Case Report Rapport de cas Metastatic anaplastic adenocarcinoma suspected to be of mammary origin in an intact male rabbit (Oryctolagus cuniculus) Noémie M. Summa, David Eshar, Heindrich N. Snyman, Brandon N. Lillie Abstract — A 7-year-old, intact male, pet dwarf rabbit (Oryctolagus cuniculus) was presented for a ventral abdominal subcutaneous mass. Histolopathology of the resected mass was suggestive of a mammary adenocarcinoma. Six months later, the rabbit died from severe dyspnea. Necropsy showed recurrence of the original mass with hepatic and pulmonary metastasis of the anaplastic adenocarcinoma, suspected to be of mammary origin. Résumé — Adénocarcinome anaplasique métastatique, suspecté d’origine mammaire, chez un lapin mâle intact (Oryctolagus cuniculus). Un lapin nain de compagnie mâle intact âgé de 7 ans (Oryctolagus cuniculus) a été présenté pour une masse sous-cutanée abdominale ventrale. L’histopathologie de la masse reséquée était en faveur d’un adénocarcinome mammaire. Six mois plus tard, le lapin est mort de dyspnée grave. La nécropsie a montré la récurrence de la masse originale avec des métastases hépatiques et pulmonaires de l’adénocarcinome anaplasique, suspecté d’origine mammaire.
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ammary adenocarcinomas are common in 3- to 4-year-old multiparous female rabbits, and have been associated with prolactin-producing pituitary adenomas or uterine hyperplasia and adenocarcinomas (1–3). However, to the authors’ knowledge, this tumor has never been reported before in an intact male rabbit. Mammary adenocarcinomas are rarely reported in male dogs, cats, specific male mice strains, rats, guinea pigs, and men (4–9). The purpose of this report is to present the unusual clinical and pathological findings of a metastatic anaplastic adenocarcinoma of suspected mammary origin in an intact male rabbit.
Case description A 7-year-old, 1.1 kg, intact male, pet dwarf rabbit (Oryctolagus cuniculus) was referred to the Avian and Exotics Service at the Ontario Veterinary College Health Sciences Centre for removal The Ontario Veterinary College Health Sciences Centre (Summa, Eshar) and Department of Pathobiology (Snyman, Lillie), University of Guelph, Guelph, Ontario N1G 2W1. Address all correspondence to Dr. Noémie Summa; e-mail: [email protected] Dr. Summa’s current address is Companion Avian and Exotic Animal Medicine, University of California, Davis. Dr. Eshar’s current address is Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506, USA. Dr. Snyman’s current address is Animal Health Centre, Ministry of Agriculture, Abbotsford, British Columbia V3G 2M3, Canada. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 55 / MAY 2014
(Traduit par Isabelle Vallières)
of a subcutaneous abdominal mass. The mass had appeared 5 mo earlier and had progressively grown since then. No other abnormalities were noticed by the owners. Blood tests [complete blood (cell) count (CBC) and biochemistry] were performed by the referring veterinarian and the results were unremarkable other than a mild increase in plasma potassium concentration [6.3 mmol/L, reference interval (RI): 3.5 to 6.2 mmol/L] (10) and mild hypoglycemia (4.3 mmol/L, RI: 5.5 to 8.6 mmol/L) (10). Both were considered artefactual secondary to hemolysis of the blood sample and glucose consumption by erythrocytes, respectively. Radiographs were declined by the owners. On physical examination, the rabbit was bright, alert, and responsive and all vital signs were within normal ranges. A 10 mm 3 18 mm, irregular, round, tan, and firm subcutaneous mass could be palpated in the region of the left second inguinal mammary gland. The mass was not adherent to adjacent tissues and no peripheral lymphadenomegaly was detected. Except for bilateral cataracts, no other physical abnormalities were noted. The main differential diagnoses included subcutaneous abscess or neoplasia. A fine-needle aspirate (FNA) of the subcutaneous mass was obtained. The cytological sample was of poor cellularity (, 10%) and was obscured by a background of erythrocytes, cellular debris, and fragmented nuclei. The interpretation was that of a non-diagnostic sample with hemorrhage, although infection could not be ruled out. An excisional biopsy of the subcutaneous mass was then scheduled. The rabbit was sedated with buprenorphine (Vetergesic; Champion Alstoe Animal Health, Whitby, Ontario), 0.03 mg/kg body weight (BW), IM, and midazolam (Midazolam Sandoz; Sandoz Canada, Burlington, Ontario), 0.8 mg/kg BW, IM. An intravenous catheter was placed in the right cephalic vein and hydration was maintained using lactated ringers solution at 10 mL/kg BW per hour. The rabbit was induced with sevoflurane (SevoFlo; 475
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200 mm
50 mm
Figure 1. Histological images of the the subcutaneous mammary nodule in a male dwarf rabbit (a and b). The mass was composed of solid cords and varisized islands (150 to 450 mm) of epithelial cells (solid arrow) and occasional well-differentiated acini and ducts (solid star) that often contained small amounts of either coarse granular to streaky basophilic luminal mucous and debris or homogenous eosinophilic protein rich fluid and cellular debris. Large polygonal to columnar epithelial cells often palisading along interspersed 5 to 10 mm thick fibrous connective tissue trabeculae were observed (b). Numerous, often bizarre, mitotic figures were observed (open arrows) and both mutlinucleation and nuclear gigantism were common (open star). Hematoxylin and eosin (H&E).
