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TP53 EXPRESSING SQUAMOUS CELL CARCINOMA OF THE TONSIL IN ACAPTIVE POLAR WOLF (CANIS LUPUS ARCTOS)Author(s): Jens P. TeifkeDr. Med. Vet. Habil., P.D., Dipl. A.C.V.P., Christiane V. LöhrDr. Med. Vet.,Ph.D., Dipl. A.C.V.P., Christoph LangnerD.V.M.Source: Journal of Zoo and Wildlife Medicine, 36(3):538-542. 2005.Published By: American Association of Zoo VeterinariansDOI: http://dx.doi.org/10.1638/04-019.1URL: http://www.bioone.org/doi/full/10.1638/04-019.1

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Journal of Zoo and Wildlife Medicine 36(3): 538–542, 2005Copyright 2005 by American Association of Zoo Veterinarians

TP53 EXPRESSING SQUAMOUS CELL CARCINOMA OF THETONSIL IN A CAPTIVE POLAR WOLF (CANIS LUPUS ARCTOS)

Jens P. Teifke, Dr. Med. Vet. Habil., P.D., Dipl. A.C.V.P., Christiane V. Lohr, Dr. Med. Vet.,Ph.D., Dipl. A.C.V.P., and Christoph Langner, D.V.M.

Abstract: The gross and histopathologic findings for a primary tonsillar squamous cell carcinoma in a captive 11-yr-old male polar wolf (Canis lupus arctos) are described. The carcinoma had metastasized to regional lymph nodesof the pharynx, the precardial mediastinum, and the lungs. Tumor suppressor protein TP53 was detected by immuno-histochemistry in the nuclei of poorly differentiated, cytokeratin-positive cells of the primary neoplasm and the me-tastases. Canine oral papillomavirus DNA was not detectable by polymerase chain reaction (PCR).

Key words: Canis lupus arctos, immunohistochemistry, metastasis, polar wolf, squamous cell carcinoma, tonsil.

BRIEF COMMUNICATION

Malignant neoplasms of the oral and pharyngealcavity are common in all domestic animal species.Oral squamous cell carcinoma (SCC) has been de-scribed in cattle and horses, but it is seen primarilyin small carnivores. SCC is the most common oralneoplasm in aged domestic cats, in which the tonsilor the ventrolateral surface of the tongue is usuallyaffected.5 In the dog, the frequency of SCC is onlyexceeded by malignant melanomas in the oral cav-ity. In older canids, the epithelium of the tonsillarcrypts is the primary site of SCC development, al-though the gingiva and lateral margins of thetongue may also be involved.5 Reports of oral, andespecially tonsillar, SCC in free-living or captivenondomestic canid and felid species are rare.2,8

Compared with prevalence in industrial cities, ca-nine tonsillar SCC is uncommon in rural areas7; airpollution in an urban setting including householdenvironmental tobacco smoke exposure also maycontribute to tumor development in cats.10 In hu-mans, tonsillar SCC may be associated with humanpapillomavirus infection, tobacco use and environ-mental tobacco smoke, and elevated levels of ex-pression of TP53.1,4 The TP53 tumor suppressorgene encodes the nuclear phosphoprotein TP53(previously p53), which is one of the most com-monly mutated genes in human cancer.6 TP53 func-tions as a transcription factor that controls the ex-pression of many cell cycle–related genes. Wild-type TP53 can up-regulate transcription of the cy-

From the Friedrich-Loeffler-Institut, Federal ResearchInstitute for Animal Health, Boddenblick 5A, 17493Greifswald-Insel Riems, Germany (Teifke); the Depart-ment of Biomedical Science, College of Veterinary Med-icine, Oregon State University, Oregon 97331-4804, USA(Lohr); and the Zoological Park Stralsund, 18437 Stral-sund, Germany (Langner). Correspondence should be di-rected to Dr. Teifke.

clin-dependent kinase inhibitor CDKN1A or theapoptosis-triggering genes GADD45 or BAX. Mu-tant or inactive TP53 has a prolonged half-life, ac-cumulates in the nucleus or cytoplasm of thestressed cell, and may act as a proto-oncogene, re-sulting in cellular proliferation and eventually ma-lignant transformation. Functional inactive TP53may be detected by immunohistochemical meth-ods.3

Gross, microscopic, immunohistologic, and poly-merase chain reaction (PCR) findings in a metas-tasizing squamous cell carcinoma that developed inthe tonsil of an 11-yr-old male polar wolf (Canislupus arctos) are described. The wolf was kept formore than 10 yr in a 4,000 m2 outdoor enclosureof the Zoological Park Stralsund, Germany. The an-imal was clinically normal until fall 2002, when itbecame progressively more depressed and anorec-tic, and it began to stagger and have difficulty walk-ing. It died 1 mo later.

At necropsy, the 32-kg animal had a slightly en-larged firm right tonsil (3.5 3 1.0 3 0.7 cm). Theoverlying mucosa was gray-tan and granular withseveral white, slightly raised nodules up to 0.5 cmin diameter (Fig. 1a). On cut surface, the tonsillartissue was diffusely replaced by a white, firm, poor-ly delineated mass. The left tonsil and the remain-der of the oral cavity were unremarkable. The rightmandibular and medial retropharyngeal lymphnodes were moderately enlarged and measured ap-proximately 3 times 3 times 2 cm with multiple,tan to white, well-demarcated, 2 times 1.5 times 1cm, firm, solid foci. Within the precardial medias-tinum there was a 7 times 6 times 6 cm large, en-capsulated, slightly lobulated, well-vascularizedmass that replaced the cranial mediastinal lymphnodes (Fig. 1b). On section, the tan to gray tissuewas lobulated and had a central, dark red cavity, 3times 2 times 1 cm. Scattered within the parenchy-

539TEIFKE ET AL.—TP53 EXPRESSING TONSILLAR CARCINOMA IN WOLF

Figure 1. a. Squamous cell carcinoma, right tonsil, polar wolf. The right tonsil has a grayish granular surface withseveral slightly elevated whitish nodules (arrow). b. Metastases of the tonsillar squamous cell carcinoma to the cranialmediastinal lymph nodes (arrowhead) and lungs (arrow) of a polar wolf. H indicates heart within the pericardium; T,trachea. Note locally extensive anthracosis.

