Case History

Melanomas are malignant neoplasm orig- inating from melanocytes (Nishiya et al., 2016). They have been described in mam- malians, reptiles and  fishes  (Kimberly  et al 2015; Rahmati-Holasoo et al., 2015; Nishiya et al., 2016). These tumors were less commonly reported in birds. How- ever, they have been identified in a vari-  ety of avian species, including African  grey parrot (Psittacus erithacus erithacus) (Shrader et al., 2016), thick-billed parrot (Rhynchopsitta pachyrhyncha) (Guthrie et al., 2010), zebra finch (Taeniopygia gut- tata) (Irizarry-Rovira et al., 2007), seagull (Larus fuscus) (Costagliola et al., 2001), mandarin duck  (Aix  galericulata)  (Reid  et al., 1993), merlin (Falco columbarius) (Barlow and Girling, 2004), rock hopper penguin (Eudyptes chrysocome)  (Duncan et al., 2014), macaroni penguin (Eudyptes chrysolophus) (Duncan et al., 2014), and Humboldt penguin (Spheniscus humboldti) (Duncan et al., 2014). In this report, we de- scribe cutaneous malignant melanoma in a mallard duck (Anas  platyrhynchos).

Clinical Presentation

In August 2018, a 2-year-old female mal- lard duck (Anas platyrhynchos), at the Qaz- vin nature village was observed with a large palpable and firm mass on the ventral por- tion of the neck (Fig. 1a). Within the previ- ous 1-month period the mass had become distinct and grew larger. No other physical abnormalities were seen. On presentation, the bird was quiet and alert. Appetite and stool appearance were normal. The duck was fed a diet of seeds and plants. Final-  ly, surgical removal of the neoplasm was elected.  The  bird  was  anesthetized   with

Diazepam/Ketamine. A skin incision was made on the ventral surface of the mass and blunt dissection was performed to separate the mass. The incision was sutured by a simple interrupted suture pattern. The mass was removed for histopathological evalu- ation and the bird recovered uneventfully. Enrofloxacin (10 mg/kg IM, once daily for 5 days) was administered IM.

Diagnostic Testing

On gross examination, the mass was firm, black and 10 × 7/5 × 3 cm in size. Tissue samples of the mass were fixed in 10% neutral buffered formalin and routinely processed, dehydrated and embedded in paraffin wax, sectioned at 5 μm thickness (Rotary Microtome RM2 145; Leica) and stained with hematoxylin-eosin (H&E). Additional sections were probed immuno- histochemically for S-100 and Melan-A as described previously (Ramos-Vara et al., 2000). Histopathologically, the mass was mainly composed of infiltrative, pigment- ed melanocytes that extended from superfi- cial to deep dermal regions and had effaced normal tissue architecture (Fig. 1b-c).  In the superficial dermis, the neoplastic me- lanocytes were arranged in various sizes of nests and lobules (Fig. 1b). The neoplastic cells coalesced in the deep dermis, forming dense sheets separated by collagenous stro- ma (Fig. 1c).

Microscopic examination of the sections revealed anaplastic, epithelioid, round to oval to polygonal cells, ranging in size from 18 µm to 90 µm in diameter (giant cells), with eosinophilic cytoplasm containing variable amounts of brown to black    gran-

Fig 1. (a) The mass on the ventral portion of the neck. (b-f): Histopathological sections of Melan ma, hmatoxylin and eosin. (b) Variably sized nests of neoplastic cells in the dermis (4x). (c) Dense sheets of the neoplastic melanocytes in the deep dermis. S: stroma (10x) (d) The cytoplasm of cells containing variable amounts of brown to black granules of melanin (40x). (e) Round (large arrowhead) to oval (small arrow) to pleomorphic (large arrow) nuclei with four prominent nucle- oli (small arrowhead) (60x). (f). Pleomorphic cells (arrow and arrowhead). Inset: multinucleated giant cell (arrow)   (60x).

Fig . 2.       (a-b): Histopathological sections of Melanoma, hematoxylin and eosin. (a) multinucleated giant cell (arrow) (60x). (b)

The mitotic figure (arrowhead) is lesser that 10 in HPF (60x). (c-d): IHC, Immunoreactivity for Melan-A (c) and S-100 (d) in the cytoplasm of the neoplastic cells (40x)

ules of melanin (Fig. 1d). The nuclei were round to oval to pleomorphic, ranging from 8 µm to 45 µm in diameter with one to four prominent nucleoli (Fig. 1e). Markedly multinucleated, pleomorphic and  bizarre- ly shaped cells were also seen. Scattered multinucleated giant cells contained up to   4 nuclei (Fig. 1f, 2a). The number of mitot- ic figures per 10 high-power fields varied from 3-5 throughout the entire tumor (Fig. 2b). The mass showed more than 80% im- munoreactivity for S-100 and Melan-A in the cytoplasm of the neoplastic cells. The histopathologic and IHC findings support- ed a final diagnosis of cutaneous malignant melanoma.   The   mallard   duck  suddenly

died and internal tissues of the bird were not available for examination.