Abbott Laboratories, Saint-Laurent, Quebec), 8% in oxygen by facemask. Tracheal intubation with a 2.0 mm diameter tracheal tube was unsuccessful and the rabbit was maintained with sevoflurane 3% to 5% in oxygen by facemask. The rabbit was placed in dorsal recumbency and the surgical site was prepared using a routine aseptic protocol. A 3-cm elliptical skin incision was made around the mass, which was completely removed. Subcutaneous tissues were apposed using 4-0 Polydioxanone (PDS II; polydioxanone; Ethicon, Somerville, New Jersey, USA) in a simple continuous pattern and the skin was closed using 4-0 PDS with an intradermal suture in a simple continuous pattern. The mass was fixed in 10% neutral buffered formalin and submitted for histopathology. Sedation was partially reversed with flumazenil (Flumazenil Sandoz; Sandoz Canada), 0.03 mg/kg BW, SQ, and the rabbit recovered uneventfully from the procedure. Meloxicam (Metacam; Boehringer Ingelheim, Burlington, Ontario), 0.5 mg/kg BW, SQ, and penicillin G (Penicillin G Procaine; Pfizer Animal Health, Kirkland, Quebec), 50 000 IU/kg BW, SQ, were administrated. The rabbit was sent home the next day with meloxicam, 0.5 mg/kg BW, PO, q12h for 7 d. Histopathology of the resected mass revealed an infiltrative, unencapsulated, multi-lobular, densely cellular neoplasm expanding the subcutaneous tissue and deep dermis. The mass was composed of solid cords and islands ranging from 150 mm to 450 mm of large polygonal to columnar epithelial cells often palisading along interspersed 5 mm to 10 mm thick fibrous connective tissue trabeculae with occasional scattered acinar structures (Figures 1a, 1b). Acini were often filled with basophilic mucus, cellular debris and homogenous eosinophilic protein rich fluid. Cell margins were distinct and cells contained a large amount of finely granular eosinophilic cytoplasm with 476
large oval nuclei, coarsely stippled chromatin, and an occasional prominent central basophilic nucleolus. Nuclear gigantism and multinucleation were common, with up to five-fold anisokaryosis and up to 22 nuclei per neoplastic cell. The mitotic rate was high, with an average of 7.2 and a maximum of 15, occasionally bizarre, mitotic figures per 4003 high power field (HPF) (Figure 1b). Often large central foci of necrosis and hemorrhage were scattered throughout the mass. Numerous small satellite nodules were observed around the periphery of the main mass. There was no histological evidence of lymphatic invasion or obstruction; however, neoplastic cells extended to within 30 mm of both the deep and lateral surgical margins. In order to evaluate the epithelial phenotype of this neoplasm immunohistochemistry (IHC) was performed to detect pancytokeratin (AE1/AE3), cytokeratin 7 (CK7), and thyroid transcription factor 1 (TTF1). Processed 4-mm sections from formalin-fixed, paraffin-embedded tissues were mounted on silane-treated glass slides, deparaffinized, and rehydrated prior to the IHC procedure and antigen retrieval was carried out as outlined in Table 1. Sections of normal rabbit tissues known to contain the antigen of interest as well as tissues on the test slides were used as species-specific internal controls. For negative reagent controls, duplicate sections of each control and test tissue were subjected to the same IHC procedure with substitution of non-immune rabbit serum, at similar concentration, for the specific primary antiserum/antibody. Approximately 90% of neoplastic cells showed strong positive cytoplasmic immunoreactivity for both the pancytokeratin and CK7 antibodies. The IHC procedures for TTF1 did not result in consistent labelling of the external and internal species-specific positive control tissues; as such, TTF1 expression could not be evaluated. The interpretation was that of a poorly differentiated, anaplastic, CVJ / VOL 55 / MAY 2014
Table 1. Antibodies and their source, pretreatment and visualization methods used in immunohistochemistrya Antigen
Antibody source, clone, and dilution
Epitope retrieval method
Platform
Pan-Cytokeratin Dako — mouse monoclonal pH 6 AE1 AE3, 1:100
Goat anti-mouse horseradish peroxidase-labelled polymer (EnVision 1, Dako)
Dako Autostainer
Cytokeratin 7 Dako — mouse monoclonal, Enzyme OV-TL 12/30, 1:100 (proteinase K, Dako)
Goat anti-mouse horseradish peroxidase-labelled polymer (EnVision 1, Dako)
Dako Autostainer
a Processed
4-mm sections from formalin-fixed, paraffin-embedded tissues were mounted on silane-treated glass slides, deparaffinized, and rehydrated prior to immunochemistry. epitope retrieval.
b Heat-induced
Figure 2. Subcutaneous tissue and mammary chain in a male dwarf rabbit observed at necropsy. A 10 mm 3 18 mm, wellcircumscribed, firm, tan, subcutaneous mass (star) was present within the subcutaneous tissue along the left mammary chain, approximately 2 cm caudal to the axilla, and in the same location as the original tumor resection.
