Odontogenic and non-odontogenic oral tumours in non-domesticated members of the order

Word count: 20 179

Sam Boulanger Student number: 01206557

Supervisor: Prof. dr. Lieven Vlaminck Co-Supervisors: Dr. Gerhard Steenkamp Prof. dr. Sonja Boy Elke Pollaris, DVM

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Veterinary Medicine

Academic year: 2017 - 2018

Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third-party rights. Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis.

Preface

Writing this dissertation felt challenging and wouldn't be possible without the help of many. Therefore, I would like to express a word of gratitude to a few people in particular. Firstly, I would like to thank my two head supervisors, Dr. Gerhard Steenkamp and Prof. dr. Lieven Vlaminck, for all their support and feedback I got since the beginning of this little project. I consider myself very lucky being able to call them my supervisors. Their great experience in the field of veterinary dentistry and oral health gave me a lot of motivation to finish this project in a good way and I have big admiration for the work they deliver, projecting them as lead examples of my own personal professional ambition. I'm also very thankful to Elke Pollaris for the numerous hours she invested in correcting my work and for all her advice I got when I was in doubt. For the histopathological part of the project, I would like to thank Prof. dr. Sonja Boy and Prof. dr. Koen Chiers. Their help in the composition of survey questions about tumour characteristics is very much appreciated. All of these opportunities wouldn't be able without the great support of my beloved parents. As well my brother as myself always got every support to study whatever we wanted to, something which I realize isn't the case for everyone. They supported me in every possible way to fulfil my dreams and were the first to encourage me during hard moments. Last but certainly not least, a word of thank is necessary for my friend group 'Les Flutes'. Thank you for the unforgettable memories and tremendous feelings of deep friendship I experienced during six years of study. You are and always will be in the deepest of my heart.

Table of contents

Preface ...... 3 Table of contents ...... 4 Abstract ...... 6 Samenvatting ...... 6 Introduction ...... 7 Situation ...... 8 1.1 Order Carnivora ...... 8 1.2 Anatomy of oral structures ...... 9 1.3 Oral neoplasia...... 13 Strategy ...... 13 Literature review ...... 14 Oral tumours in domesticated carnivores ...... 14 1. Prevalence, differentiation and classification ...... 14 1.1 Odontogenic tumours ...... 14 1.2 Non-odontogenic tumours ...... 15 2. Pathological behaviour ...... 15 2.1 Squamous cell carcinoma ...... 15 2.2 Malignant melanoma ...... 15 2.3 Fibrosarcoma ...... 16 2.4 Papilloma ...... 16 2.5 Adenocarcinoma ...... 16 2.6 Mucoepidermoid carcinoma ...... 16 2.7 Osteoma ...... 16 2.8 Haemangioma ...... 16 2.9 Anaplastic carcinoma ...... 17 2.10 Fibromatous epulis of periodontal origin ...... 17 2.11 Amyloid-producing odontogenic tumour ...... 17 2.12 Gingival hyperplasia ...... 17 3. Symptoms and history ...... 17 4. Diagnostic approach ...... 17 5. Histological features ...... 18 5.1 Squamous cell carcinoma ...... 18 5.2 Malignant melanoma ...... 18 5.3 Fibrosarcoma ...... 18 5.4 Papilloma ...... 19 5.5 Adenocarcinoma ...... 19 5.6 Mucoepidermoid carcinoma ...... 19

5.7 Osteoma ...... 19 5.8 Haemangioma ...... 20 5.9 Anaplastic carcinoma ...... 20 5.10 Fibromatous epulis of periodontal origin ...... 20 5.11 Amyloid-producing odontogenic tumour ...... 20 5.12 Gingival hyperplasia ...... 20 6. Therapy ...... 21 6.1 Surgery ...... 21 6.2 Radiation therapy ...... 21 6.3 Chemotherapy ...... 22 7. Prognosis ...... 22 Materials and methods ...... 24 1. Survey outline ...... 24 2. Response...... 24 Results ...... 25 Discussion ...... 27 Conclusion ...... 30 References ...... 31 Appendix A: chart of the Carnivora order (ITIS, 2018) ...... 39 Appendix B: Reported cases of oral tumours in non-domesticated carnivorans described in literature, classified per tumour type ...... 40 1. Odontogenic tumours ...... 40 1.1 Amyloid-producing Odontogenic Tumour ...... 40 1.2 Calcifying epithelial odontogenic tumour ...... 40 1.3 Fibromatous epulis of periodontal origin ...... 40 2. Non-odontogenic tumours ...... 40 2.1 Papillomatosis...... 40 2.2 Squamous cell carcinoma ...... 42 2.3 Adenocarcinoma ...... 43 2.4 Mucoepidermoid carcinoma ...... 43 2.5 Anaplastic carcinoma ...... 43 2.6 Secondary carcinoma ...... 44 2.7 Malignant melanoma ...... 44 2.8 Haemangioma ...... 44 2.9 Fibrosarcoma ...... 44 Appendix C: Reported cases of oral tumours in non-domesticated carnivorans retrieved through survey ...... 45

Abstract

In this comprehensive literature review, an attempt was made to enlist all reported cases of oral tumours, as well from odontogenic as non-odontogenic origin, in members of the order Carnivora and classify them regarding different tumour types. Only cases supported by histopathological evidence were included in the study. To add up the number of collected case reports, surveys were send out to professionals, organisations and institutions experienced with carnivorans and/or (oral) tumours. A total number of 75 cases were included in the study, and consisting of multiple tumour types. Symptomatology, diagnostic approach, treatment therapy and outcome were investigated in comparison to the current knowledge of oral tumours in domestic cats and . Only a slight minority of were reported to undergo curative treatment (21%) and successful outcome was noticed in only one third of the treated individuals. The use of multimodality therapy may be a valuable option, but comprises financial and technical restraints. In a large number of cases (22 out of 75 cases), oral tumours were only identified at necropsy. In 9 cases the decision for euthanasia or palliative treatment was made based on poor prognosis or expected complications of treatment. Detection of oral tumours and their associated symptoms in an early stage should be strived for and could be achieved by solid retraining of staff members and regular examination of the oral cavity.

