Three Neoplasms in A Eurasian Otter (Lutra Lutra): Malignant Melanoma, Trichoblastoma And Mammary Gland Adenoma

Abstract

An 18-year-old female Eurasian otter died of waste. Necropsy revealed skin masses on the nose and chest, subcutaneous masses on the abdomen, and multiple nodules in the liver and lungs. Malignant melanoma was found in the nasal skin mass and had metastasized to the liver, lungs, kidneys, adrenal glands, mammary glands, and left mandibular lymph node. The tumor cells were labeled for wave proteins, melanoma, and S100. the thoracic skin mass consisted of spindle-shaped tumor epithelial cells and was diagnosed as Trichoblastoma. A subcutaneous abdominal mass was seen as a mammary adenoma. This is the first report of three tumors, primary malignant melanoma, Trichoblastoma, and mammary adenoma, in a Eurasian otter.

Keywords

Eurasian otter, malignant melanoma, spindle cell Trichoblastoma, triple neoplasms, Cistanche supplements.

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Otters are classified as a subfamily of otters in the family Mustelidae and are distributed in several countries, especially the Eurasian otter (Lutra Lutra) is a species of otter that travels throughout Europe, Asia, and Africa. The Eurasian otter lives in a wide range of aquatic habitats and has an average lifespan of about 17 years. Several types of tumors have been described in the Lutrinae, such as smooth muscle tumors and basal cell carcinomas. In the Eurasian otter, malignant melanoma, intestinal lymphoma, and hepatocellular adenoma have been reported. Previously reported malignant melanomas included multiple subcutaneous nodules that appeared in the dorsal right and left thoracic regions, metastasizing to lymph nodes and the liver. Multiple primary tumors have been reported in only one sea otter. Here, we report a case of malignant melanoma with concurrent Trichoblastoma and mammary adenoma in a Eurasian otter.

An 18-year-old female Eurasian otter born in a zoo in Japan and transported to another zoo in Miyazaki was observed to have a subcutaneous abdominal mass at the age of 16 years. Two years later, a red nose and a thoracic skin mass were observed. 2 months later, biopsies were performed on the nasal mass (2.1 × 2.6 cm) and the left mandibular lymph node (2.2 × 3.0 cm) of the anesthetized animal, and fine needle aspiration cytology was performed on the thoracic skin mass (2.2 × 2.2 cm) and the subcutaneous abdominal mass (3.8 × 4.0 cm). Three days after the biopsy, the animal developed anorexia and hypermobility and died 6 days later. The otter was transferred to the Department of Veterinary Pathology of Miyazaki University for a necropsy.

The autopsy revealed irregular black corporate mass (2.3 x 2.8 cm) bleeding and ulcers on the skin of the nose, almost covering the surface covering the nose and adhering to the skin (Figure 1). The cutting surface mass was gray and black spotted in color, and the border between the solid mass and the surrounding normal tissue was unclear (Figure 1). No nose skin penetration was observed in the developing nasal or oral cavity. Cutaneous and subcutaneous masses were seen on the lateral and ventral sides of the left thorax with diameters of 2.5 × 3.5 cm and 4.0 × 4.0 cm, respectively. Severe subcutaneous edema was seen in the peripheral area of the thoracic mass and the mass was cystic in dark gray color on the cut surface. The abdominal subcutaneous mass was cystic, contained blood, and was white on the cut surface with papillary protrusions. The mass had clear borders with the surrounding normal tissue. The left mandibular lymph node was enlarged (3.5 × 4.5 cm) and had a solid grayish-white cut surface. There were four nodules on the left lateral side of the liver, 7.0 × 9.0, 2.5 × 3.0, 2.0 × 2.0, and 1.5 × 1.0 cm in diameter, which were soft and dark red or dark green. The lobes were caudate, right median, and square, respectively. The envelope of the left hepatic nodule was rough, and fibrin and blood clots were visible. All nodules were dark red solid in section with hemorrhage in the surrounding areas and clear borders between the nodules and surrounding normal tissue. Multiple white to black nodules, 1 ~ 5 mm in diameter, were seen in the lungs. hemorrhagic ascites (~250 ml) were present in the abdominal cavity. Mucosal pallor may be seen.