solid adenocarcinoma suspected to be of mammary epithelial origin based on anatomic location, histological appearance, and expression of CK7. As CK7 is a marker of ductular origin, apocrine adenocarcinoma could not be ruled out, although this tumor diagnosis was considered less likely in this case. Given the nature of this neoplasia and with the concern of other organ involvement, whole body radiographs and abdominal ultrasonography were recommended but declined by the owners. The owners were instructed to check for local recurrence and to monitor the rabbit’s respiratory pattern. Six months later the rabbit was presented again for severe respiratory distress and died upon arrival at the hospital. The owners reported a 1-week history of weight loss and dyspnea. The body was submitted for necropsy, which revealed 2 wellcircumscribed 5 mm 3 5 mm, firm, tan-white, round nodules within the subcutaneous tissue along the left mammary chain, approximately 2 cm caudal to the axilla and within the same site as the previously resected mass (Figure 2). The thoracic cavity contained approximately 10 mL of serosanguineous fluid and approximately 40% of the pulmonary parenchyma was effaced by multiple, similar, firm raised, round to oval, 3 mm to 15 mm, often coalescing, tan-white nodules. Similar nodules were also present along the visceral and parietal pleura. Two similar, 10 mm 3 10 mm nodules were present within the CVJ / VOL 55 / MAY 2014
hepatic parenchyma; protruding 3 to 5 mm above the capsular surface of the visceral surface of the left and right medial lobes of the liver. Tissues were collected and fixed in 10% neutral buffered formalin, further dissected and embedded in paraffin wax. Sections (4 mm) were mounted on silane-treated glass slides and stained with hematoxylin and eosin (H&E). Histopathological appearance of the subcutaneous, pulmonary, visceral, and parietal pleura as well as hepatic nodules was similar to that observed in the original excisional biopsy. Immunohistochemistry, as previously described, was perfomed on pulmonary and hepatic mestatases and had similar results as those for the primary tumor. The histological diagnosis was that of recurrence of the previously diagnosed poorly differentiated anaplastic solid adenocarcinoma suspected to be of mammary origin, with disseminated pulmonary, pleural, and hepatic metastasis.
Discussion This paper reports an anaplastic adenocarcinoma located on the ventral abdomen and suspected to be of mammary origin in a male rabbit with hepatic, pulmonary, and pleural metastases. The strong suspicion of a primary mammary gland tumor was based on the histological features, immunohistochemical results, and absence of respiratory signs on the first day of presentation. Apocrine adenocarcinoma could not be completely ruled out and the axillary area is a common site for this neoplasia in dogs and cats (11). Three cases of apocrine adenocarcinoma have been reported in 1 male and 2 female rabbits based on histological appearance (12). One of these tumors was located in the axilla, the second in the perineum, and the location of the last one was unknown (12). Cutaneous and hepatic metastasis was reported in 1 of these cases (12). Histological examination of rabbit apocrine adenocarcinomas revealed a “well-demarcated proliferation of cuboidal to columnar cells with basilar-oriented, moderately pleomorphic nuclei, which formed tubules, acini, or cysts” (12). The histological appearance in the case presented here was not similar to the histological description of apocrine adenocarcinoma in these 3 rabbits, but was similar to a mammary adenocarcinoma previously described in a female New Zealand White rabbit (2). Therefore, a mammary origin of the anaplastic adenocarcinoma in this case was strongly suspected. To our knowledge, mammary adenocarcinoma has never been reported in a male rabbit. In contrast, this neoplasm has been frequently described as a progression of noninfectious cystic mastitis in 3- to 4-year old multiparous does (Oryctolagus 477
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Detection system
HIERb
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spp.) and has also been reported in Lepus spp. (1,3). Mammary gland tumors have been rarely described in male dogs (4,13), cats (5), mice (6), rats (9), guinea pigs (9), and humans (7,8). Mammary adenocarcinoma represents less than 1% and 1% to 5% of diagnosed tumors in humans and male cats, respectively, and female dogs are 62 times more likely to develop mammary gland tumors than male dogs (4,5,7,8). Endocrine disorders including estrogen excess (exogenous estrogen or liver disease) or lack of androgen (prolactinoma) have been reported as a risk factor for mammary gland tumors in rabbits, mice, cats, and humans (2,5,6,8). Prolactin secreting anterior pituitary adenomas associated with mammary dysplasia or mammary adenocarcinoma have been reported in nulliparous New Zealand white female rabbits (2,14). The hypothesis is that prolonged elevations in estrogen in does result in hyperplasia and hypertrophy of pituitary acidophils with progression to pituitary adenoma (14). In humans, prolonged hyperprolactinemia is suspected to lead to malignant breast tumors (15). Prolactin secretion has been shown to induce mammary dysplasia by promoting the proliferation, survival, differentiation, and mobility of mammary epithelium, which could progress to mammary adenocarcinoma (8,16). In addition, prolactin functions to stimulate the growth and mobility of breast cancer cells in humans (16). Hyperprolactinemia with mammary dysplasia has been described in only 1 other species of laboratory animal, the FVB/NCr mice (2). Rabbit mammary tissue has proven to be particularly responsive to prolactin compared to other laboratory mammals, which could explain the scarcity of this condition in other laboratory species (17). In this case, the pituitary gland was grossly normal; however, pituitary adenoma could not be definitively ruled out on gross examination alone as pituitary acidophil adenomas with no change in pituitary gland size have been described in one-third of females rabbits with this condition in 1 study (14). On histological examination, the pituitary gland was incomplete; however, it appeared unremarkable. Regular progestin injections seem to increase the risk of mammary adenocarcinoma in male cats (5) and mammary adenocarcinomas have been induced with estrogen administration in male mice of particular strains (6). Approximately 50% to 77% of epithelial mammary gland tumors express estrogen receptors in female dogs (4). In comparison, 90% of male breast cancers express estrogen receptors and 81% express progesterone receptors (8). Immunohistochemistry for progesterone- and estrogen-receptors in spontaneous mammary gland tumors has been described in rabbits in one proceeding (18). Ten mammary adenocarcinomas were used in this study; 2 mammary adenocarcinomas were estrogen-receptor-a positive, and none of these tumors expressed progesterone receptors (18). However, testing for estrogen and progesterone receptors was not performed in this case, as this procedure has yet not been routinely adopted as a diagnostic marker in mammary gland tumors for this species and as few laboratories are able to complete this procedure in rabbits. Urine and blood samples were also collected for androgens and prolactin levels but were not tested as no laboratory was found to be able to perform these tests. Testicular disorders, including undescended testes, congenital inguinal hernia, orchiectomy, orchitis, and infertility, were also 478