Assessments

Avian melanoma may originate from the skin, liver, lung, beak, eyes, and adrenal glands, and is usually reported to be malig- nant (Reid et al., 1993; Barlow and Girling, 2004; Irizarry-Rovira et al., 2007). Similar to present case, malignant melanomas tend to be fast-growing tumors, and often are pigmented (grey, brown, or black). Me- lanocytic tumors vary in size from small, pigmented macules, to larger masses which

may be 5 cm or more in diameter (Gold- schmidt and Goldschmidt, 2017). Large size of melanoma is considered poor prognostic factor (Smith et al., 2002). In dogs, melano- mas that have at least 1 cm diameter are de- scribed as malignant neoplasms (Meyrer et al., 2016). Additionally, in mandarin duck, neoplasm over 4 cm in diameter was diag- nosed as a malignant melanoma (Reid et  al., 1993). Large size of the cutaneous mass in the present study supports malignant be- havior of this neoplasia. The marked cellu- lar pleomorphism of the melanoma in our case, which warrants description of an an- aplastic malignant melanoma, is similar to other cases of avian malignant melanomas (Irizarry-Rovira et al., 2007; Goldschmidt and Goldschmidt, 2017). Also, the ma- jority of cutaneous malignant melanomas similar to this case have nuclear atypia and multiple nucleoli (Goldschmidt and Gold- schmidt, 2017). As in the present report, some avian melanomas may be pleomor- phic in histologic sections, contain bizarre neoplastic cells, and/or contain multinu- cleated cells (Irizarry-Rovira et al., 2007). Nuclear atypia is a common term used by pathologists to help classify neoplastic le- sions (i.e., benign versus malignant) (Gold- schmidt and Goldschmidt, 2017). In ani- mal, skin melanocytic neoplasms, the most reliable histologic feature for distinguish- ing malignant from benign is the mitotic in- dex (Goldschmidt and Goldschmidt, 2017). In this case, the number of mitotic figures per 10 high-power fields varied from 3-5. In the World Health Organization’s Histo- logic Classification of Epithelial and Mela- nocytic Skin Tumors of Domestic Animals (Smith et al., 2002), three or more mitotic figures per 10 high-power fields indicate malignancy.  The  majority  of   melanocyt-

ic neoplasms are easily recognized by the presence of melanin pigment in combina- tion with histologic features (Goldschmidt and Goldschmidt, 2017). S-100 remains  the most sensitive marker for melanocyt-   ic lesions, while markers such as Melan-A demonstrate relatively good specificity but not as good as S-100. However, in dogs, Melan-A is highly  sensitive  and  specif-  ic for detecting melanocytic neoplasms, whereas S-100 has poor specificity (Smed- ley et al., 2011). S-100 and Melan-A have not been reliable markers for melanocytic neoplasms in several avian species, includ- ing the domestic chicken, zebra finch, and cormorant (Phalacrocorax carbo) (Reid et al., 1993; Irizarry-Rovira et al., 2007; Wil- liams et al., 2011). Even more interesting, immunohistochemical findings similar to the melanoma diagnosed in the mallard duck were described in a seagull (Larus fuscus) and showed moderate immunore- activity for S-100 in the cytoplasm of 80% of the neoplastic cells and marked intra- cytoplasmic positivity for Melan-A in all neoplastic cells (Costagliola et al., 2011). Regardless of the species, malignant mela- nomas have similar biological behavior in that they frequently recur and have a high rate of metastasis (Reid et al., 1993; Smith et al., 2002). The regrowth of malignant melanomas occurs after surgery. We were unable to find the cause of death. However, it appears regrowth of neoplasia and metas- tasis after surgery could be reasons for the duck’s death. The etiology of spontaneous melanomas remains unknown in most spe- cies (Smith et al., 2002). The major risk factors for human melanoma include fam- ily history, skin and mucosal pigmentation characteristics, sun exposure,  particularly to ultraviolet B-rays (UVB) (Nishiya et  al.,