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Specimens were collected, fixed in 10% neutral buffered formalin, routinely paraffin-embedded, 3 μm sectioned, and stained with hematoxylin and eosin (HE). Cytokeratin-specific primary antibodies (clone AE1/AE3; Dako, Glostrup, Denmark; prepared for use), vimentin (clone V9; Dako; prepared for use), melan A (clone A103; Dako; dilution 1/50), melanoma (clone PNL2; Dako; prepared for use) and S100 (polyclonal. Dako; ready for use). Antigens were obtained by incubating cytokeratin, vimentin, and S100 with citrate buffer (pH 6.0), or melanin a and melanoma with Tris-EDTA buffer (pH 9.0) for 10 min at 105°C. Histofine Simple Stain MAX-POTM (Multi) (Nichirei Bioscience, Tokyo, Japan) was used for all primary antibodies. The chromogenic agent was 0.05% 3,3 ' -diaminobenzodiazine (Sigma-Aldrich, St. Louis, MO, USA) and 0.03% hydrogen peroxide in the tris-hydrochloric acid buffer. Hematoxylin was used as counterstaining agent. Cross-reactivity of the primary antibody with otter was confirmed by analysis of the nasal tissue and isotype IgG in this case.

Microscopic examination of the nasal dermal mass, liver, lung, kidney, adrenal gland, breast, and left mandibular lymph node revealed tumor melanocytic hyperplasia. Tumor cells were found in the dermis, invading the surrounding normal tissue without a capsule, attached at the nasal dermal-epidermal junction (Figure 2A). The tumor cell morphology was divided into two types: epithelioid or polygonal and spindle-shaped. Epithelioid or polygonal tumor cells were arranged in mucus-like bundles, cords, or nests, with single double-oval nuclei, single prominent nucleoli, and sparse cytoplasm (Figure 2B). The spindle-shaped tumor cells were arranged in flow bundles and contained a single elongated spindle-shaped nucleus and a single small nucleolus (Figure 2C). In primary and metastatic tissues, a few epithelioid or polygonal tumor cells contained melanin, whereas spindle-shaped tumor cells did not contain melanin. Cellular anisotropy was moderate and the nucleoplasmic ratio was high. The mitotic sign frequency was 2 or 3/high magnification fields. There were foci of tumor cells in the liver, lung, kidney, adrenal gland, and breast, and foci of tumor cells in the lymph or blood vessels (Figure 3). Normal lymph node structure was replaced by tumor cell hyperplasia and necrosis in the left mandibular lymph node. four liver nodes and several lung nodes were dominated by tumor melanocytic hyperplasia and hemorrhage within the liver nodes. In the nasal skin mass, liver, lung, kidney, adrenal, breast, and left mandibular lymph nodes, all tumor cells were vimentin-positive, epithelioid or polygonal tumor cells were melanoma-positive and S100 negative (Figures 3 and 4A). The spindle-shaped tumor cells in the nasal skin mass were S100 positive and melanoma negative (Figure 4B). Neither cell type was labeled by cytokeratin and melanin A. Normal melanocytes in the skin of this case were positive for wave proteins, melanoma, and melanin A and negative for S100.

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The thoracic skin mass consisted of spindle-shaped to round epithelial tumor cells that extended into the subcutaneous tissue in the dermis. The tumor cells formed multiple cysts in a whorled arrangement, not differentiated towards the hair follicle triad, keratinous horn cysts, and hair keratinization (Figures 5A and 5B). The cells had little cytoplasm and contained only a small amount of melanin, and the nuclei were elongated and shuttle-shaped. Degenerated inflammatory cells and necrotic debris were seen in the center of the cysts, while melanin-rich macrophages were seen in the interstitial and cyst centers. Tumor cells were labeled with cytokeratin, but not with other antibodies (Figure 5C). Based on the histological and immunohistochemical findings, a diagnosis of spindle cell hairy blastoma was made.

Subcutaneous abdominal mass showing papillary hyperplasia of the breast epithelium in the subcutaneous tissue. The mass was encapsulated, well-defined, and did not invade the surrounding normal tissue. In squamous metaplasia, the tumor cells showed papillary or tubular hyperplasia (Figure 6A). Cellular anisotropy was minimal, and mitotic figures were rare. The tumor cells were positive for keratin and negative for wave proteins (Figure 6B). Normal mammary glands were seen around the tumor tissue. Therefore, the abdominal mass was diagnosed as a simple adenoma of the mammary gland with squamous metaplasia.

