Introduction

Mammary tumors are among the most common neoplastic processes in female dogs and 50% of them are malignant (Morrison 1998, Withrow & Mac Eween 2001). Prognosis is directly related to factors such as tumor size, lymph node involvement, presence of distant metastasis, histological type, histological grade, nuclear differentiation level, invasion level, intravascular growth and presence of estrogen receptors. Large tumors (> 3.0 cm) with lymph node metastases indicate a worse prognosis; approximately 80% of patients show recurrence six months after treatment, leading to short overall and disease-free survival (Cavalcanti & Cassali 2006, Withrow & Mac Eween 2001). Cox-2 is an inducible enzyme that interferes with tumor development and angiogenesis. During cancer progression, in participates in arachidonic acid metabolism, generating prostaglandins that can mediate various mechanisms including cellular proliferation, apoptosis, immune system modulation and angiogenesis (Grosch et al 2006). In human medicine, Cox-2 expression in colon and breast cancer is increased under preneoplastic conditions and in more aggressive neoplasias. Carcinomas with increased Cox-2 expression have been correlated with worse prognosis for women with breast cancer (Costa et al 2002). Malignant canine mammary tumors express Cox-2 more strongly than benign tumors, irrespective of histological type. However, the precise implication of this for animal survival has not yet been studied (Dore et al 2003). Angiogenesis is essential for the growth and metastasis of major solid tumors. High levels of angiogenic factors and histological evidence of increased tumor neovascularization through microvessel density measurement are considered to have important prognostic value in human medicine for various solid tumors (Folkman 1974, Sorenmo 2003). Many authors correlate the capillary microvessel index with prognosis in human breast cancer (Gasparini et al 1998, Gasparini 2000, Heimann et al 1998, Leek 2001) and recent studies have reported similar results in veterinary medicine (Queiroga & Lopes 2002). The aim of the present study was to evaluate Cox-2 expression and microvessel density in canine mammary carcinomas.

Materials and Methods

Mammary tumor samples from female dogs were obtained after surgical removal in the Veterinary Teaching Hospital of the Federal University of Minas Gerais, Brazil. The samples were fixed in 10% neutral formalin and embedded in paraffin. Histological diagnosis was conducted on HE-stained sections according to the World Health Organization's diagnostic criteria for dog mammary gland tumors (Misdorp et al 1999). We used human diagnostic criteria and human classification in order to compare lesions in the two species (Dutra et al 2004). After revision by two pathologists, 46 malignant tumor samples (27 carcinomas in mixed tumors, 11 tubular carcinomas and 8 solid carcinomas) were selected for immunohistochemical and survival analysis. The tubular and solid carcinomas were combined to form the ductal carcinoma group and the carcinomas in mixed tumors formed the metaplastic group. Sections of 4μm were cut from one representative block for each case and collected on gelatin-coated slides. The slides were incubated at 4°C for 16 h with a primary rabbit monoclonal anti-human Cox-2 antibody (SP21, 1:10, Lab Vision) and an anti-human Cd31 mouse monoclonal (JC70A, 1:50, DAKO) (Milanta et al 2006), followed by the EnVision polymer HRP and Envision+ (DAKO, SA, Denmark) for 1 h at 37°C. The sections were stained with the chromogen 3.3-diaminobenzidine tetrahydrochloride (DAB Substrate System, Lab Vision), incubated for 10 min and counterstained with Mayer's hematoxylin. Positivity for Cox-2 was indicated by cytoplasmic staining. The number of Cox-2 positive cells was evaluated semiquantitatively with the distribution score defined by the estimated percentage of positive cells in five fields (Dore et al 2003, Heller et al 2005). CD31 positivity was indicated by membrane staining. Microvessels were counted in the three most vascularized areas, known as hot spots, in 200 magnification fields, from which the median was obtained (Weidner et al 1991). To determine whether Cox-2 staining intensity, distribution score, total score, staining indices and microvessel density differed by histological subtype of tumor (metaplastic carcinoma vs. ductal carcinoma), a Mann-Whitney test was performed. An unpaired Student's t-test was performed to detect statistically significant differences in microvessel density between metaplastic and ductal carcinomas. The tests were two-sided, and a P value of less than 0.05 was considered statistically significant. The correlation between Cox-2 staining and microvessel density or tumor type was assessed using a Spearman's rank correlation coefficient test (Sampaio 1998).

Results

Of the 46 cases analyzed, 27 were metaplastic carcinomas and 19 ductal carcinomas. The results showed that Cox-2 was not expressed in normal canine mammary gland. Immunohistochemical analysis revealed some degree of positivity in 100% (46/46) of the tumors examined. In both metaplastic carcinomas and ductal carcinomas, Cox-2 expression was located in the tumor cell cytoplasm. Immunohistochemistry for Cox-2 revealed that 74% (20/27) of the metaplastic carcinomas had a final score of 0-5 and 26% (7/27) of 6-12. Of the 19 ductal carcinomas, 53% (10/19) showed a Cox-2 final score of 0-5 and 47% (9/19) of 6-12. Ductal carcinomas stained more intensely for Cox-2 than metaplastic carcinoma (P = 0.0221). However, analysis of the distributions and total scores showed no statistical significance among tumor types (P = 0.0914). CD31 immunohistochemistry revealed positive staining in 46/46 (100%) of the tumors. CD31 expression did not differ significantly among tumor types (P = 0.747). Cox-2 protein expression correlated positively with CD31 staining (r = 0.3742, p = 0.0104) but did not correlate significantly with tumor type.