reported as risk factors of mammary adenocarcinoma in humans (8). In this case, apart from a mild increase in septal connective tissue, gross and histological apearance of the testicles were unremarkable. Genetic predispositions for mammary adenocarcinoma including mutations and Klinefelter’s syndrome (XXY) have been reported in men (7,8). Some breeds of dogs (e.g., poodles, cocker spaniels) are also over-represented in both genders for mammary gland tumors (4). Reported rabbit breeds with mammary adenocarcinoma in females included Belgian, EnglishBelgian and pure and crossbred New Zealand whites (3). In humans, other risk factors for breast cancers include trauma with breast injury, radiation exposure, and lifestyle (obesity, alcohol, diet, low level of physical activity) (7,8). None of these can be attributed to this case. In female rabbits, cystic mastitis often progresses to mammary adenocarcinoma and these 2 disorders have a similar clinical presentation often characterized by a variety of stages of pathology (1,3). The affected glands are generally not painful but are swollen or firm, occasionally with discolored and enlarged teats (1,3,17); a clear to serosanguinous discharge or milk can be expressed from the distended nipples (1,3,14). The condition can be restricted to a single mammary gland or be bilaterally diffuse (2). This differs from the current case in which the tumor was a unique, non-painful, multilobular, round, tan and firm subcutaneous mass. Fine-needle aspiration and cytology are recommended as a diagnostic test in cases of mammary masses in rabbits (1). Both tests were performed but fine-needle aspiration did not provide a definitive diagnosis and surgical resection and histopathology was indicated. Upon confirmation of mammary adenocarcinoma, standard pre-surgical investigation should be performed as metastasis to regional lymph nodes, lungs, kidney, liver, adrenal glands, pancreas, ovary, and bone marrow have been reported (1,3). In this case, screening imaging tests were declined by the owner and metastasis could not be ruled out, even if no lymphatic invasion was noticed on histopathological examination of the resected mass and no regional lymph nodes were enlarged. Prognosis with mammary adenocarcima without treatment is poor in female rabbits as metastasis to organs noted above is frequent and mammary adenocarcinomas are commonly associated with uterine tumors (1). In male guinea pigs, mammary adenocarcinomas are locally invasive but rarely metastasize (9). Mammary carcinoma in male cats presents with a similar aggressive clinical course to the disease in females, with similar local tumor recurrence in up to 45% of cases (5). In male dogs, mammary gland tumors are usually benign and have a good prognosis with surgery (4). In humans, when adjusted for age at diagnosis and stage of disease, outcomes for male and female patients with breast cancers is similar (8). The most important prognostic factors in men with breast cancer are tumor size and lymph node status, which are also reported as prognostic factors in female cats and dogs (4,5,8). In this case, prognosis was guarded with a high probability of local recurrence. Because of the risk of metastasis, surgical removal of mammary adenocarcinoma with prescreening tests is recommended (1). In female CVJ / VOL 55 / MAY 2014
Acknowledgments We thank J. DeLay, S. Lapos, and the Animal Health Laboratory (AHL) histotechnology team for assistance with immunohistochemical studies. CVJ
References 1. Klaphake E, Paul-Murphy J. Section II: Rabbit — Disorders of the reproductive and urinary systems. In: Quesenberry KE, Carpenter JW, eds. Ferets, Rabbits, and Rodents Clinical Medicine and Surgery. 3rd ed. St. Louis, Missouri: Elsevier Saunders, 2012:217–231. 2. Sikoski P, Trybus J, Cline JM, et al. Cystic mammary adenocarcinoma associated with a prolactin-secreting pituitary adenoma in a
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New Zealand White Rabbit (Oryctolagus cuniculus). Comp Med 2008; 58:297–300. 3. Heatley JJ, Smith AN. Spontaneous neoplasms of lagomorphs. Vet Clin North Am Exot Anim Pract 2004;7:561–577. 4. Saba CF, Rogers KS, Newman SJ, Mauldin GE, Vail DM. Mammary gland tumors in male dogs. J Vet Intern Med 2007;21:1056–1059. 5. Skorupski KA, Overley B, Shofer FS, Goldschmidt MH, Miller CA, Sorenmo KU. Clinical characteristics of mammary carcinoma in male cats. J Vet Intern Med 2005;19:52–55. 6. Bonser GM. The effect of oestrone administration on the mammary glands of male mice of two strains differing greatly in their susceptibility to spontaneous mammary carcinoma. J Pathol Bacteriol 1936;42: 169–181. 7. Donegan WL, Perez-Mesa CM. Carcinoma of the male breast. A 30-year review of 28 cases. Arch Surg 1973;106:273–279. 8. Gomez-Raposo C, Zambrana Tevar F, Sereno Moyano M, Lopez Gomez M, Casado E. Male breast cancer. Cancer Treat Rev 2010;36:451–457. 9. Greenacre CB. Spontaneous tumors of small mammals. Vet Clin North Am Exot Anim Pract 2004;7:627–651. 10. Rabbit Reference Ranges for Common Analytes (Comprehensive Diagnostic and Mammalian Liver Profiles). For VetScan and VetScan VS2. Laboratories AVR: Abaxis, Inc. © Abaxis 2011. 11. Goldschmidt MH, Hendrick MJ. Tumors of the skin and soft tissues. In: Meuten DJ, ed. Tumors in Domestic Animals. 4th ed. Ames, Iowa: Blackwell Publishing, 2002:70–73. 12. von Bomhard W, Goldschmidt MH, Shofer FS, Perl L, Rosenthal KL, Mauldin EA. Cutaneous neoplasms in pet rabbits: A retrospective study. Vet Pathol 2007;44:579–588. 13. Mulligan RM. Feminization in male dogs — A syndrome associated with carcinoma of the testis and mimicked by the administration of estrogens. Am J Pathol 1944;20:865–875. 14. Lipman NS, Zhao ZB, Andrutis KA, Hurley RJ, Fox JG, White HJ. Prolactin-secreting pituitary-adenomas with mammary dysplasia in New Zealand White rabbits. Lab Anim Sci 1994;44:114–120. 15. Sato T, Muto I, Hasegawa M, et al. A rare case of invasive ductal carcinoma with hyperprolactinemia. Breast Cancer 2007;14:302–306. 16. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin in mammary carcinoma. Endocr Rev 2003;24:1–27. 17. Percy DH, Barthold SW. Rabbit. In: Percy DH, Barthold SW, eds. Pathology of Laboratory Rodents and Rabbits. 3rd ed. Ames, Iowa: Blackwell Publishing, 2007:253–307. 18. Bacci B, Ressel L, Erika A, et al. Phenotypic characterization of spontaneous mammary tumours in pet rabbits. J Comp Pathol 2010;143: 350–350.
