Malignant mammary tumors: Biologic behavior, prognostic factors, and therapeutic approach in cats

Mammary tumors are the third most common feline cancer,1-3 accounting for 10.3% to 12% of all diagnosed tumors.1,2,4 Female cats are more frequently affected than males, with 25.4 out of 100,000 queens developing mammary tumors.5 Mammary neoplasia in male cats is rare; less than 1% of cats with mammary neoplasia are male.6 Regardless of a cat's sex, mammary tumors most commonly arise in older cats (average age 10 to 12 years). However, the diagnosis of feline mammary tumors is not restricted to geriatric patients, as mammary cancer in cats as young as 9 months old has been reported.1,6-11

BIOLOGIC BEHAVIOR

Most cats have four sets of mammae, and malignant tumors most commonly arise from the thoracic and inguinal glands.7,12 Malignant mammary tumors readily spread to ipsilateral regional lymph nodes, and sites of local metastases are dictated principally by lymphatic drainage patterns. Thoracic mammary gland tumors drain to the axillary lymph nodes, and inguinal mammary gland tumors drain to the superficial inguinal lymph nodes. The cranial and caudal abdominal mammary glands can drain bidirectionally, so tumors arising from these sites may spread to both axillary and superficial inguinal lymph nodes. Additionally, mammary tumors developing within the thoracic glands or the cranial or caudal abdominal glands may also drain to the cranial sternal lymph nodes.

Recent evidence suggests that mammary tumors may not spread to adjacent mammary glands or contralateral lymph nodes through the lymphatics, as no interglandular lymphatic connections have been identified.13 However, because both mammary chains share a common venous system, the seeding of neoplastic cells between contralateral mammary chains is anatomically possible.7

Common metastatic sites include the lungs (diffuse or nodular metastasis) (Figure 1) and regional lymph nodes, but metastases may also occur in the liver, spleen, brain, and bone.4-8,14-17 Regional lymph node metastasis is reported to be present in more than 25% of cats at diagnosis.4,12 At necropsy, 76% of cats have pulmonary metastasis and 40% have pleural metastasis,12 with up to 93% of cats having one or more sites of metastasis (lymph nodes, lungs, pleura, liver).18

Figure 1. A lateral thoracic radiograph of a female cat presenting for evaluation of inappetence, behavioral changes, and rapid breathing. Radiographic findings are consistent with diffuse pulmonary metastasis (diffuse infiltrates) from a relatively large (> 3 cm) primary mammary tumor (white arrow).

PATHOLOGY

About 90% of feline mammary tumors are malignant.6,7,15,19 Most are carcinomas or adenocarcinomas, with the most common histologic patterns being tubular, papillary, and solid carcinomas.6-8,20 Tumors are graded as well-differentiated (Grade I), moderately differentiated (Grade II), and poorly differentiated (Grade III) based on histologic features including tubule formation, nuclear and cellular atypia, and mitotic index.21 Histologically, invasion into the lymphatic or vascular system or both is common—noted in 27% to 57% of tumor samples.12,22,23 Additionally, infiltration of cancer cells into the surrounding stroma occurs at an even greater frequency, with a reported rate of 42% to 88%.12,23 Although most feline mammary tumors are of epithelial origin, malignant transformation may occur in mesenchymal tissues resulting in the development of mixed mammary tumors and sarcomas.8,21

ETIOLOGY AND RISK FACTORS

The etiology of feline mammary tumors remains poorly defined, but several known risk factors have been recognized (Table 1).

Table 1 Risk Factors for Mammary Cancer in Cats

Biological carcinogens

Biological carcinogens, such as viruses, have been evaluated in feline mammary tissues. Although viral particles have been identified within the tissues, their presence has not been directly linked with tumor development.24,25

Genetic factors

Familial or genetic predispositions for developing feline mammary carcinoma have also been investigated. Both Siamese and Persian cats appear to develop mammary neoplasia frequently, representing up to 34% and 16% of the affected population, respectively.7,16,26,27 In addition to Siamese cats being overrepresented, mammary neoplasia appears to occur at a younger age (9 years for Siamese vs. 14 years for non-Siamese) in this popular Oriental breed.6