Samenvatting

In deze literatuurstudie werd getracht alle gerapporteerde gevallen van orale tumoren, van zowel odontogene als niet-odontogene oorsprong, in dieren behorend tot de orde Carnivora in kaart te brengen en in te delen volgens type tumor. Enkel gevallen ondersteund door histopathologisch onderzoek werden ingebrepen in de studie. Om bijkomende cases te verzamelen werd een zelfgecreeërde enquête opgestuurd naar professionelen en organisaties ervaren met Carnivora en/of orale tumoren. In totaal werden 75 cases verzameld met tumoren behorende tot verschillende types. Symptomatologie, diagnostische aanpak, behandeling en uitkomst werden onderzocht en vergeleken met de huidige kennis in orale tumoren van de gedomesticeerde hond en kat. Enkel in een kleine minderheid van de gevallen werd curatieve behandeling beschreven (21%) en succesvolle behandeling werd in acht genomen in een derde van de gevallen. Het gebruik van combinatietherapie kan een waardevolle optie betekenen, maar gaat gepaard met de nodige financiële en technische beperkingen. In een groot aantal cases (22 van 75 cases) werd een orale tumor pas geïdentificeerd na autopsie. In 9 gevallen werd geopteerd voor euthanasie of palliatieve behandeling ingesteld gebaseerd op slechte prognose of verwachte complicaties bij behandeling. Er moet gestreefd worden naar detectie van orale tumoren en bijhorende symptomen in een vroeg stadium, wat zou kunnen verwezenlijkt worden door middel van doelgerichte bijscholing van personeel en door regelmatig onderzoek van de mondholte.

6

Introduction

A great diversity of neoplasms has been reported in captive wild carnivores (Effron et al., 1977; Lombard and Witte, 1959; Owston et al., 2008) but only few of them originate from the oral cavity. This in contrast with domestic carnivores, where oral tumours represent 3-12% of the tumours in cats (Stebbins et al., 1989) and 5-7% in dogs (Frew and Dobson, 1992).

Odontogenic tumours in the oral cavity of the small domestic animals are rare (Fiani et al., 2011; Gardner, 1992; Walsh et al., 1987) and infrequently observed in wild Carnivora. Although odontogenic tumours are considered rare, various cases have been reported in animals throughout the last couple of decades. Further on, different histological types have been identified and classified. (Walsh et al., 1987)

For a long time, neoplasia was not regarded as a major threat concerning wildlife conservation. Recent discoveries about a certain number of neoplastic diseases with the ability to severely decrease population numbers have raised the awareness of the impact of cancer on conservation outcomes. (Curry et al., 2000; Gulland et al., 1996; Hawkins et al., 2006; Maccubbin et al., 1985; Martineau et al., 2002; McAloose and Newton, 2009)

A good example of the impact of certain neoplastic diseases on population numbers is devil facial tumour disease (DFTD), a contagious cancer in Tasmanian devils (Sarcophilus harrisii). This transmissible allograft cancer can spread through direct contact with diseased cells and behaves as a focal or multicentric neuroendocrine tumour. It can cause severe soft tissue malformations with a predisposition for the head and neck area. Metastasis, mostly in local lymph nodes and lungs, is found in up to 65% of affected animals. (Loh et al., 2006) Affected animals have a mortality rate of 100% due to complications caused by tumor growth or metastasis. Since the first observation of the disease in 1996, Tasmanian devil populations have declined by 53%. (McCallum et al., 2007) Mathematical modeling of this disease estimates a population decline of 90% in 60% of territories with affected animals and a reduction of the entire Tasmanian devil population by 70% over the next ten years. (Hawkins et al., 2006; Lachish et al., 2007; Pyecroft, 2007) The International Union for the Conservation of Nature and Natural Resources (IUCN) has enlisted the Tasmanian devil as endangered in 2008, while extinction of the Tasmanian devil is a possibility in the near future. Disappearance of this could interfere with Tasmania's ecosystem and would make DFTD as the first contagious cancer causing the extinction of a species. (Hollings et al., 2015)

In two studies, captive wildlife and domestic animals show similar rates of neoplasia. (Effron et al., 1977; Lombard and Witte, 1959) A variety of tumour cases have been described for captive wildlife and even less frequent tumour types have been detected in these animals. (McAloose and Newton, 2009) Occasionally, management practices in zoos, aquariums or sanctuaries can play a role in neoplasia development. The former use of megestrol acetate for contraception in exotic felids is linked with an increased risk of mammary carcinoma. (McAloose et al., 2007) Subcutaneous microchip implementation in zoo animals has been associated with soft tissue sarcomas in a few species. (Pessier, 1999; Siegal-Willott et al., 2007)

Although quite some studies have been done on oral neoplasia in domestic carnivores, odontogenic and non-odontogenic tumours in wildlife carnivores are slightly documented. A limited number of cases have been published since the major development of veterinary dentistry in 1970’s. (Bernstein and Schelling, 1999; Bossart, 1990; Broughton et al., 1970; Dadone et al., 2014; Dorso et al., 2008; Fecchio et al., 2015; Joslin et al., 2000; Kang et al., 2006; Lam et al., 2013; McNulty et al., 2000; Mylniczenko et al., 2005; Nelson et al., 2013; Samuel et al., 1978; Sato et al., 2002; Sladakovic et al., 2016; Sundberg et al., 2000; Wolfe and Spraker, 2007; Yanai et al., 2003) Due to their familiarity with humans, oral diseases of domestic carnivores are detected in an earlier stage. Oral problems of captive wild carnivores are mostly not detected by a clinician until the oral disease is so aggravated or chronically evaluated to a point of oral inability and depletion of strength. (Fagan et al., 1998) Almost