Although other types of tumors have been reported in Eurasian otters, Trichoblastoma and mammary adenoma have not been reported. In addition, multiple tumors in otters are very rare, although cholangiocarcinoma, smooth muscle tumor, and pheochromocytoma have been reported in sea otters. In the present case, three tumors were observed. As this is the first report of a Trichoblastoma or mammary adenoma in a Eurasian otter, the occurrence of such a case is of value.

Melanoma originates from melanocytes and is one of the tumors of the hair skin, mouth, eyes, and feet. They have been reported to be common in several domestic animals such as dogs, cats, horses, and pigs, as well as in wild animals such as the horned horse (Connochaetes taurinus), the white tiger (Panthera tigris) and the rhesus monkey (Macaca mulatta). In dogs, melanomas have cellular variants, such as balloon and spindle types. Several criteria for malignancy of melanoma in dogs are well known, for example, mitotic figures above 3/10 high per field indicate malignancy. In a previous case of malignant melanoma in an 11-year-old Eurasian otter, tumor cells presented as subcutaneous nodules in the right and left thoracic vertebrae and metastasized to the lymph nodes and liver. Malignant melanoma in animals can metastasize to local lymph nodes and organs via lymphatic vessels. In the present case, primary tumor cells were found in the nasal mass and metastasized to the liver, lung, kidney, adrenal gland, breast, and left mandibular lymph node via lymphatic vessels or blood vessels. The tumor melanocytes may have initially metastasized to the left mandibular lymph node because the lymph node structure was almost completely replaced by tumor cells. Since the animal had hemorrhagic ascites and multiple friable liver nodes, the cause of death may have been anemia due to hemorrhage from the liver nodes and wasting due to cachexia caused by the malignancy.

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Melanocytic tumors in animals usually react with wave proteins, melanin A, PNL2, S100, TRP-1, and TRP-2. In humans, HMB-45 and tyrosinase markers are also useful. In the present case, vimentin, melanoma, and S100 immunolabeling were performed on tumor melanocytes, but not on melanin A. On the other hand, normal melanocytes were positive for both melanoma and melanin A. Based on these results, we suspect that melanin A expression in otter tumor melanocytes may be reduced or lost. In humans and dogs, melanin A is less responsive to several types of melanoma compared to other melanocyte markers. Using several antibodies, such as wave protein, melanoma A, melanoma, and S100, it may be important to diagnose malignant melanoma in Eurasian otters.

Spindle cell Trichoblastoma, previously classified as a basal cell tumor in cats, is most commonly seen in cats. In the present case, the tumor cell morphology of the thoracic skin mass included long spindle-shaped nuclei with sparse cytoplasm and only a small amount of melanin. In addition, the tumor exhibited multifocal cystic lesions and a whorl-like appearance. These findings were similar to those of the spindle cell type of Trichoblastoma in cats. Since no trochoidal differentiation of hair follicles, keratinous horn cysts, and hair keratinization were seen in this case, the possibility of hair epithelioma was excluded.

Mammary tumors are common in female domestic animals, but rare in otters. In this case, the tumor appeared in the subcutaneous region of the abdomen. Epithelial tumor cells between the normal mammary glands did not show any malignant features. A new classification of mammary tumors in dogs [6] was reported by Goldschmidt et al. Based on the above findings and diagnostic criteria, a diagnosis of mammary adenoma was made

Appendices

References

1. Acharjyo, L. N. and Mishra, C. G. 1983. A note on the longevity of two species of Indian otters in captivity. J. Bombay Nat. Hist. Soc. 80: 636.

2. Adetunji, S. A., Krecek, R. C., O’Dell, N., Prozesky, L., Steyl, J. and Arenas-Gamboa, A. M. 2018. Melanoma in golden and king wildebeests (Connochaetes taurinus). J. Zoo Wildl. Med. 49: 134–142.

3. Bae, I. H., Pakhrin, B., Jee, H., Shin, N. S. and Kim, D. Y. 2007. Hepatocellular adenoma in a Eurasian otter (Lutra lutra). J. Vet. Sci. 8: 103–105.