Sex hormones

The role of sex hormones in mammary neoplasia development remains to be clearly elucidated, but several studies underscore the probable involvement of estrogen and progesterone in mammary gland tumorigenesis. Long-term progestin administration and endogenous progesterone increase the risk of both benign and malignant mammary tumor development, while intermittent or occasional progestin administration has no effect.5,11,28,29 Furthermore, although the relevance to tumor development could not be determined in a recent clinical evaluation, eight of 22 male cats with mammary tumors were reported to have received progestin treatments.10

Reproductive status

In addition to progesterone, intact sexual status also influences the incidence of mammary tumor development, with early ovariohysterectomy providing a protective effect.6,28 A recent study showed that intact cats were 2.7 times more likely to develop mammary carcinoma, and the age of queens at neutering was important. Spaying before 6 months, 12 months, and 24 months of age results in a 91%, 86%, and 11% risk reduction in mammary tumor development, respectively.30 However, spaying after two years of age does not alter the risk of developing mammary tumors, and parity has no effect.11,30

Estrogen and progesterone receptor expression

Estrogen receptor (ER) and progesterone receptor (PR) expressions in mammary carcinomas are routinely evaluated in human breast cancer patients, with ER- and PR-positive tumors being associated with a more favorable prognosis. Almost one in eight women will develop breast cancer, and it is the second-leading cause of cancer death in women.31

Given the biologic similarities of human and feline breast carcinomas, recent studies have investigated ER and PR status in feline mammary tumors. Compared with 76% ER expression in normal tissue and 25% to 40% ER expression in dysplastic and benign tumors, feline mammary carcinomas are predominantly ER-negative (56% to 90%).31-33 However, PR expression is variable in normal tissues as well as in benign lesions and malignant mammary carcinomas.33-35 Collectively, these findings demonstrate that ER and PR expression varies in feline mammary carcinomas, perhaps reflecting the undifferentiated state and, hence, aggressive biologic behavior of mammary tumors in cats.

HER2 expression

In human breast carcinoma, the human epidermal growth factor receptor 2 gene (HER2/neu) is overexpressed in 10% to 40% of patients.23 This overexpression of the HER2 receptor is permissive for uncontrolled cell growth and facilitates tumor development. In human breast carcinoma patients, increased HER2 expression confers a poor prognosis and may predict limited response to hormonal therapy. The overexpression of the HER2 protein is variable in canine mammary tumors (37% to 73% positive) but confers a poor prognosis.36,37

Because feline mammary tumors generally behave aggressively, identifying HER2 overexpression in these tumors may account for the poor prognosis associated with this tumor type. Recent investigations have demonstrated HER2 expression to be low (25%) in benign mammary tumors and absent in nontumor samples.38 In contrast, 41% to 90% of feline mammary carcinomas express HER2.22,23,38

In addition to its identification in mammary carcinomas, HER2 expression appears prognostic for survival time. Queens with greater HER2 expression treated with surgery alone had a shorter median survival time (14.6 months) than HER2-negative queens (18.7 months). Interestingly, HER2 was not correlated with histologic subtype, tumor grade, or lymphatic invasion, suggesting that HER2 expression may serve as a prognostic variable independent of other histologic criteria in cats with mammary tumors.23

CLINICAL SIGNS

Cats with mammary neoplasia often present with multiple tumors, and bilateral mammary gland involvement may be identified in up to 40% of patients at diagnosis.4,8 Bleeding and ulceration of the affected mammary glands may be observed in 18% to 25% of cats with large or long-standing tumors (Figure 2).4,7,14 Pet owners may note nonspecific signs such as weight loss, inappetence, and lethargy. Less commonly, exercise intolerance, dyspnea, or cyanosis caused by diffuse pulmonary metastasis may be the presenting complaint.15

Figure 2. A geriatric female cat suffering from painful and ulcerative bilateral mammary gland carcinoma. (Photo courtesy of Craig Clifford.)

DIAGNOSIS AND STAGING

A palpable mass that is not freely movable or attached to underlying structures and is associated with the mammary chain is highly suggestive of a mammary tumor; however, your differential diagnoses should include mastitis and mammary fibroepithelial hyperplasia, a benign condition in young, cycling females or animals receiving progestin therapy (Figure 3). Mammary fibroepithelial hyperplasia can result in marked nonpainful enlargement of all mammary tissue and is treated effectively with ovariohysterectomy or discontinuation of progestin therapy.