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rabbits with mammary adenocarcinoma, ovariohysterectomy with partial or total mastectomy is the standard treatment (1,3). In one study, male cats with mammary adenocarcinoma that underwent a simple gland mastectomy did not have a longer disease-free interval than those that underwent a lumpectomy only (5). In men, standard treatment of early-stage disease is mastectomy with axillary clearance, in association with hormonal therapy with an estrogen receptor antagonist (Tamoxifen) as 90% of breast cancers in men express estrogen receptors (8). In locally advanced disease, chemotherapy prior to mastectomy is recommended, with post-mastectomy radiotherapy and hormonal therapy. In metastatic breast cancers, hormonal therapy is often the first approach (8). Alternative adjuvant therapies, like aromatase inhibitor, have also been described with controversial effectiveness (8). Many antineoplastic medications are tested on rabbits for human research and alternative therapies may provide adjuvant treatments in the future (1). This case report describes a metastatic anaplastic adenocarcinoma suspected to be of mammary origin in a male dwarf rabbit. As this is the first report of this neoplasm in an intact male rabbit, the information presented here may aid practitioners in the diagnosis of similar cases presented with ventral abdominal masses.
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ammary adenocarcinomas are common in 3- to 4-year-old multiparous female rabbits, and have been associated with prolactin-producing pituitary adenomas or uterine hyperplasia and adenocarcinomas (1–3). However, to the authors’ knowledge, this tumor has never been reported before in an intact male rabbit. Mammary adenocarcinomas are rarely reported in male dogs, cats, specific male mice strains, rats, guinea pigs, and men (4–9). The purpose of this report is to present the unusual clinical and pathological findings of a metastatic anaplastic adenocarcinoma of suspected mammary origin in an intact male rabbit.
Case description A 7-year-old, 1.1 kg, intact male, pet dwarf rabbit (Oryctolagus cuniculus) was referred to the Avian and Exotics Service at the Ontario Veterinary College Health Sciences Centre for removal The Ontario Veterinary College Health Sciences Centre (Summa, Eshar) and Department of Pathobiology (Snyman, Lillie), University of Guelph, Guelph, Ontario N1G 2W1. Address all correspondence to Dr. Noémie Summa; e-mail: [email protected] Dr. Summa’s current address is Companion Avian and Exotic Animal Medicine, University of California, Davis. Dr. Eshar’s current address is Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506, USA. Dr. Snyman’s current address is Animal Health Centre, Ministry of Agriculture, Abbotsford, British Columbia V3G 2M3, Canada. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 55 / MAY 2014
(Traduit par Isabelle Vallières)
of a subcutaneous abdominal mass. The mass had appeared 5 mo earlier and had progressively grown since then. No other abnormalities were noticed by the owners. Blood tests [complete blood (cell) count (CBC) and biochemistry] were performed by the referring veterinarian and the results were unremarkable other than a mild increase in plasma potassium concentration [6.3 mmol/L, reference interval (RI): 3.5 to 6.2 mmol/L] (10) and mild hypoglycemia (4.3 mmol/L, RI: 5.5 to 8.6 mmol/L) (10). Both were considered artefactual secondary to hemolysis of the blood sample and glucose consumption by erythrocytes, respectively. Radiographs were declined by the owners. On physical examination, the rabbit was bright, alert, and responsive and all vital signs were within normal ranges. A 10 mm 3 18 mm, irregular, round, tan, and firm subcutaneous mass could be palpated in the region of the left second inguinal mammary gland. The mass was not adherent to adjacent tissues and no peripheral lymphadenomegaly was detected. Except for bilateral cataracts, no other physical abnormalities were noted. The main differential diagnoses included subcutaneous abscess or neoplasia. A fine-needle aspirate (FNA) of the subcutaneous mass was obtained. The cytological sample was of poor cellularity (, 10%) and was obscured by a background of erythrocytes, cellular debris, and fragmented nuclei. The interpretation was that of a non-diagnostic sample with hemorrhage, although infection could not be ruled out. An excisional biopsy of the subcutaneous mass was then scheduled. The rabbit was sedated with buprenorphine (Vetergesic; Champion Alstoe Animal Health, Whitby, Ontario), 0.03 mg/kg body weight (BW), IM, and midazolam (Midazolam Sandoz; Sandoz Canada, Burlington, Ontario), 0.8 mg/kg BW, IM. An intravenous catheter was placed in the right cephalic vein and hydration was maintained using lactated ringers solution at 10 mL/kg BW per hour. The rabbit was induced with sevoflurane (SevoFlo; 475
R A P P O R T D E CA S
200 mm
50 mm
Figure 1. Histological images of the the subcutaneous mammary nodule in a male dwarf rabbit (a and b). The mass was composed of solid cords and varisized islands (150 to 450 mm) of epithelial cells (solid arrow) and occasional well-differentiated acini and ducts (solid star) that often contained small amounts of either coarse granular to streaky basophilic luminal mucous and debris or homogenous eosinophilic protein rich fluid and cellular debris. Large polygonal to columnar epithelial cells often palisading along interspersed 5 to 10 mm thick fibrous connective tissue trabeculae were observed (b). Numerous, often bizarre, mitotic figures were observed (open arrows) and both mutlinucleation and nuclear gigantism were common (open star). Hematoxylin and eosin (H&E).