7

all oral tumours that have been seen in exotic carnivores are described in their domestic relatives. (Wiggs and Bloom, 2003) As such, the clinical approach of oral tumours in non-domesticated carnivores could be based on the current knowledge in domestic animals. An increased knowledge of the presentation, diagnosis and treatment options of oral neoplasm in exotic carnivores would benefit anyone who’s involved in their management or health care. (Fagan et al., 1998)

To date no study has attempted to classify and compare oral tumours described within members of the order Carnivora. Considering the limited number of reported cases, the primary aim of this study project is to create a comprehensive literature review where collected cases are analysed by their classification. This literature review could serve as a reference for wildlife practitioners when dealing with oral neoplasia. Treatment options and prognosis could be reviewed or considered based on their clinical descriptions in the literature.

A second objective of this study is to add extra cases through mining of University of Pretoria’s database as well as questionnaires to wildlife facilities/veterinarians. Situation 1.1 Order Carnivora The order Carnivora (from Latin carō "flesh" and vorāre "to devour"), belonging to the Class of Mammalia within the animal kingdom, includes 12 families and over 280 species. Members of this group are referred as carnivorans, not to be confused with the general term “carnivores” which comprises any meat-eating organism. Most animals belonging to the order Carnivora are strictly meat eaters, although a substantial number of them also feed on vegetation and are thus omnivorous (f.ex. arctos ssp.) or herbivorous (f.ex. melanoleuca). Due to evolutional specialization concentrating on the functionality of devouring meat, carnivorans have developed characteristic skull structures and dentition, which are further on described in “Anatomy of oral structures”.

Members of the order Carnivora are split into two suborders: (cat-like) and (- like). Extinct animals are not included in the following description. Feliformia are divided into six families: , , Herpestidae, Hyaenidae, and Nandiniidae. Eupleridae are a group of carnivorans endemic to Madagascar and strongly related to the Herpestidae. Herpestidae are mainly consisting out of moongooses (f.ex. Cynictis penicillata, edwardsii), (f.ex. Suricata suricatta), and dwarf (f.ex. obscurus). Felidae is the best known family within Feliformia and animals belonging to this family are the strictest carnivores of all terrestrial families in the order Carnivora. They can be split up in and . Pantherinae are often referred in popular language as "big cats" and comprise amongst other species the famous ( leo), tiger (Panthera tigris), jaguar (Panthera onca), leopard (Panthera pardus) and snow leopard (Panthera uncia). The other subfamily of Felidae is Felinae or non- pantherine cats, in which the (f.ex. Lynx lynx), ocelot ( pardalis), (Leptailurus serval), cheetah ( jubatus) or domestic cat ( silvestris catus) can be found. Hyaenidae has three genera: Crocuta (Spotted Hyaena or Crocuta crocuta), Hyaena (f.ex. Hyaena brunnea) and Proteles ( or Proteles cristatus). Although behaviour and morphology are similar to Caniformia, Hyaenidae are phylogenetically closer to Viverridae and Felidae. This is represented in grooming, defecating habits, parental behaviour, scent marking and mating, but for their hunting methods and feeding habits they are more similar to Caniformia. Of all the families of the Feliformia, Viverridae are the most primitive and are obviously less specialised in comparison to Felidae. Best known animals in this family are civets (f.ex. Civettictis civetta) and genets (f.ex. Genetta genetta). Nandiniidae consist of only one living species, the (Nandinia binotata). The suborder Caniformia counts nine suborders: , , , , Odobenidae, Otariidae, Phocidae, and Ursidae. Contrary to Feliformia, Caniformia also comprise semi-aquatic members within the suborder (Odobenidae, Otariidae, Phocidae, Lutrinae, Mustela lutreola, Neovison vison). Ailuridae only comprise the , the sole living representative of this family. Canidae can further on be grouped into thirteen genera with a certain number of

8

monotypic genera (Atelocynus, Cerdocyon, Chrysocyon, Cuon, Dusicyon, Lycaon, , Otocyon, ). The genus consists out of some well-known species such as the grey wolf (Canis lupus), golden jackal (Canis aureus), coyote (Canis latrans) and domesticated dog (Canis lupus familiaris). Another large genus is (true , f.ex. Vulpes vulpes, Vulpes zerda). The Ursidae family or is divided into four monotypic genus (Ailuropoda, Helarctos, Melursus and Tremarctos) and the genus Ursus, in which can be found the American black (Ursus americanus), brown bear (Ursus arctos), polar bear (Ursus maritimus) and Asian black bear (Ursus thibetanus). Phocidae, also known as earless seals or true seals, counts thirteen genera. Well known members of this family are the harbour or common seal ( vitulina), (f.ex. Mirounga angustirostris) and (Erignathus barbatus). Phocidae has to be distinguished from Otariidae, also called eared seals referring to the small external ear flaps visible on the head. Seven genera are identified in the Otariidae family which includes for example the California ( californianus) and brown ( pusillus). The Odobenidae family has only one living species: Odobenus rosmarus. Mustelidae is a diverse family and the largest one in the order Carnivora. It is divided into two subfamilies: Lutrinae; basically grouping -like carnivorans such as the Eurasian otter ( lutra), North American river otter ( canadensis) and (Enhydra lutris) and . This subfamily contains further on a variety of fifteen genera. The biggest genera are Martes (f.ex. Martes americana, Martes martes) and Mustela. Mustela contains seventeen species including polecats (f.ex. Mustela putorius), ermines (f.ex Mustela erminea), (f.ex Mustela nivalis) and minks (f.ex. Mustela lutreola). Other notable genera are (f.ex. Meles meles), Mellivora (Mellivora capensis), Neovison (f.ex. Neovison vison) and Taxidea (Taxidea taxus). The last two families are Mephitidae -often referred as (f.ex. mephitis) - and Procyonidae, a new world family generally omnivorous and including raccoons (f.ex. lotor), (Potos flavus), ringtails ( astutus), olinguitos ( neblina), olingos (Bassaricyon gabbii), ( narica) and cacomistles (Bassariscus sumichrasti).