Figure 3. A young pregnant queen with benign fibroepithelial hyperplasia, presenting with massive bilateral mammary gland enlargement, pain, and ulceration. This condition is often mistaken for mammary cancer.

Obtain a thorough history, including information regarding the duration of clinical signs, rate of growth, spay status, age at ovariohysterectomy, and the use of progestins. During the physical examination, identify the tumor size and location, number of affected glands, evidence of tumor ulceration and fixation to underlying tissues, lymph node enlargement, and evidence of distant metastasis.21 Make a definitive diagnosis with fine-needle aspiration biopsy and cytology (Figure 4) or incisional biopsy. Cytologic examination may yield false negative results, so perform a histologic examination to confirm malignancy.

Figure 4. Cytologic examination of pleural effusion collected from a female cat with mammary adenocarcinoma. Carcinoma cells are characterized by ballooning cytoplasm (Wrights-Giemsa stain; 500X).

Clinical staging with a modified TNM (tumor, nodes, metastasis) staging system (Table 2) can help you determine the prognosis and help guide your treatment decisions. Perform a complete blood count, serum chemistry profile, urinalysis, three-view thoracic radiographic examination, abdominal ultrasonographic examination, and cytologic examination of regional lymph node aspirates.

Table 2 Staging of Feline Mammary Tumors*

TREATMENT

A combination of surgery and chemotherapy is standard for treating malignant mammary tumors in cats, but palliative care, such as with radiation therapy or analgesics, is also important.

Surgery

Bilateral or unilateral radical mastectomy (as indicated based on lymphatic drainage) is the recommended surgical treatment for feline mammary carcinoma. Bilateral mastectomy often requires two separate staged surgeries to allow for adequate closure and healing of each surgical site (Figure 5). Radical surgical intervention results in an increased disease-free interval (575 to 1,300 days) compared with more conservative surgical approaches (300 to 325 days).4,39

Figure 5. An 8-year-old female Siamese cat recovering from the second surgery of a staged bilateral mastectomy performed 10 days earlier.

Although surgery is often beneficial for controlling local and regional disease, given the aggressive metastatic behavior of feline mammary carcinomas, surgery alone should not be considered curative. In fact, surgery alone achieves only a one-year survival rate of 47% to 50% and two-year survival rate of 32% in cats.9,23,40,41 Local tumor recurrence at the surgery site or regional metastasis has been reported to occur at a median of 5.5 months in 51% to 66% of cats undergoing surgical resection of mammary carcinomas.15,19

Less aggressive palliative surgery may be considered in patients with advanced disease.

Chemotherapy

The results of several studies suggest that systemic adjuvant chemotherapy may be useful for treating mammary carcinoma after surgical excision as well as for providing pain relief in cats with nonresectable tumors. However, most clinical studies to date have been limited by their retrospective nature, small sample size, patient selection bias, variability in treatment protocols, and stage of disease treated. These confounding factors prevent discernment in treatment efficacy among various chemotherapy regimens.

Doxorubicin-based protocols are most common. In one study, single-agent doxorubicin improved the median survival time in cats with more advanced disease (stage III) compared with historical controls.42 Similarly, another retrospective study evaluating surgery and adjuvant doxorubicin reported a disease-free interval and median survival time of 255 and 448 days, respectively.43

Combining doxorubicin with cyclophosphamide has also been evaluated in both adjuvant and palliative settings. For this combination, the reported response rate for nonresectable or metastatic disease is 35% to 50%, with complete remission in 21% of patients.44,45 In addition, cats that responded to combined doxorubicin and cyclophosphamide therapy had a longer median survival time (150 to 180 days) than did those patients unresponsive to therapy (75 to 86 days).44,45 Despite the apparent efficacy of combination doxorubicin and cyclophosphamide, this treatment protocol can cause transient gastrointestinal side effects and other adverse complications, including azotemia, cardiac abnormalities, leukopenia, and anemia.44,45

Another antineoplastic agent that has been evaluated to treat mammary carcinoma belongs to the platinum family. The adjuvant use of carboplatin has been anecdotally reported to provide a median duration of remission of 436 days and median survival time of 535 days, with 40% of cats alive at two years.46 The single-agent use of carboplatin appears to be well-tolerated, with only mild to moderate hematologic and gastrointestinal toxicity noted in treated cats.46

In summary, given the high metastatic potential of feline mammary carcinomas, the adjuvant use of systemic chemotherapy after radical mastectomy is recommended to maximize survival times. Additionally, systemic chemotherapy may have a limited role for the palliative management of macroscopic, nonresectable primary tumors, but future prospective studies should be performed.