Discussion and Conclusions

We suggest that microvessel density and angiogenesis could be a sensitive indicator of malignancy in veterinary medicine. The increased expression of VEGF in canine mammary tumors indicates a higher risk of development and dissemination of the disease. In this study, we chose to compare the three histological types of canine mammary tumors. Carcinomas in mixed tumors are characterized by carcinomatous transformation of epithelial components in benign mixed tumors, corresponding morphologically to the matrix-producing human female breast carcinoma. Tubular and solid carcinomas, simple or complex, correspond morphologically to ductal carcinomas in women (Cavalcanti & Cassali 2006, Misdorp et al 1999, Wargotz & Norris 1989). We suggest that the small difference between Cox-2 scores in the ductal carcinoma group may be related to the differentiated behavior of these tumors, confirming the association between the Cox-2 expression and disease aggressiveness. Our results demonstrate that increased microvessel density and increased Cox-2 expression were correlated in the canine mammary tumors studied.

References

1.  Morrison WB. 1998. Canine and feline mammary tumors, p. 591-598. In: Morrison W.B., Cancer in dogs and cats; medical and surgical management, 1st ed. Lippincott Willians & Wilkins, Philadelphia.

2.  Withrow SJ, Mac Eween EG. 2001. Tumors of the mammary gland, pp. 455-77. In: Withrow SJ & Mac Eween EG. Small Animal Clinical Oncology, 3rd ed. W. B. Saunders Company, Philadelphia.

3.  Cavalcanti MF, Cassali GD. 2006. Fatores prognósticos no diagnóstico clínico e histopatológico dos tumores de mama em cadelas--revisão. Clínica Veterinária 11:56-64.

4.  Grosch S, Maier TJ, Schiffmann S, Geisslinger G. 2006. Cyclooxygenase-2 (Cox-2). Independent anticarcinogenic effects of selective Cox-2 inhibitors. J. Natl. Cancer Inst. 98:736-747.

5.  Costa C, Soares R, Reis Filho JS, Leitão D, Amendoeira I, Schmitt FC. 2002. Cyclo-oxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer. J. Clin. Pathol. 55:429-434.

6.  Doré M, Lanthier I, Sirois J. 2003. Cyclooxygenase-2 expression in canine mammary tumors. Vet. Pathol. 40:207-212.

7.  Folkman J. 1974. Tumor angiogenesis factor. Cancer Res. 344:2109-2113.

8.  Sorenmo K. 2003. Canine mammary gland tumors. Vet. Clin. N. Am. Small Anim. Pract. 33:573-596.

9.  Gasparini G, Locopo N, Fanelli M. 1998. Clinical significance of angiogenic factors in breast cancer. Breast Cancer Res. Treat. 52:159-173.

10. Gasparini G. 2000. Prognostic value of vascular endothelial growth factor in breast cancer. Oncologist 5:37-44.

11. Heimann R, Ferguson D, Gray S, Hellman S. 1998. Assessment of intratumoral vascularization (angiogenesis) in breast cancer prognosis. Breast Cancer Res. Treat. 52:147-158.

12. Leek RD. 2001. The prognostic role of angiogenesis in breast cancer. Anticancer Res. 21:4325-4331.

13. Queiroga F, Lopes C. 2002. Tumores mamários caninos, pesquisa de novos fatores prognósticos. RPCV 97:119-127.

14. Misdorp W, Else RW, Hellmén E, Lipscomb TP. 1999. Histological classification of mammary tumors of the dog and the cat, 2nd series, v. VII. Washington DC, Arm. Forc. Inst. Pathol. American Registry of Pathology and the World Health Organization Collaborating Center for Worldwide Reference on Comparative Oncology, p.1-59.

15. Dutra AP, Granja NVM, Schimitt FC, Cassali GD. 2004. C-erb-2 expression and nuclear pleomorphism in canine mammary tumors. Braz. J. Med. Biol. Res. 37:1-9.

16. Millanta F, Citi S, Della Santa D, Porciani M, Poli A. 2006. Cox-2 expression in canine and feline invasive mammary carcinomas: correlation with clinicopathological features and prognostic molecular markers. Breast Cancer Res. Treat. 98:115-120.

17. Heller DA, Clifford CA, Goldschimidt MH, Holt DE, Shofer FS, Smith A, Sorenmo KU. 2005. Cyclooxygenase-2 expression is associated with histologic tumor type in canine mammary carcinoma. Vet. Pathol. 42:776-80.

18. Weidner N, Semple JP, Welch WR, Folkman J. 1991. Tumor angiogenesis and metastasis correlation in invasive breast cancer. New England J. Med. 334:1-8.

19. Sampaio IBM. 1998. Estatística aplicada à experimentação animal, 1a ed. Ed. Fundação de Ensino e Pesquisa em Medicina Veterinária e Zootecnia, Belo Horizonte, Brazil, 221 p.

20. Wargotz ES, Norris HJ. 1989. Metaplastic carcinomas of the breast I matrix-producing carcinoma. Hum. Pathol. 20:628-635.