1.2 Anatomy of oral structures

1.2.1 Oral cavity The oral cavity stretches out from the lips to the palatoglossal arch and is enclosed dorsally and ventrally by mucosal lining inside the lips and cheeks. The cavity's outer vestibule is limited by lips and cheeks and forms a small separated space from the teeth and gingiva. (Barnes et al., 2005) It includes the lips, hard palate (the bony front portion of the roof of the mouth), soft palate (the muscular back portion of the roof of the mouth), front two-thirds of the tongue, gingiva (gums), buccal mucosa (the inner lining of the lips and cheeks), and floor of the mouth under the tongue. (NCI, 2018)

1.2.2 Dentition Carnivoran teeth are divided into incisors, canines, premolars and molars. The characteristic sharp canines are used for the apprehension, killing and butchering of their prey. They have another important role in the retainment of the tongue and the positioning of the lips. The incisors are relatively small in comparison to the canines, premolars and molar. The main functions of these teeth are grooming, nibbling, biting and cutting. Incisors are single rooted teeth normally aligned in a scissor occlusion. Very prominent teeth in a carnivoran dentition are the carnassials, which are the upper fourth premolars and the lower fourth molars. They act as a pair of scissors when the jaw is closing due to their knife-like edges and are essential to cut off slices of meat. Other premolars are generally used for shearing, cutting and holding and contain one to three roots depending on their anatomical localisation and inter-species differences.(Harvey et al., 1990; Hillson, 2005)

9

Figure 1: skull of a wolf (Canis lupus): characteristic for carnivore dentition are the well-developed carnassials and canine teeth (Matthews and Preston, 2004)

The basic dental formula of carnivores is I 3/3, C 1/1, PM 4/4, M 3/3, but adaptations in the diverse diets of carnivorans have led to many variations on this formula. Some few species have adapted to highly specialised diets: panda bears (Ailuropoda melanoleuca) feed on bamboo, (Proteles cristata) on termites, (Odobenus rosmarus) on molluscs and crab-eating seals (Lobodon carcinophaga) on krill. Omnivores such as bears have carnassials with a slightly different morphology from strict carnivores. (Hillson, 2005) They are also used to, but not perfectly suited for, the devouring of solid plant material. This results in the swallowing of large, bit-sized pieces or crushed food that is not chewed carefully. (Feldhamer, 2007) An example of dentition adaptation within the same genus can be found in polar bears (Ursos arctos). Polar bears are, in comparison to other bears strictly carnivore with feeding habits based on seal flesh and blubber. This results in reduced molars and premolars, since vegetable food don't have to be grinded. (Sacco and Van Valkenburgh, 2004)

Figure 2: skull of a bear (Ursidae): Adaptations include undeveloped carnassial teeth and broad flat molars (Matthews and Preston, 2004)

1.2.3 Anatomy of the tooth and periodontium The tooth contains a crown which can be detected visually because of its exposition above the gingival margin and a root covered by periodontal tissues and situated below the gum level. The crown is coated in enamel, while the embedded root is covered by cement. The borderline between cement and enamel is at the cementoenamel junction. (Harvey et al., 1990) Enamel formation is initiated by ameloblasts before dental eruption. After eruption tooth damage can only be restored by remineralisation of the tooth (Nanci and Ten Cate, 2008), a process interaction between phosphorus, calcium and fluoride ions which finally results in ion deposition into crystal voids in the non-cavitated lesion.(Featherstone, 2004) The cement is formed by cementoblasts that are present in the parodontal ligament, which is the connective tissue between cement and alveolar bone.

10

Beneath this tooth surface lays the dentine which is the greater and main part of the tooth. Dentine is produced (primary dentine) and provided lifelong (secundary dentine) by odontoblasts. These cells are of neural crest origin and are lining the pulp chamber as well as a connexion with numerous microtubules that are being part of the dentine (70 000 per mm² in average). The pulp chamber contains dental pulp, which is composed of blood vessels, nerves and lymphatic tissue in a mesenchymal tissue matrix. The pulp chamber is large while the dentine layer is rather thin in juvenile animals, and due to continuously remodelling of dentine by odontoblasts it results in narrowing of the pulp chamber and widening of the dentine particularly during the first 24 months of age. The dental apex receiving the nerves, blood vessels and lymphatics will gradually close in a similar period of time.(Harvey et al., 1990)

The base of the dental crown forms a bulge just above the dental cervix and just above the gingival attachment. This bulge allows a good adaptation and protection of the gingiva during mastication; alimentary particles are deviated from the gingival sulcus. This gingival sulcus consists of a fine and shallow space separating the free gingival margin of the tooth. The epithelial attachment is situated at the end of this margin and inserts the gingiva on the tooth. (Hennet and Boutoille, 2013)

Enamel Crown Dentine

Dental pulp Dentine Gingival sulcus

Neck Gingiva

Parodontal ligament Root

Radicular cement Lamina cribriforme

Alveolar bone Apical delta

Figure 3: Anatomical representation of at tooth (first mandibular molar) and adjacent tissues in the dog (Hennet and Boutoille, 2013)

Enamel

Dentine

Gingival sulcus

Epithelium of the sulcus

Epithelial attachment Gingival epithelium

Alveolar bone

Parodontal ligament

Muco-gingival junction

Figure 4: Anatomical representation of the periodontium (Hennet and Boutoille, 2013)