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Figure 2. a. Squamous cell carcinoma, tonsil, polar wolf. Tonsillar tissue is infiltrated and replaced by cords ofpoorly differentiated carcinoma cells. Frequent tumor cells express cytoplasmic cytokeratins (arrow). Immunohisto-chemistry avidin-biotin-complex (ABC) method, anti-cytokeratin mouse monoclonal antibodies AE1, AE3, Ks13.1mixture, hematoxylin counterstain. Bar 5 175 mm. b. Lymph node metastasis of tonsillar squamous cell carcinoma,polar wolf. Pleomorphic carcinoma cells show TP53 immunoreactivity as dark nuclear staining (arrow). TP53 negativecells (arrowheads). Immunohistochemistry of TP53 using the ABC method, mouse monoclonal antibody BP53–12 (Ab-3, Oncogene Science Inc., Uniondale, New York 11553, USA) diluted 1:150 in Tris buffered saline, Mayer’s hematox-ylin counterstain. Bar 5 75 mm.

541TEIFKE ET AL.—TP53 EXPRESSING TONSILLAR CARCINOMA IN WOLF

ma, which had large areas of fine black stippling(anthracosis), and subpleurally in all lung lobeswere multiple white, sharply demarcated nodulesup to 1 cm in diameter (Fig. 1b).

Tissue samples were formalin-fixed and paraffinembedded; 5-mm sections were mounted oncharged slides and routinely stained with hematox-ylin and eosin (H&E). Histopathologic examinationof the tonsil revealed a nonencapsulated, poorly de-marcated mass composed of cords and nests ofsquamous epithelial cells embedded in desmoplas-tic stroma, which were invading and obliteratingthe pre-existing lymphoreticular tissue. In some ar-eas, central necrosis of the cords of neoplastic cellsformed clefts that sometimes resulted in pseudoa-cinar structures. Neoplastic cells were medium-sized to large and polygonal with distinct bordersand intercellular bridges. Moderate to largeamounts of eosinophilic, occasionally fibrillar, cy-toplasm was present. Nuclei were 15–30 mm in di-ameter (moderate anisokaryosis) and round to ovalor crimped (moderate pleomorphism) with vacuo-lated euchromatin and up to two medium to large,central, eosinophilic nucleoli. On average there wasone mitotic figure per 3400 field. Multifocally, sin-gle, round neoplastic cells had hypereosinophiliccytoplasm and had retracted from the surroundingtumor cells (keratinization). The masses in the ret-ropharyngeal lymph nodes, precardial mediasti-num, and lung were composed of anaplastic cellsthat lacked squamous epithelial cell differentiationwith rare single cell keratinization. A broad spec-trum of cytokeratins (CK 1–8, 10, 13–16, 19; anti-cytokeratin mouse monoclonal antibodies AE1,AE3, Ks13.1 mixture; Linaris Biological Products,Wertheim-Bettingen, Germany) was detected byimmunohistochemistry in the cytoplasm of the neo-plastic cells, demonstrating their epithelial origin(Fig. 2a). Immunohistochemistry for cytokeratinwas performed as described previously.12 The pat-tern of metastasis of the tonsillar carcinoma of thiscase was similar to that seen in domestic canids andtwo other reports in wolves.7,9,11

The advanced age of the wolf and its history to-gether with locally extensive pulmonary anthraco-sis suggest the potential that air pollution in an ur-ban environment may have played a role in the tu-mor development. Specific immunoreactivity toTP53 occurs in about one third of cutaneous andapproximately 20% of tonsillar SCC in dogs.14 Theavidin-biotin-complex (ABC) method was used todetect overexpression or accumulation of TP53;mouse anti-TP53 monoclonal antibody BP53-12was used as the primary antibody.13 Strong immu-noreactivity to TP53 was shown in the nuclei of

about 25% of the poorly differentiated carcinomacells, but not in the surrounding normal or stromalcells (Fig. 2b). The lack of immunostaining in stro-mal cells confirms the specificity and excludes thepossibility that it may result from the demaskingprocedures. Therefore a possible role for TP53 inthe pathogenesis of the SCC in this wolf is stronglysuggested.

In humans, a high percentage of oral SCC is as-sociated with papillomavirus infection. However,canine oral papilloma viral DNA was not detectedby PCR in the SCC in this wolf, leading us to con-clude that the TP53 accumulation was not papillo-mavirus-dependent. A similar independence ofTP53 overexpression and presence of human pap-illomavirus has been shown for some of the headand neck SCC in humans.1 The results of this in-vestigation suggest that TP53 may be overex-pressed and involved in the development of oralSCC in canid species, although only a single caseis described here.

Acknowledgment: We would like to thank Ga-briele Czerwinski for excellent technical assistance.

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14. Teifke, J. P., C. V. Lohr, and H. Shirasawa. 1998.Detection of canine oral papillomavirus DNA in canineoral squamous cell carcinomas and p53 overexpressingskin papillomas of the dog using the polymerase chainreaction and non-radioactive in situ hybridization. Vet.Microbiol. 60: 119–130.

Received for publication 2 February 2004