**1. Introduction**

In humans, a few types of tumors are identified as odontogenic ghost cell lesions (OGCL) of the maxilla and mandible. This includes calcifying odontogenic cysts (COC), dentinogenic ghost cell tumors (DGCT), and ghost cell odontogenic carcinoma (GCOC) [1]. DGCT is a benign but locally infiltrative neoplasm of odontogenic epithelium. It is a rare tumor in humans with very limited reports. A ghost cell is an enlarged epithelial cell having an eosinophilic cytoplasm with a faint nucleus outline or no nucleus [2]. It is associated with a marked aberrant keratinization. DGCT has been described as a rare form of ghost cell lesion, accounting for 3–5% of all cases involving ghost cell lesions [3,4].

For OGCL in humans, the prognosis and recurrence rate may differ according to the type of tumor. For COC, prognosis is considered excellent and the recurrence rate is low. When recurrence of COC occurs, it typically involves elderly persons [5]. Recurrence in

**Citation:** Salleh, A.; Zainuddin, Z.Z.; Tarmizi, R.M.M.; Yap, C.K.; Jeng, C.-R.; Zamri-Saad, M. Dentinogenic Ghost Cell Tumor in a Sumatran Rhinoceros. *Animals* **2021**, *11*, 1173. https://doi.org/10.3390/ ani11041173

Academic Editors: Alejandro Suárez-Bonnet and Gustavo A. Ramírez Rivero

Received: 29 January 2021 Accepted: 16 April 2021 Published: 20 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

young persons is rarely reported [6]. For DGCT, reports on recurrence rates range between 33% and 73% [7]. Surgical removal of DGCT involving an extensive procedure usually results in a low recurrence rate, while simple enucleation of the tumor usually results in a higher recurrence rate. Recurrence may occur within 5 to 10 years [8]. GCOC has a 73% five-year survival rate, and recurrence is reported to be common [9].

To our knowledge, DGCT has never been documented in animals. This article reports the first case of DGCT in an animal.

#### **2. Description of the Case**

A female Sumatran rhinoceros (*Dicerorrhinus sumatrensis*) weighing 508 kg and estimated to be between 25 and 30 years old was managed in a one-hectare forested paddock at the Tabin Wildlife Reserve, Sabah, Malaysia. In January 2017, it showed signs of difficulty in mastication, especially chewing on larger stems. Subsequently, in February 2017, it developed a 5 cm left unilateral, infraorbital and maxillary swelling with epiphora. It was treated with oral flunixin meglumine (Banamine® at 1500 mg per day for 3 days, and oral amoxicillin and clavulanate potassium (AugmentinTM) for five consecutive days. However, within a month, the swelling rapidly developed into a firm mass measuring about 15 cm in diameter, which later ruptured to discharge a mucopurulent exudate. In addition to wound cleaning twice a day, the rhinoceros was treated with oral amoxicillin and clavulanate potassium (AugmentinTM) at 25 mg/kg for 5 days, and parenteral dexamethasone (Dexadreson®) at 0.1 mg/kg intramuscularly for 3 days. Despite the treatment, the wound did not show any improvement and eventually became a 5 cm over-granulated open wound with blood-tinged nasal discharge from the left nostril. At this point, the appetite and body weight were slightly reduced, while the right jaw was predominantly used for mastication.

*Staphylococcus* sp. was isolated from the swab sample of the open wound, while an antibiotic sensitivity test showed resistance to amoxicillin-clavulanate acid but susceptibility to enrofloxacin and cephalosporin. In April 2017, a radiograph revealed a unilocular radiolucent area surrounding the 2nd and 3rd maxillary cheek teeth, suggestive of a cys<sup>t</sup> (Figure 1A). This cys<sup>t</sup> was connected to the paranasal sinuses by an oronasal fistula. A radiopaque fragment was noted dorsal to the 3rd maxillary cheek tooth, indicating a fracture of alveolar bone. The rhinoceros was orally treated with dexamethasone, AugmentinTM, lactated Ringer's solution, dextrose, Duphalyte, vitamin K, iron supplement, and phenylbutazone.

Dental extraction surgery was performed with peri-operative treatment comprising flunixin meglumine and enrofloxacin. Three cheek teeth (1st, 2nd, and 3rd cheek teeth) were successfully extracted in the surgery. All the extracted teeth had yellowish expansile solid masses around the roots. However, the oronasal fistula was not examined, as it could not be reached through the alveolar opening. For post-operative treatment, phenylbutazone, enrofloxacin, ceftiofur, and oral rinse were administered.

Thirty minutes after the recovery from anesthesia, the animal regained normal appetite. Wound cleaning, mouth wash, and parenteral enrofloxacin once daily, every other day were continued. However, the open wound, nasal discharge, and epiphora persisted. Thus, the antibiotic was changed to ceftiofur on day 8 after dental extraction. The bodyweight increased to 512 kg 7 days after the surgery. The intraoral granulation tissue eventually subsided. However, between May and June 2017, the animal showed occasional epistaxis and dyspnea, while the cutaneous mass aggressively grew larger. The rhinoceros was euthanized by intravenous administration of detomidine, ketamine, and pentobarbitone.