11

The periodontium consists of gingiva, periodontal ligament, cement and alveolar bone. Its primary function is to assure attachment and support of the tooth. The gingiva surrounds the alveolar bone and the teeth. Two parts can be distinguished: the free gingival margin and the attached gingiva that is bound to the cement at the level of the dental cervix and to alveolar bone. The gingiva is separated from alveolar mucus by the mucogingival junction. The gingival sulcus is aligned internally by non-keratinised epithelium which allows the diffusion of sulcus fluid. This fluid is rich with immunoglobulins (Ig G in majority, Ig G and Ig M), plasma proteins (albumine, fibrinogen ...), antibacterial substances (protease, lysosym ...), antioxidants (ascorbic acid, α-Tocopherol ...) and cellular elements (neutrophils, lymphocytes, granulocytes, monocytes, desquamated epithelial cells and bacteria). It protects the periodontal ligament and epithelial attachment against bacterial infection and generated oxidative reactions. The periodontal ligament plays a key role in the periodontium and has several functions: fixation of the tooth in the alveolar bone, shock absorber of moving teeth, sensory mechanisms, formation and resorption of cement and adjacent bone. It consists of collagen fibres and elastic fibres that connect cement and lamina dura (internal cortex) of alveolar bone. Aside from fibres, the periodontal ligament also contains lymphatic and blood vessels, nerves, fibroblasts, mesenchymal cells capable of forming clastic or blast cells (fibroblasts, osteoblasts, cementoblasts, osteoclasts, cementoclasts). Alveolar bone is the part of maxilla or mandibula that forms and supports dental alveoli. The alveolar process is composed simultaneously with dental development and eruption and is resorbed progressively when the tooth disappears. It consists of compact bone situated in the periphery and spongy bone in the centre. It possesses two cortices: an external cortex covered with periosteum connecting with the mandibular or maxillary cortex, and an internal cortex forming the osseous margin of the alveolus where fibres of the periodontal ligament are attaching. (Hennet and Boutoille, 2013)

1.2.4 Anatomy of the skull Carnivorans have varied skull forms. The transverse glenoid is mostly well developed and the jaw is orientated in dorso-ventral direction. Jaws in carnivorans are unequal: the maxillar molar occlusal zone is wider than the mandibular counterpart, and the mandibula is shorter than the upper jaw. This phenomenon is described as anisognathism and is also present in bovine and equine species. (Wiggs and Bloom, 2003) Well-developed temporomandibular joints allowing high occlusive forces, wide vertical excursion but limited lateral excursion are required with the aim of entrapping pray and applying shearing forces. (Wiggs and Bloom, 2003) The temporal muscle enforces the jaw as the primary force and an obvious part of the surface of the skull is the sagittal crest, which is connected to the temporal muscle. Characteristic for carnivorans is the strong zygomatic arch and relatively large cranium. Carnivorans are considered to be intelligent animals. Most have brains of larger size; the turbinals and auditory bullae are large, while the ear structure contains a higher complexity than most other animals. (Stains, 1984; Vaughan et al., 2000)

b a c

Figure 5: Skull of an African lion (Panthera leo): a/ temporomandibular joint b/ sagittal crest c/ zygomatic arch (Matthews and Preston, 2004)

12

1.3 Oral neoplasia

1.3.1 Definitions Neoplasia of the oral cavity can be separated into two groups depending on the origin of the tumour: odontogenic and non-odontogenic tumours.(Theodorou et al., 2003) Neoplastic lesions developed from mesenchymal, ectomesenchymal and/or epithelial origin that still are, or have been, part of the tooth forming structures are categorised as odontogenic tumours. Consequently, these tumours are mainly observed in the soft tissue overlying tooth-bearing areas or alveolar mucosae in teeth-lacking region, or within the maxillofacial skeleton. Odontogenic tumours can develop at any age of an individual. (Barnes et al., 2005) The term non-odontogenic indicates that the tumour is composed of cellular constituents whose primary purpose is to form structures in the oral cavity with the exception of teeth or 6 tooth-related structures during odontogenesis, or the remnants of these structures after odontogenesis.

1.3.2 'Epulis' The term ‘epulis’ must be considered as a pure clinical descriptive term and mustn’t be associated with odontogenic or non-odontogenic origin alone. A histological review of 129 dogs appearing with epulides done by Verstraete et al. (1992) suggested that the majority of these cases can be classified as focal fibrous hyperplasia (43,5%), peripheral ameloblastoma (17,5%), peripheral odontogenic fibroma (16,9%) and pyogenic granuloma (1,95%). Further on, some of other odontogenic tumours (1,95%) and non-odontogenic tumours (18,2%) such as fibrosarcoma and squamous cell carcinoma which are not traditionally associated with the clinical appearance of an epulis, were diagnosed. (Verstraete et al., 1992) Therefore, the appearance of epulis-like lesions is advised to be accompanied by histopathological examination. For many years the nomination of benign gingival masses has been a point of discussion. A classification scheme created by Dubielzig et al. (1979) divided them into three categories: fibromatous epulis, ossifying epulis and acanthomatous epulis. Fibromatous and ossifying epulis were grouped under the human peripheral odontogenic fibroma (POF) by another group of authors because of similarities between the two veterinary tumours and the human POF. (Gardner, 1982, 1996; Verstraete et al., 1992) This nomination also led to the separation of reactive lesions provoked by plaque and calculus which were classified as focal fibrous hyperplasia (FFH). (Fiani et al., 2011b) This distinction between reactive lesions (FFH) and neoplastic lesions (POF) is important. Although POF is considered as the current term for the neoplastic lesions, fibromatous epulis of periodontal ligament origin is preferred by other authors. It is reported to reflect the stroma as having features of the periodontal ligament, while this would not be the case for the cellular fibrous stroma of POF. (Dubielzig et al., 1979; Head et al., 2002) To avoid further confusion in this classification scheme, fibromatous epulis of periodontal ligament mentioned by Head et al. (2003) is used in this dissertation to classify lesions formerly known as 'fibromatous epulis' or 'ossifying epulis'. The formerly known acanthomatous epulis is now named as canine acanthomatous ameloblastoma (CAA) because of the microscopic and clinical similarities with human ameloblastoma without specifying a subtype. (Fiani et al., 2011b) Strategy