Other treatment options

Although surgery and systemic chemotherapy are considered the cornerstones of therapy, other treatment modalities may also benefit feline cancer patients.

Attempting to harness the host's immune response against feline mammary carcinoma has been attempted, but response to treatment with biologic response modifiers has been poor.47,48

Palliative radiation therapy likely has a role in providing pain relief from nonresectable, ulcerative mammary carcinomas, as well as in delaying local regrowth from residual microscopic disease.

The judicious use of analgesics should be considered for patients suffering from large bulky disease, ulcerated tumors, or painful metastases, such as to bone. Although many analgesics are considered off-label for use in cats, medications such as nonsteroidal anti-inflammatory drugs and opioids may be beneficial for the supportive management of these advanced-stage patients (see "Understanding and recognizing cancer pain in dogs and cats" and "Treating cancer pain in dogs and cats" in the May 2005 issue of Veterinary Medicine).

PROGNOSIS

Without treatment, cats are likely to die of their disease within a year.5,39 The most important prognostic factors include tumor size, extent of surgery (when comparing radical with conservative surgery), histologic grade, and lymph node metastasis (Table 3).

Table 3 Selected Prognostic Factors for Mammary Cancer in Cats

Tumor size

Tumor size has been the most important prognostic factor for recurrence and survival in most studies.12,16,39,48,49 In one sentinel investigation, cats with smaller tumors treated with surgery alone had longer disease-free intervals and median survival times than did cats with tumors larger than 2 cm in diameter (Table 3).39 A comparable relationship is also observed in male cats with mammary carcinoma, with a median survival time of 14 months for tumors less than 2 cm in diameter and a median survival time of less than two months for tumors greater than 3 cm.10 These collective findings advocate early surgical intervention in any cat presenting with a mammary mass.

Metastasis

Clinical evidence of distant metastasis is a negative prognostic factor.12,26 Pulmonary metastasis is the most common cause of mammary-carcinoma–related deaths, and cats presenting with advanced lung involvement at diagnosis have a reported median survival time of only one month (Figure 6).8,16 The location of metastatic disease is a significant prognostic factor for both disease-free interval and median survival time, with cats with metastatic disease in regional lymph node, pulmonary, and pleural anatomic sites having a median survival time of 1,543, 332, and 188 days, respectively.43 In addition to the negative impact of distant metastasis, it has been anecdotally reported that cats with histologic evidence of lymphatic invasion have a median survival time of seven months compared with 18 months in cats without lymphatic invasion.50 Likewise in male cats with mammary carcinoma, lymphatic invasion is also a negative prognostic factor, with a median survival time of 195 days vs. 863 days in tumors positive or negative for lymphatic invasion, respectively.10

Figure 6. A lateral thoracic radiograph of a cat presenting for evaluation of dyspnea of one weeks duration. Past pertinent history included surgical resection of a mammary tumor nine months earlier. Radiographic findings are consistent with moderate to severe pleural fluid accumulation and multiple soft tissue metastatic lesions (white arrows).

Histologic criteria

Histologic grade appears to be inversely correlated with prognosis and survival time. Although low-grade tumors are less common (13% prevalence), most cats with low-grade tumors survive for more than one year, while only 10% of cats with high-grade tumors are alive one year after surgical resection alone.41 Histologic subtype is prognostic for disease-free interval, with papillary or tubular, ductular, and anaplastic tumors having a disease-free interval of greater than 1,131, 306, and 95 days, respectively.43

In addition to tumor grade, the prognostic utility of other histologic criteria has been evaluated individually. Proliferative indices, including proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizer regions (AgNORs), and Ki-67 nuclear antigen, have been evaluated in feline mammary carcinomas. PCNA was higher in malignant tumors, reflecting greater mitotic activity.51 In two other clinical studies, increased AgNOR counts in malignant mammary tumors correlated with shorter postsurgical survival times.9,26 However, the relevance of Ki-67 nuclear antigen in feline mammary malignancies is unclear, as there is an inconsistent relationship between Ki-67 expression and either clinical outcome or grade.40,52,53 Thus, feline mammary tumors with greater mitotic activities, as reflected by greater PCNA and AgNOR counts, may possess more aggressive biologic behaviors.