4. Bartlett, S. L., Imai, D. M., Trnpkiewicz, J. G., Garner, M. M., Ogasawara, S., Stokol, T., Kiupel, M., Abou-Madi, N. and Kollias, G. V. 2010. Intestinal lymphoma of granular lymphocytes in a fisher (Martes pennanti) and a Eurasian otter (Lutra lutra). J. Zoo Wildl. Med. 41: 309–315.

5. Frazier, K. S., Herron, A. J., Hines, M. E. 2nd. and Altman, N. H. 1993. Immunohistochemical and morphologic features of an intradermal nevocellular nevus (benign intradermal junctional melanocytes) in a rhesus monkey (Macaca mulatta). Vet. Pathol. 30: 306–308.

6. Goldschmidt, M., Peña, L., Rasotto, R. and Zappulli, V. 2011. Classification and grading of canine mammary tumors. Vet. Pathol. 48: 117–131.

7. Goldschmidt, M. H. and Goldschmidt, K. H. 2017. Epithelial and melanocytic tumors of the skin. pp. 88–141. In: Tumors in Domestic Animals, 5th ed. (Meuten, D. J. ed.), John Wiley & Sons, Ames.

8. Goldschmidt, M. H., Dunstan, R. W., Stannard, A. A., Tscharner, C. V., Walder, E. J. and Yager, J. A. 1998. Histological Classification of Epithelial and Melanocytic Tumors of the Skin of Domestic Animals. pp. 18–101. In: World Health Organization International Histologic Classification of Tumors of Domestic Animals, 2nd ed, Vol. 3 (Schulman, F. Y. ed.), Armed Forces Institute of Pathology, Washington, D.C.

9. Jungbluth, A. A. 2008. Serological reagents for the immunohistochemical analysis of melanoma metastases in sentinel lymph nodes. Semin. Diagn. Pathol. 25: 120–125.

10. Mason, C. F. and Macdonald, S. M. 1986. LUTRA LUTRA. pp. 7–45. In: Otters: Ecology and Conservation (Mason, C. F. ed.), Cambridge University Press, Cambridge.

11. Nakamura, K., Tanimura, H., Katsuragi, K., Shibahara, T. and Kadota, K. 2002. Differentiated basal cell carcinoma in a Cape clawless otter (Aonyx capensis). J. Comp. Pathol. 127: 223–227.

12. Ramos-Vara, J. A. and Miller, M. A. 2011. Immunohistochemical identification of canine melanocytic neoplasms with antibodies to melanocytic antigen PNL2 and tyrosinase: comparison with Melan A. Vet. Pathol. 48: 443–450.

13. Rao, A. T., Achaejyo, L. N. and Mohanty, A. K. 1991. Malignant melanoma in a white tiger. Indian J. Vet. Pathol. 15: 113–114.

14. Smedley, R. C., Spangler, W. L., Esplin, D. G., Kitchell, B. E., Bergman, P. J., Ho, H. Y., Bergin, I. L. and Kiupel, M. 2011. Prognostic markers for canine melanocytic neoplasms: a comparative review of the literature and goals for future investigation. Vet. Pathol. 48: 54–72.

15. Smith, S. H., Goldschmidt, M. H. and McManus, P. M. 2002. A comparative review of melanocytic neoplasms. Vet. Pathol. 39: 651–678.

16. Spangler, W. L. and Kass, P. H. 2006. The histologic and epidemiologic bases for prognostic considerations in canine melanocytic neoplasia. Vet. Pathol. 43: 136–149.

17. Stetzer, E., Williams, T. D. and Nightingale, J. W. 1981. Cholangiocellular adenocarcinoma, leiomyoma, and pheochromocytoma in a sea otter. J. Am. Vet. Med. Assoc. 179: 1283–1284.

18. Weber, H. and Mecklenburg, L. 2000. Malignant melanoma in a Eurasian otter (Lutra lutra). J. Zoo Wildl. Med. 31: 87–90.

19. Williams, T. D. and Pulley, L. T. 1981. Leiomyomas in two sea otters, Enhydra lutris. J. Wildl. Dis. 17: 401–404.

Naoyuki FUKE ¹, Nguyen Van DIEP ^1,2, Takuya HIRAI ¹, Mathurot SUWANRUENGSRI ¹, Uda Zahli IZZATI ¹ and Ryoji YAMAGUCHI ¹

1. Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan

2. Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Trau Quy, Gia Lam, Hanoi 10000, Vietnam