percentage was significantly higher in malignant tumors < 5 cm in di-ameter, 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 mam-mary 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 surgi-cal 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.

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Comparison of survival in dogs with malignant mammary gland tumors on the basis of hormone re-ceptor expression—Kaplan-Meier survival analysis and log-rank tests were used to investigate the relation-ship 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 ex-pression (52/63 [82.5%]), compared with the propor-tion 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 vari-ous 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 like-lihood 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 indi-cated that expression of ERa or PR was significantly associated with tumor size, clinical stage, and metas-tasis to lymph nodes or distant metastasis, which was similar to results in another report.6 Furthermore, hor-mone 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 hor-mone receptor expression plays an important role in

predicting the biological behavior of breast cancer in humans and mammary gland neoplasia in dogs. How-ever, in the study reported here, analysis of survival curves revealed that only PR expression was signifi-cantly 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 obser-vations 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 find-ings 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%). Fur-thermore, 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 differentia-tion 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 ex-pression 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 be-cause PR expression is modulated by ERa and ERa ex-pression 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. How-ever, 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 func-tional 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 can-cers, 51.5% of patients whose tumors had expression of both ER and PR responded to tamoxifen treatment; how-ever, only 16.7% of patients whose tumors had expres-sion 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,

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]).

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PR expression may play an important role for use in pre-dicting 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 pyo-metra, 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 signifi-cantly 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 sta-tus. Furthermore, expression of PR was associated with clinical stage, tumor size, metastasis status, tubule for-mation, and mitotic index. Of the hormone receptors evaluated, only PR was significantly associated with postoperative survival in dogs with malignant mamma-ry 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. Clone 1D5, DakoCytomation, Glostrup, Denmark.b. Clone PR10A9, Immunotech, Marseille, France.c. EnVision+Dual Link system, DakoCytomation, Glostrup, Denmark.d. HRP(DAB+), DakoCytomation, Glostrup, Denmark.e. IHC Select immunoperoxidase secondary detection system,

Chemicon (Millipore), Temecula, Calif.f. Muto Pure Chemicals Co, Tokyo, Japan.g. SPSS, version 15.0, SPSS Inc, Chicago, Ill.

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