Finally, similar to human breast cancer patients, the histologic identification of HER2 protein expression in cats with mammary carcinoma has also been identified as a negative prognostic factor.23

Molecular markers

Other factors associated with malignant transformation and clinical outcome have been evaluated, including various molecular markers. Both vascular endothelial growth factor (VEGF), an angiogenic factor involved in new blood vessel formation, and tumor microvessel density are routinely used to assess neoplastic angiogenesis. While mammary tumor microvessel density is not associated with survival or prognosis, increased mammary carcinoma VEGF expression has been associated with a worse clinical outcome.27 Aberrant expressions of other molecular markers that do not appear to provide direct prognostic information in feline mammary carcinoma include reduced E-cadherin, overexpression of bcl-2, and mutations in p53.54-58 These diagnostic tools are being evaluated in a research setting.

SUMMARY

Malignant mammary neoplasia is a common tumor affecting cats, with a well-described clinical course and documented risk factors. Although the efficacies of adjunctive treatments such as chemotherapy, radiation therapy, and immunotherapy are being investigated, the mainstay of current treatment is aggressive surgical intervention. Accepted prognostic factors include tumor size, extent of surgery, histologic grade, and presence of metastatic disease. Current research focuses on delineating the biologic pathways involved in malignant transformation in the hopes of providing new preventive, diagnostic, and therapeutic options for our feline patients.

Jackie Wypij, DVM

Timothy M. Fan, DVM, DACVIM (oncology, internal medicine)

Louis-Philippe de Lorimier, DVM, DACVIM (oncology)

Department of Veterinary Clinical Medicine

College of Veterinary Medicine

University of Illinois

Urbana, IL 61802

REFERENCES

1. Schmidt RE, Langham RF. A survey of feline neoplasms. J Am Vet Med Assoc 1967;151:1325–1328.

2. Dorn CR, Taylor DO, Frye FL, et al. Survey of animal neoplasms in Alameda and Contra Costa Counties, California. I. Methodology and description of cases. J Natl Cancer Inst 1968;40:295-305.

3. Patnaik AK, Liu SK, Hurvitz AI, et al. Nonhematopoietic neoplasms in cats. J Natl Cancer Inst 1975;54:855-860.

4. Hayes AA, Mooney S. Feline mammary tumors. Vet Clin North Am Small Anim Pract 1985;15:513-520.

5. Dorn CR, Taylor DO, Schneider R, et al. Survey of animal neoplasms in Alameda and Contra Costa Counties, California. II. Cancer morbidity in dogs and cats from Alameda County. J Natl Cancer Inst 1968;40:307-318.

6. Hayes HM Jr, Milne KL, Mandell CP. Epidemiological features of feline mammary carcinoma. Vet Rec 1981;108:476-479.

7. Hayden DW, Nielsen SW. Feline mammary tumours. J Small Anim Pract 1971;12:687-698.

8. Weijer K, Head KW, Misdorp W, et al. Feline malignant mammary tumors. I. Morphology and biology: some comparisons with human and canine mammary carcinomas. J Natl Cancer Inst 1972;49:1697-1704.

9. Castagnaro M, Casalone C, Ru G, et al. Argyrophilic nucleolar organiser regions (AgNORs) count as indicator of post-surgical prognosis in feline mammary carcinomas. Res Vet Sci 1998;64:97-100.

10. Skorupski KA, Overley B, Shofer FS, et al. Clinical characteristics of mammary carcinoma in male cats. J Vet Intern Med 2005;19:52-55.

11. Misdorp W, Romijn A, Hart AA. Feline mammary tumors: a case-control study of hormonal factors. Anticancer Res 1991;11:1793-1797.