Abbott Laboratories, Saint-Laurent, Quebec), 8% in oxygen by facemask. Tracheal intubation with a 2.0 mm diameter tracheal tube was unsuccessful and the rabbit was maintained with sevoflurane 3% to 5% in oxygen by facemask. The rabbit was placed in dorsal recumbency and the surgical site was prepared using a routine aseptic protocol. A 3-cm elliptical skin incision was made around the mass, which was completely removed. Subcutaneous tissues were apposed using 4-0 Polydioxanone (PDS II; polydioxanone; Ethicon, Somerville, New Jersey, USA) in a simple continuous pattern and the skin was closed using 4-0 PDS with an intradermal suture in a simple continuous pattern. The mass was fixed in 10% neutral buffered formalin and submitted for histopathology. Sedation was partially reversed with flumazenil (Flumazenil Sandoz; Sandoz Canada), 0.03 mg/kg BW, SQ, and the rabbit recovered uneventfully from the procedure. Meloxicam (Metacam; Boehringer Ingelheim, Burlington, Ontario), 0.5 mg/kg BW, SQ, and penicillin G (Penicillin G Procaine; Pfizer Animal Health, Kirkland, Quebec), 50 000 IU/kg BW, SQ, were administrated. The rabbit was sent home the next day with meloxicam, 0.5 mg/kg BW, PO, q12h for 7 d. Histopathology of the resected mass revealed an infiltrative, unencapsulated, multi-lobular, densely cellular neoplasm expanding the subcutaneous tissue and deep dermis. The mass was composed of solid cords and islands ranging from 150 mm to 450 mm of large polygonal to columnar epithelial cells often palisading along interspersed 5 mm to 10 mm thick fibrous connective tissue trabeculae with occasional scattered acinar structures (Figures 1a, 1b). Acini were often filled with basophilic mucus, cellular debris and homogenous eosinophilic protein rich fluid. Cell margins were distinct and cells contained a large amount of finely granular eosinophilic cytoplasm with 476
large oval nuclei, coarsely stippled chromatin, and an occasional prominent central basophilic nucleolus. Nuclear gigantism and multinucleation were common, with up to five-fold anisokaryosis and up to 22 nuclei per neoplastic cell. The mitotic rate was high, with an average of 7.2 and a maximum of 15, occasionally bizarre, mitotic figures per 4003 high power field (HPF) (Figure 1b). Often large central foci of necrosis and hemorrhage were scattered throughout the mass. Numerous small satellite nodules were observed around the periphery of the main mass. There was no histological evidence of lymphatic invasion or obstruction; however, neoplastic cells extended to within 30 mm of both the deep and lateral surgical margins. In order to evaluate the epithelial phenotype of this neoplasm immunohistochemistry (IHC) was performed to detect pancytokeratin (AE1/AE3), cytokeratin 7 (CK7), and thyroid transcription factor 1 (TTF1). Processed 4-mm sections from formalin-fixed, paraffin-embedded tissues were mounted on silane-treated glass slides, deparaffinized, and rehydrated prior to the IHC procedure and antigen retrieval was carried out as outlined in Table 1. Sections of normal rabbit tissues known to contain the antigen of interest as well as tissues on the test slides were used as species-specific internal controls. For negative reagent controls, duplicate sections of each control and test tissue were subjected to the same IHC procedure with substitution of non-immune rabbit serum, at similar concentration, for the specific primary antiserum/antibody. Approximately 90% of neoplastic cells showed strong positive cytoplasmic immunoreactivity for both the pancytokeratin and CK7 antibodies. The IHC procedures for TTF1 did not result in consistent labelling of the external and internal species-specific positive control tissues; as such, TTF1 expression could not be evaluated. The interpretation was that of a poorly differentiated, anaplastic, CVJ / VOL 55 / MAY 2014
Table 1. Antibodies and their source, pretreatment and visualization methods used in immunohistochemistrya Antigen
Antibody source, clone, and dilution
Epitope retrieval method
Platform
Pan-Cytokeratin Dako — mouse monoclonal pH 6 AE1 AE3, 1:100
Goat anti-mouse horseradish peroxidase-labelled polymer (EnVision 1, Dako)
Dako Autostainer
Cytokeratin 7 Dako — mouse monoclonal, Enzyme OV-TL 12/30, 1:100 (proteinase K, Dako)
Goat anti-mouse horseradish peroxidase-labelled polymer (EnVision 1, Dako)
Dako Autostainer
a Processed
4-mm sections from formalin-fixed, paraffin-embedded tissues were mounted on silane-treated glass slides, deparaffinized, and rehydrated prior to immunochemistry. epitope retrieval.