Research was primarily conducted using online databases specialised in medical or veterinary topics. The following search engines were used: PubMed, Embase, Elsevier sciencedirect, Web of Science and the AAZV & EAZA annual meeting databases. Some journals were also hand-searched for relevant articles. These journals were Journal of Zoo and Wildlife Medicine, Journal of Veterinary Dentistry, Journal of Comparative Pathology and Journal of Wildlife Diseases. The content of articles was looked after the presence of oral tumours supported by histopathology in non-domesticated carnivorans. Bibliographies of those papers that match the inclusion criteria were evaluated to identify any further, relevant references.

Until today, 64 cases of oral tumours in non-domesticated members of the order Carnivora have been retrieved from literature. A detailed list with description of species name, age and sex of the animal, histopathological diagnosis, oral localisation, author(s), journal name and year of publication can be

13

found under 'Appendix A'. To collect additional cases, self-created surveys were drafted and send to several organisations/institutions frequently dealing with non-domesticated carnivorans or examination of oral tumours in animals. Literature review

Oral tumours in domesticated carnivores

1. Prevalence, differentiation and classification 1.1 Odontogenic tumours Figures about the prevalence of odontogenic tumours are generally rare and few retrospective studies exist. (Boehm et al., 2011; Fiani et al., 2011b; Schmidt et al., 2010; Verstraete et al., 1992) A retrospective case study of 1390 canine and 317 feline oral tumours by Boehm et al. (2011) revealed that 18% of the canine and 3,2% of the feline oral neoplasia turned out to be of odontogenic origin. The most common odontogenic tumour in dogs (67%) and cats (40%) was odontogenic fibroma. Ameloblastoma was the second most frequent observed odontogenic tumour in dogs (30%), while for cats this was ameloblastic fibroma (20%). In this study, all discovered odontogenic tumours in cats were of benign origin, while in dogs only 3 cases representing 1,2 % of odontogenic tumours were malignant. Another retrospective study by Schmidt et al. (2010) in 112 dogs up to the age of 12 months reported a prevalence of 38% for tumours of odontogenic origin within discovered oral tumours. 50% of the tumours of odontogenic origin were classified as fibromatous epulis of periodontal ligament origin (or peripheral odontogenic fibroma), followed by, in descending order, unspecified epulis/odontogenic tumours (24%), acanthomatous ameloblastoma (14%), ameloblastoma (7%) and odontoma (5%). No specific figures about prevalence in malignity were mentioned.

No differentiation is made between benign and malignant odontogenic tumours according to the veterinary WHO-classification of 2003. The differentiation of malignancy is therefore classified following the latest human WHO-classification. (Barnes et al., 2005) Benign neoplasms are divided into epithelial, epithelial and ectomesenchymal, and ectomesenchymal. Malignant neoplasms consist of two categories: odontogenic carcinomas and sarcomas. According to a retrospective study of 250 domestic dogs and 10 domestic cats, only 1,2% of the odontogenic tumours were defined as malignant after histopathological examination. (Boehm et al., 2011)

The classification of odontogenic tumours has been largely discussed throughout the last decades. (Gardner, 1992; Poulet et al., 1992; Walsh et al., 1987) A broad classification is one which divides tumours into three groups depending on whether the tumours are able to induce a stromal reaction – inductive and non-inductive – and depending on the origin of the tumour cells – epithelial or mesenchymal. (Gorlin et al., 1961; Walsh et al., 1987) Critics stated that this classification was tending to be confusing and was leading to such paradoxes as tumours that have large or even predominant mesenchymal components could still be classified as epithelial tumours. (Gardner, 1992) An alternative was suggested by separating epithelial, mesenchymal and mixed types of odontogenic tumours. (Gardner, 1992; Thoma and Goldman, 1946) Since 1976 the World Health Organisation (WHO) is publishing its own classification for odontogenic tumours of domestic animals. Their first classifications were very similar to the human-ones presented in 1971. (Boehm et al., 2011; Pindborg et al., 1971) In 2003, a new WHO-classification was presented for veterinary use only. (Boehm et al., 2011; Head et al., 2003) In this classification, odontogenic tumours are divided into 6 groups: tumours of odontogenic epithelium without odontogenic mesenchyme, tumours of odontogenic epithelium with odontogenic mesenchyme, tumours composed primarily of odontogenic ectomesenchyme, tumours derived from the tissues of the periodontal ligament, cysts of the jaw and finally tumour-like lesions. In this paper, the WHO-classification will be used as a reference to classify all collected cases.

14

1.2 Non-odontogenic tumours Oral tumours account for approximately 10% of all tumours in domestic cats, and approximately 90% of these oral tumours are malignant. Squamous cell carcinoma (SCC) is the most commonly encountered malignant oral tumour in domestic cats, making 60-70% of malignant oral tumours. Fibrosarcomas are the second most frequent oral neoplasia’s and are considered to represent 10-15% of oral tumours. Other notable non-odontogenic tumours encountered in the same survey are adenocarcinoma, osteosarcoma and salivary adenocarcinoma. (Stebbins et al., 1989) Benign tumours are mostly at least as common as malignant tumours in the dog. (Lascelles et al., 2011)

The most common malignant non-odontogenic tumours of the mandible and maxilla in dogs are, in descending order, malignant melanoma, squamous cell carcinoma and fibrosarcoma. Other malignant oral tumours include osteosarcoma, chondrosarcoma, anaplastic sarcoma, multilobular osteochondrosarcoma, intraosseus carcinoma, myxosarcoma, haemangiosarcoma, lymphoma, mast cell tumour, and transmissible venereal tumour. (Lascelles et al., 2011)

Following the classification proposed by Head et al. (2003) tumours of the upper alimentary tract are divided into eight histological types: epithelial tumours, neuroendocrine tumours, melanocytic tumours, mesenchymal tumours, granular cell tumours, tumours of bone, tumours of hematopoietic and related tissues and tumourlike lesions.