12. Weijer K, Hart AA. Prognostic factors in feline mammary carcinoma. J Natl Cancer Inst 1983;70:709-716.

13. Raharison F, Sautet J. Lymph drainage of the mammary glands in female cats. J Morphol 2006;267:292-299.

14. Ogilvie GK. Recent discoveries: mammary cancer and the cat, in Proceedings. 10th Annu North Am Vet Conf 1996.

15. Nielsen SW. The malignancy of mammary tumors in cats. North Am Vet 1952;33:245-252.

16. Ito T, Kadosawa T, Mochizuki M, et al. Prognosis of malignant mammary tumor in 53 cats. J Vet Med Sci 1996;58:723-726.

17. Waters DJ, Honeckman A, Cooley DM, et al. Skeletal metastasis in feline mammary carcinoma: case report and literature review. J Am Anim Hosp Assoc 1998;34:103-108.

18. Hahn KA, Bravo L, Avenell JS. Feline breast carcinoma as a pathologic and therapeutic model for human breast cancer. In Vivo 1994;8:825-828.

19. Hayes A. Feline mammary gland tumors. Vet Clin North Am 1977;7:205-212.

20. Bostock DE. Canine and feline mammary neoplasms. Br Vet J 1986;142:506-515.

21. Misdorp W. Tumors of the mammary gland. In: Mueten DJ, ed. Tumors in domestic animals. 4th ed. Ames: Iowa State Press, 2002;575-606.

22. Winston J, Craft DM, Scase TJ, et al. Immunohistochemical detection of HER-2/neu expression in spontaneous feline mammary tumours. Vet Comp Oncol 2005;3:8-15.

23. Millanta F, Calandrella M, Citi S, et al. Overexpression of HER-2 in feline invasive mammary carcinomas: an immunohistochemical survey and evaluation of its prognostic potential. Vet Pathol 2005;42:30-34.

24. Calafat J, Weijer K, Daams H. Feline malignant mammary tumors. III. Presence of C-particles and intracisternal A-particles and their relationship with feline leukemia virus antigens and RD-114 virus antigens. Int J Cancer 1977;20:759-767.

25. Weijer K, Calafat J, Daams JH, et al. Feline malignant mammary tumors. II. Immunologic and electron microscopic investigations into a possible viral etiology. J Natl Cancer Inst 1974;52:673-679.

26. Preziosi R, Sarli G, Benazzi C, et al. Multiparametric survival analysis of histological stage and proliferative activity in feline mammary carcinomas. Res Vet Sci 2002;73:53-60.

27. Millanta F, Lazzeri G, Vannozzi I, et al. Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas. Vet Pathol 2002;39:690-696.

28. Misdorp W. Progestagens and mammary tumours in dogs and cats. Acta Endocrinol (Copenh) 1991;125 Suppl 1:27-31.

29. Misdorp W, Romijn A, Hart AA. The significance of ovariectomy and progestagens in the development of mammary carcinoma in cats. [Dutch] Tijdschr Diergeneeskd 1992;117:2-4.

30. Overley B, Shofer FS, Goldschmidt MH, et al. Association between ovariohysterectomy and feline mammary carcinoma. J Vet Intern Med 2005;19:560-563.

31. Dolinsky, C. Breast cancer: The basics. Available at: www.oncolink.org/types/article.cfm?c=3&s=5&ss=33&id=8320. Accessed Aug 16, 2005.

32. Hamilton JM, Else RW, Forshaw P. Oestrogen receptors in feline mammary carcinomas. Vet Rec 1976;99:477-479.

33. Millanta F, Calandrella M, Bari G, et al. Comparison of steroid receptor expression in normal, dysplastic, and neoplastic canine and feline mammary tissues. Res Vet Sci 2005;79:225-232.

34. de las Mulas JM, van Niel M, Millan Y, et al. Immunohistochemical analysis of estrogen receptors in feline mammary gland benign and malignant lesions: comparison with biochemical assay. Domest Anim Endocrinol 2000;18:111-125.

35. de las Mulas JM, van Niel M, Millan Y, et al. Progesterone receptors in normal, dysplastic and tumourous feline mammary glands. Comparison with oestrogen receptors status. Res Vet Sci 2002;72:153-161.

36. Dutra AP, Granja NV, Schmitt FC, et al. c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors. Braz J Med Biol Res 2004;37:1673-1681.

37. Ahern TE, Bird RC, Bird AE, et al. Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines. Am J Vet Res 1996;57:693-696.