b Heat-induced
Figure 2. Subcutaneous tissue and mammary chain in a male dwarf rabbit observed at necropsy. A 10 mm 3 18 mm, wellcircumscribed, firm, tan, subcutaneous mass (star) was present within the subcutaneous tissue along the left mammary chain, approximately 2 cm caudal to the axilla, and in the same location as the original tumor resection.
solid adenocarcinoma suspected to be of mammary epithelial origin based on anatomic location, histological appearance, and expression of CK7. As CK7 is a marker of ductular origin, apocrine adenocarcinoma could not be ruled out, although this tumor diagnosis was considered less likely in this case. Given the nature of this neoplasia and with the concern of other organ involvement, whole body radiographs and abdominal ultrasonography were recommended but declined by the owners. The owners were instructed to check for local recurrence and to monitor the rabbit’s respiratory pattern. Six months later the rabbit was presented again for severe respiratory distress and died upon arrival at the hospital. The owners reported a 1-week history of weight loss and dyspnea. The body was submitted for necropsy, which revealed 2 wellcircumscribed 5 mm 3 5 mm, firm, tan-white, round nodules within the subcutaneous tissue along the left mammary chain, approximately 2 cm caudal to the axilla and within the same site as the previously resected mass (Figure 2). The thoracic cavity contained approximately 10 mL of serosanguineous fluid and approximately 40% of the pulmonary parenchyma was effaced by multiple, similar, firm raised, round to oval, 3 mm to 15 mm, often coalescing, tan-white nodules. Similar nodules were also present along the visceral and parietal pleura. Two similar, 10 mm 3 10 mm nodules were present within the CVJ / VOL 55 / MAY 2014
hepatic parenchyma; protruding 3 to 5 mm above the capsular surface of the visceral surface of the left and right medial lobes of the liver. Tissues were collected and fixed in 10% neutral buffered formalin, further dissected and embedded in paraffin wax. Sections (4 mm) were mounted on silane-treated glass slides and stained with hematoxylin and eosin (H&E). Histopathological appearance of the subcutaneous, pulmonary, visceral, and parietal pleura as well as hepatic nodules was similar to that observed in the original excisional biopsy. Immunohistochemistry, as previously described, was perfomed on pulmonary and hepatic mestatases and had similar results as those for the primary tumor. The histological diagnosis was that of recurrence of the previously diagnosed poorly differentiated anaplastic solid adenocarcinoma suspected to be of mammary origin, with disseminated pulmonary, pleural, and hepatic metastasis.
Discussion This paper reports an anaplastic adenocarcinoma located on the ventral abdomen and suspected to be of mammary origin in a male rabbit with hepatic, pulmonary, and pleural metastases. The strong suspicion of a primary mammary gland tumor was based on the histological features, immunohistochemical results, and absence of respiratory signs on the first day of presentation. Apocrine adenocarcinoma could not be completely ruled out and the axillary area is a common site for this neoplasia in dogs and cats (11). Three cases of apocrine adenocarcinoma have been reported in 1 male and 2 female rabbits based on histological appearance (12). One of these tumors was located in the axilla, the second in the perineum, and the location of the last one was unknown (12). Cutaneous and hepatic metastasis was reported in 1 of these cases (12). Histological examination of rabbit apocrine adenocarcinomas revealed a “well-demarcated proliferation of cuboidal to columnar cells with basilar-oriented, moderately pleomorphic nuclei, which formed tubules, acini, or cysts” (12). The histological appearance in the case presented here was not similar to the histological description of apocrine adenocarcinoma in these 3 rabbits, but was similar to a mammary adenocarcinoma previously described in a female New Zealand White rabbit (2). Therefore, a mammary origin of the anaplastic adenocarcinoma in this case was strongly suspected. To our knowledge, mammary adenocarcinoma has never been reported in a male rabbit. In contrast, this neoplasm has been frequently described as a progression of noninfectious cystic mastitis in 3- to 4-year old multiparous does (Oryctolagus 477
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spp.) and has also been reported in Lepus spp. (1,3). Mammary gland tumors have been rarely described in male dogs (4,13), cats (5), mice (6), rats (9), guinea pigs (9), and humans (7,8). Mammary adenocarcinoma represents less than 1% and 1% to 5% of diagnosed tumors in humans and male cats, respectively, and female dogs are 62 times more likely to develop mammary gland tumors than male dogs (4,5,7,8). Endocrine disorders including estrogen excess (exogenous estrogen or liver disease) or lack of androgen (prolactinoma) have been reported as a risk factor for mammary gland tumors in rabbits, mice, cats, and humans (2,5,6,8). Prolactin secreting anterior pituitary adenomas associated with mammary dysplasia or mammary adenocarcinoma have been reported in nulliparous New Zealand white female rabbits (2,14). The hypothesis is that prolonged elevations in estrogen in does result in hyperplasia and hypertrophy of pituitary acidophils with progression to pituitary adenoma (14). In humans, prolonged hyperprolactinemia is suspected to lead to malignant breast tumors (15). Prolactin secretion has been shown to induce mammary dysplasia by promoting the proliferation, survival, differentiation, and mobility of mammary epithelium, which could progress to mammary adenocarcinoma (8,16). In addition, prolactin functions to stimulate the growth and mobility of breast cancer cells in humans (16). Hyperprolactinemia with mammary dysplasia has been described in only 1 other species of laboratory animal, the FVB/NCr mice (2). Rabbit mammary tissue has proven to be particularly responsive to prolactin compared to other laboratory mammals, which could explain the scarcity of this condition in other laboratory species (17). In this case, the pituitary gland was grossly normal; however, pituitary adenoma could not be definitively ruled out on gross examination alone as pituitary acidophil adenomas with no change in pituitary gland size have been described in