2. Pathological behaviour 2.1 Squamous cell carcinoma Squamous cell carcinoma (SCC) can appear anywhere in the oral cavity and shows a particular local invasiveness in bone tissue, with severe and extensive invasion in the cat. Even if precise causes of the disease have not been determined yet, a number of environmental or contributing factors have been described such as flea collars, high intake of canned food or canned tuna and exposure to household tobacco smoke. (Bertone et al., 2003; Snyder et al., 2004) Their metastatic rate in cats at diagnosis is rather low, but there is still some discussion about the true metastatic potential because long-term follow-up for metastasis is complicated by the difficult control of local disease. Results of few studies on metastasis in cats with oral SCC support the belief that the metastatic rate is low and that local disease is rather the cause of death than metastatic effects. (Hutson et al., 1992; Northrup N .C. et al., 2006; Postorino Reeves et al., 1993; Withrow and MacEwan, 2001) In dogs, non-tonsillar SCC was reported to have a metastatic rate of approximately 20% with rates depending on localisation in the oral cavity: rostral located oral tumours had lower metastatic rate than the caudal tongue and tonsil. (Theon et al., 1997b)

2.2 Malignant melanoma Although malignant melanoma is the most frequent oral tumour in dogs, it is uncommon in cats. (Farrelly et al., 2004) Oral melanoma is known to be a highly malignant tumour with high metastatic rate to the lymph nodes and lungs. (Kudnig et al., 2003; Overley et al., 2001; Williams and Packer, 2003) Reported in up to 80% of dogs, metastasis is depending on site, size and stage. Use of the clinical staging system composed by the World’s Health Organization (WHO) may benefit in prognostic interpretation of oral melanoma. (Blackwood and Dobson, 1996; Hahn et al., 1994; Kudnig et al., 2003; Overley et al., 2001; Owen, 1980; Proulx et al., 2003) Histopathological diagnosis of malignant melanoma can be challenging: in one third of all melanoma cases amelanotic melanoma is present. If dealing with undifferentiated or anaplastic sarcoma or epithelial cancer, underlying presence of malignant melanoma should be checked. (Liptak and Withrow, 2007) Malignant melanoma shows high immunoreactivity and therefore active research in the molecular approaches of treatment, especially in genetic immunotherapy, is intensively conducted. (Alexander et al., 2005; Bergman et al., 2004; Bergman et al., 2006; Bergman et al., 2003; Dow et al., 1998; Elmslie et al., 1994; Elmslie et al., 1995; Hogge et al., 1998; MacEwen et al., 1999; Macewen et al., 1986; Moore et al., 1991; QuintinColonna et al., 1996)

15

2.3 Fibrosarcoma On histology fibrosarcoma tends to have a benign appearance and histological diagnosis provided by pathologists often includes “fibroma” and “low-grade fibrosarcoma” due to doubt. This appearance frequently involves the hard palate and dental arcade between the canine and carnassial teeth of large-breed dogs and has been described as “histologically low-grade but biologically high-grade” fibrosarcoma. (Ciekot et al., 1994) Fibrosarcoma requires an aggressive treatment, particularly when fast tumour growth, recurrence or bone invasion is present. Despite its high local invasion, metastasis to the lungs and regional lymph nodes in dogs was reported in less than 30% of cases. (Hahn et al., 1994; Kosovsky et al., 1991; Schwarz et al., 1991a, b; Todoroff and Brodey, 1979; Wallace et al., 1992)

2.4 Papilloma Papilloma is caused by a viral agent (papovavirus) and transmitted horizontally by direct contact; however indirect spread is possible because of their ability to survive in the environment. (Roden et al., 1997) Papillomatosis mostly affects young animals. Spontaneous regression of the lesions within 4 to 8 weeks is seen in most affected animals. Significant side effects are uncommon, but occasionally marked involvement of the lesions can cause dysphagia or respiration problems. (Liptak and Withrow, 2007) Because of their self-limiting nature and easy visual detection, prevalence records may vary and are difficult to determine. The influence of papillomavirus in the development of cancer has been reported multiple times in cats and dogs, but the precise role of papillomavirus in cancer processes is still unclear. (Altamura et al., 2016; Luff et al., 2016; Munday, 2014; Munday and Kiupel, 2010; Munday et al., 2011; Thomson et al., 2016) A predisposition for oral SCC in individuals affected longer than 18 months has been suggested. (Regalado, 2016)

2.5 Adenocarcinoma Adenocarcinoma is an uncommon malignant tumour usually originating from palate and salivary gland. It has local invasive growth and metastasis to regional lymph nodes is common.