38. De Maria R, Olivero M, Iussich S, et al. Spontaneous feline mammary carcinoma is a model of HER2 overexpressing poor prognosis human breast cancer. Cancer Res 2005;65:907-912.

39. MacEwen EG, Hayes AA, Harvey HJ, et al. Prognostic factors for feline mammary tumors. J Am Vet Med Assoc 1984;185:201-204.

40. Castagnaro M, De Maria R, Bozzetta E, et al. Ki-67 index as indicator of the post-surgical prognosis in feline mammary carcinomas. Res Vet Sci 1998;65:223-226.

41. Castagnaro M, Casalone C, Bozzetta E, et al. Tumor grading and the one-year post-surgical prognosis in feline mammary carcinomas. J Comp Pathol 1998;119:263-275.

42. Mauldin GE, Mooney SC, Patnaik AK. Adjuvant doxorubicin for feline mammary adenocarcinoma, in Proceedings. 14th Annu Vet Cancer Soc Conf 1994.

43. Novosad CA, Bergman PJ, O'Brien MG, et al. Retrospective evaluation of adjunctive doxorubicin for the treatment of feline mammary gland adenocarcinoma: 67 cases. J Am Anim Hosp Assoc 2006;42:110-120.

44. Jeglum KA, deGuzman E, Young KM. Chemotherapy of advanced mammary adenocarcinoma in 14 cats. J Am Vet Med Assoc 1985;187:157-160.

45. Mauldin GN, Matus RE, Patnaik AK, et al. Efficacy and toxicity of doxorubicin and cyclophosphamide used in the treatment of selected malignant tumors in 23 cats. J Vet Intern Med 1988;2:60-65.

46. Dhaliwal RS, Kitchell BE, Hintermeister J. Efficacy of carboplatin in feline mammary carcinoma (abst), in Proceedings. 20th Annu Am Coll Vet Intern Med 2002.

47. Fox LE, MacEwen EG, Kurzman ID, et al. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine for the treatment of feline mammary adenocarcinoma—a multicenter randomized double-blind study. Cancer Biother 1995;10:125-130.

48. MacEwen EG, Hayes AA, Mooney S, et al. Evaluation of effect of levamisole on feline mammary cancer. J Biol Response Mod 1984;3:541-546.

49. Viste JR, Myers SL, Singh B, et al. Feline mammary adenocarcinoma: tumor size as a prognostic indicator. Can Vet J 2002;43:33-37.

50. Ozawa S, Minami T, Utsunomiya H, et al. Study on histopathological prognostication of mammary gland tumor in cat (97 cases), in Proceedings. 14th Annu Vet Cancer Soc Conf 2002.

51. Preziosi R, Sarli G, Benazzi C, et al. Detection of proliferating cell nuclear antigen (PCNA) in canine and feline mammary tumours. J Comp Pathol 1995;113:301-313.

52. Millanta F, Lazzeri G, Mazzei M, et al. MIB-1 labeling index in feline dysplastic and neoplastic mammary lesions and its relationship with postsurgical prognosis. Vet Pathol 2002;39:120-126.

53. Dias Pereira P, Carvalheira J, Gartner F. Cell proliferation in feline normal, hyperplastic and neoplastic mammary tissue—an immunohistochemical study. Vet J 2004;168:180-185.

54. Dias Pereira P, Gartner F. Expression of E-cadherin in normal, hyperplastic and neoplastic feline mammary tissue. Vet Rec 2003;153:297-302.

55. Madewell BR, Gandour-Edwards R, Edwards BF, et al. Topographic distribution of bcl-2 protein in feline tissues in health and neoplasia. Vet Pathol 1999;36:565-573.

56. Murakami Y, Tateyama S, Rungsipipat A, et al. Immunohistochemical analysis of cyclin A, cyclin D1 and P53 in mammary tumors, squamous cell carcinomas and basal cell tumors of dogs and cats. J Vet Med Sci 2000;62:743-750.

57. Nasir L, Krasner H, Argyle DJ, et al. Immunocytochemical analysis of the tumour suppressor protein (p53) in feline neoplasia. Cancer Lett 2000;155:1-7.

58. Mayr B, Blauensteiner J, Edlinger A, et al. Presence of p53 mutations in feline neoplasms. Res Vet Sci 2000;68:63-70.