one-third of females rabbits with this condition in 1 study (14). On histological examination, the pituitary gland was incomplete; however, it appeared unremarkable. Regular progestin injections seem to increase the risk of mammary adenocarcinoma in male cats (5) and mammary adenocarcinomas have been induced with estrogen administration in male mice of particular strains (6). Approximately 50% to 77% of epithelial mammary gland tumors express estrogen receptors in female dogs (4). In comparison, 90% of male breast cancers express estrogen receptors and 81% express progesterone receptors (8). Immunohistochemistry for progesterone- and estrogen-receptors in spontaneous mammary gland tumors has been described in rabbits in one proceeding (18). Ten mammary adenocarcinomas were used in this study; 2 mammary adenocarcinomas were estrogen-receptor-a positive, and none of these tumors expressed progesterone receptors (18). However, testing for estrogen and progesterone receptors was not performed in this case, as this procedure has yet not been routinely adopted as a diagnostic marker in mammary gland tumors for this species and as few laboratories are able to complete this procedure in rabbits. Urine and blood samples were also collected for androgens and prolactin levels but were not tested as no laboratory was found to be able to perform these tests. Testicular disorders, including undescended testes, congenital inguinal hernia, orchiectomy, orchitis, and infertility, were also 478
reported as risk factors of mammary adenocarcinoma in humans (8). In this case, apart from a mild increase in septal connective tissue, gross and histological apearance of the testicles were unremarkable. Genetic predispositions for mammary adenocarcinoma including mutations and Klinefelter’s syndrome (XXY) have been reported in men (7,8). Some breeds of dogs (e.g., poodles, cocker spaniels) are also over-represented in both genders for mammary gland tumors (4). Reported rabbit breeds with mammary adenocarcinoma in females included Belgian, EnglishBelgian and pure and crossbred New Zealand whites (3). In humans, other risk factors for breast cancers include trauma with breast injury, radiation exposure, and lifestyle (obesity, alcohol, diet, low level of physical activity) (7,8). None of these can be attributed to this case. In female rabbits, cystic mastitis often progresses to mammary adenocarcinoma and these 2 disorders have a similar clinical presentation often characterized by a variety of stages of pathology (1,3). The affected glands are generally not painful but are swollen or firm, occasionally with discolored and enlarged teats (1,3,17); a clear to serosanguinous discharge or milk can be expressed from the distended nipples (1,3,14). The condition can be restricted to a single mammary gland or be bilaterally diffuse (2). This differs from the current case in which the tumor was a unique, non-painful, multilobular, round, tan and firm subcutaneous mass. Fine-needle aspiration and cytology are recommended as a diagnostic test in cases of mammary masses in rabbits (1). Both tests were performed but fine-needle aspiration did not provide a definitive diagnosis and surgical resection and histopathology was indicated. Upon confirmation of mammary adenocarcinoma, standard pre-surgical investigation should be performed as metastasis to regional lymph nodes, lungs, kidney, liver, adrenal glands, pancreas, ovary, and bone marrow have been reported (1,3). In this case, screening imaging tests were declined by the owner and metastasis could not be ruled out, even if no lymphatic invasion was noticed on histopathological examination of the resected mass and no regional lymph nodes were enlarged. Prognosis with mammary adenocarcima without treatment is poor in female rabbits as metastasis to organs noted above is frequent and mammary adenocarcinomas are commonly associated with uterine tumors (1). In male guinea pigs, mammary adenocarcinomas are locally invasive but rarely metastasize (9). Mammary carcinoma in male cats presents with a similar aggressive clinical course to the disease in females, with similar local tumor recurrence in up to 45% of cases (5). In male dogs, mammary gland tumors are usually benign and have a good prognosis with surgery (4). In humans, when adjusted for age at diagnosis and stage of disease, outcomes for male and female patients with breast cancers is similar (8). The most important prognostic factors in men with breast cancer are tumor size and lymph node status, which are also reported as prognostic factors in female cats and dogs (4,5,8). In this case, prognosis was guarded with a high probability of local recurrence. Because of the risk of metastasis, surgical removal of mammary adenocarcinoma with prescreening tests is recommended (1). In female CVJ / VOL 55 / MAY 2014
Acknowledgments We thank J. DeLay, S. Lapos, and the Animal Health Laboratory (AHL) histotechnology team for assistance with immunohistochemical studies. CVJ
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rabbits with mammary adenocarcinoma, ovariohysterectomy with partial or total mastectomy is the standard treatment (1,3). In one study, male cats with mammary adenocarcinoma that underwent a simple gland mastectomy did not have a longer disease-free interval than those that underwent a lumpectomy only (5). In men, standard treatment of early-stage disease is mastectomy with axillary clearance, in association with hormonal therapy with an estrogen receptor antagonist (Tamoxifen) as 90% of breast cancers in men express estrogen receptors (8). In locally advanced disease, chemotherapy prior to mastectomy is recommended, with post-mastectomy radiotherapy and hormonal therapy. In metastatic breast cancers, hormonal therapy is often the first approach (8). Alternative adjuvant therapies, like aromatase inhibitor, have also been described with controversial effectiveness (8). Many antineoplastic medications are tested on rabbits for human research and alternative therapies may provide adjuvant treatments in the future (1). This case report describes a metastatic anaplastic adenocarcinoma suspected to be of mammary origin in a male dwarf rabbit. As this is the first report of this neoplasm in an intact male rabbit, the information presented here may aid practitioners in the diagnosis of similar cases presented with ventral abdominal masses.