**Figure 1.** Radiographic, gross, and routine histopathological findings in a Sumatran rhinoceros with a dentinogenic ghost cell tumor. (**A**) Left-lateral view radiograph taken in April 2017 showing a unilocular radiolucent cys<sup>t</sup> (arrow) at the left maxilla. (**B**) The infraorbital mass in June 2017 measuring 21 cm × 30 cm. a: anterior horn, b: posterior horn, c: left upper eyelid. (**C**) Nests of neoplastic squamous cells (long arrows) surrounded by substantial compact fibrous stroma. Note the formation of keratin pearls (short arrows) embedded in a dentinoid material. HE (hematoxylin and eosin), bar = 100 μm. (**D**) Numerous islands of dentinoid material (long arrows) surrounded by loose and vascularized stroma (short arrows). HE, bar = 100 μm. (**E**) Neoplastic cells showing pleomorphism with basaloid- or stellate-reticulum-like appearance arranged in a nest. Vesicular nuclei can be observed. HE, bar = 20 μm (**F**) Ghost cell (long arrows) at the center of dentinoid material with cementum-like appearance (short arrow). HE, bar = 20 μm.

During the post-mortem examination, a tissue mass was visible around the dental extraction site, with the remaining 2nd and 3rd molars having enormous amounts of the expansile solid mass around the crowns and roots. The skin around the open wound was edematous and swollen. The infraorbital mass measured 21 × 30 cm (Figure 1B), with several open wounds of 1 to 7 cm in diameter. The mass extended ventrally and dorsal into the eyes. The size and color of the left masseter muscles were darker compared to the opposite side, suggestive of degenerative changes. Fistula between the maxilla and infraorbital mass was noted, while the left parotid gland was gritty with whitish spots. No metastasis to either adjacent or distant organs was observed. Samples from the infraorbital mass, left parotid gland, and left masseter muscle were collected and fixed in 10% neutral-buffered formalin, routinely processed, and stained with hematoxylin and eosin (HE), special histochemical Masson's trichrome and Von Kossa stains, and immunohistochemistry was conducted for detection of cytokeratin AE1/AE3, epithelial membrane antigen (EMA), p53, and S-100.

In the infraorbital mass, islands of neoplastic epithelium of various sizes were observed embedded or infiltrated in substantial amounts of either compact or loose fibrous stroma (Figure 1C). In some areas, the stroma was extensively loose with increased vascularization (Figure 1D). The neoplastic cells showed an infiltrative growth pattern arranged in strands, unsuccessful anastomosing, or medusa-like patterns. A long trabecular arrangemen<sup>t</sup> of tumor cells was observed. Multifocal squamous metaplasia or keratin-like material deposition was noted in the centers of the tumor islands. The tumor cells could be seen surrounding and embedded in numerous islands of dentinoid material. In addition, accumulation of pale eosinophilic ghost cells and a whirl-like arrangemen<sup>t</sup> of keratin-like material infiltrating the dentinoid material were noticeable. At high magnification, the neoplastic cells showed pleomorphism with a basaloid- or stellate-reticulum-like appearance with vesicular nuclei, usually arranged in a nest (Figure 1E). Ghost cells, characterized by large, eosinophilic cells that contained either the outline of a nucleus or no nucleus, were present at the cementum-like appearance of the dentinoid materials (Figure 1F). Mitoses were occasionally seen. The mitotic count, determined using a previously described method, was a low count of 2 [10]. The dentinoid was further confirmed by positive staining using Von Kossa stain to indicate the presence of calcium, and blue staining by Masson's trichrome stain. Most of the keratin and ghost cells lacked calcium, as indicated by the negative staining by Von Kossa stain (Figure 2A) and red staining by Masson's trichrome stain (Figure 2B). Some keratin pearls were observed without dentinoid formation, but they were surrounded by substantial amounts of neoplastic epithelial cells. The left masseter muscle was mildly degenerated but showed no evidence of invasion by neoplastic cells, while the left parotid gland was severely calcified.

The neoplastic epithelial cells showed intense intracytoplasmic immunodetection of cytokeratin AE1/AE3 but were negative for EMA and S-100. All keratin pearls, including those found inside the dentinoid material, and most of the ghost cells, showed intense staining with cytokeratin AE1/AE3 (Figure 2C), mild staining against S-100 (Figure 2D), and negative against p53 and EMA.

The differential diagnoses for this case included ghost cell odontogenic carcinoma (GCOC), dentinogenic ghost cell tumor (DGCT), craniopharyngioma, primary intraosseous squamous cell carcinoma (PIOSCC), squamous cell carcinoma (SCC), and ameloblastoma. The radiology and histopathology examinations established the diagnosis of DGCT.

**Figure 2.** Special histochemical and immunohistochemistry findings in a Sumatran rhinoceros with a dentinogenic ghost cell tumor. (**A**) Brown stain of Von Kossa indicating the presence of calcium in the dentinoid material (long arrow), while most keratin pearls were devoid of calcium (short arrows). Von Kossa, bar = 100 μm. (**B**) Bone tissue stained in blue and keratin stained in red with Masson's trichrome. Masson's trichrome, bar = 50 μm. (**C**) Intense intracytoplasmic staining for cytokeratin AE1/AE3 in the neoplastic squamous cells (long arrows) and keratin pearls (short arrows). AE1/AE3, bar = 50 μm. (**D**) Mild positive staining for S-100 in the ghost cells and keratin (arrows) located inside the dentinoid material. S-100, bar = 50 μm.