2.6 Mucoepidermoid carcinoma Mucoepidermoid carcinoma can be defined as a combination of squamous epidermoid cells, mucus- producing cells and intermediate type cells. Due to this characteristic several sections of the tumour have to be closely examined until all three components are observed. Pathological behaviour of this tumour varies from low grade (well differentiated) to high grade (poorly differentiated). The degree of malignancy depends on the degree of differentiation of the mucous cells, the number of well- differentiated epidermoid cells, anaplasia and the growth pattern. This growth pattern may vary from broad “pushing” invasion to infiltrative growth, while all cells are poorly encapsulated. (Head et al., 2002)

2.7 Osteoma Osteoma is a benign tumour and consists of normal mature compact bone and/or trabecular bone on histological appearance. (Fiani et al., 2011a; Goldschmidt and Thrall, 1985; Goudar et al., 2011; Misdorp and Vanderheul, 1976) It is a progressively slow growing mass and no clinical signs occur until it interferes with occlusal functioning of the jaw or nearby structures and tissues. (Dalambiras et al., 2005; Ogbureke et al., 2007) Predisposed localisations of oral osteoma are skull and maxillofacial bones. (Goldschmidt and Thrall, 1985) Osteoma has not been reported with metastasis, malignant transformation, bone destruction or bone lysis. (Baba and Catoi, 2007; Gassel and Huber, 2002; Woldenberg et al., 2005)

2.8 Haemangioma Haemangioma is a benign tumour of vascular endothelial origin occurring in a variety of sites. Despite their benign character, they can induce severe anaemia due to heavy blood loss. (Schoofs, 1997; Widmer and Carlton, 1990)

16

2.9 Anaplastic carcinoma Anaplastic carcinoma is a malignant epithelial tumour without further squamous or glandular differentiation. (Head et al., 2003) It is a very malignant, diffusely infiltrating and extensively metastasizing cancer causing death in a short period. Anaplastic carcinoma is not only highly invasive but also predominantly scirrhous which is not the usual feature in human anaplastic carcinoma. (Misdorp et al., 1973)

2.10 Fibromatous epulis of periodontal origin Fibromatous epulis of periodontal origin is a benign gingival proliferation appearing similar on macroscopical appearance to gingival hyperplasia. They are slow-growing and firm masses usually covered by intact epithelium and having a predilection for maxillary premolar teeth. (Yoshida et al., 1999)

2.11 Amyloid-producing odontogenic tumour Amyloid-producing odontogenic tumor (APOT) is distinguished by the proliferation of odontogenic epithelium along with intercellular deposition of amyloid materials. APOTs usually are reported with expansile growth, form solid or cystic masses, and typically behave as a benign tumour. They may show local invasion, incorporate teeth, and may progressively destroy the jaw and facial bones. Recurrence occasionally occurs following excision. (Dubielzig, 2002)

2.12 Gingival hyperplasia Gingival hyperplasia is a reactive proliferation of epithelium and may appear focal, multiple focal or generalized. Generalized hyperplasia may be induced by plaque accumulation, but also certain drugs (diphenylhydantoine, cyclosporine, amlodipine) can be the cause of these gingival enlargements. Treatment consists of removing the underlying cause or surgical excision in more developed cases. (Verhaert, 2001)

3. Symptoms and history In domestic cats and dogs, the presence of a mass in the mouth is usually noticed by the owner. Other symptoms that may occur in animals with an oral tumour are increased salivation, dysphagia or pain when opening the mouth, bloody oral discharge, facial swelling or exopthalmos, swollen lymph nodes in the cervical region and epistaxis. The combination of loose teeth and a general good dentition has to raise the clinician's attention for possible neoplastic bony processes. (Liptak and Withrow, 2007) Paraneoplastic syndromes are quite rare for tumours, although hypercalcemia and hyperglycemia have been reported. (Hutson et al., 1992; Padgett et al., 1997)

4. Diagnostic approach When an oral tumour is suspected to be in connection with bone tissue, radiographic examination under general anaesthesia is recommended. Useful projections are intra-oral, open mouth, lateral and ventrodorsal or dorsoventral. Bone lysis can be evaluated, keeping in mind that cortical destruction has to be 40% or more before being radiographically visible. However, bone invasion can’t be excluded by normal radiographs. Radiographs will facilitate clinical staging of the tumour and resection width when surgery is recommended. Involvement and dislocation of associated teeth can be revealed through intraoral radiographs. Possible secondary pathological jaw fractures can also be identified. The use of computer tomography (CT) can be a helpful indicator to determine more detailed information for staging or tumour extension into bone, nasal cavity, orbit or caudal pharynx because of its higher sensitivity. (Liptak and Withrow, 2007) Palpation of the regional lymph nodes for enlargement or asymmetry is advised as a possible predictor of metastasis; however it isn’t an accurate predictor. (Williams and Packer, 2003) These regional lymph nodes consist of the mandibular, parotid and medial retropharyngeal lymph node. Aspiration of the lymph nodes should be undertaken in all suspected cases of oral cancer, regardless of size or mobility. (Herring et al., 2002; Williams and Packer, 2003) Valuable staging information can be

17

obtained by en bloc resection of the lymph nodes, even though therapeutic effects are uncertain. (Herring et al., 2002; Smith, 1995) Golden standard in the diagnostic approach of oral tumours is biopsy. Incisional biopsies include large samples of healthy tissue at the edge and deeper areas of the lesion. It is recommended to biopsy through an intraoral incision, skin biopsies should never be undertaken because of the risk of tumour spreading. Excisional biopsies can be performed when dealing with smaller lesions, but awaiting biopsy results is strongly advised due to the possibility of an additional treatment plan. Aspiration or cytological touch of the tumour mass was believed not to provide useful information considering necrosis and inflammation of tissue. (Liptak and Withrow, 2007) Nevertheless, a recent study in 85 dogs and 29 cats reported a great agreement in diagnostic accuracy between cytological (fine needle aspiration, fine-needle insertion and impression smear) and histopathological diagnosis. It was suggested that cytological examination of oral tumours is an appropriate and a reliable diagnostic tool. (Bonfanti et al., 2015)

5. Histological features

5.1 Squamous cell carcinoma The typical histologic characteristics of SCC in cats consist of irregular cords and islands of pleomorphic squamous epithelial cells with abundant eosinophilic cytoplasm, prominent intercellular bridges and occasional formation of keratin pearls. (Bilgic et al., 2015; Stebbins et al., 1989) Extensive squamous differentiation is seen in many squamous cell carcinomas, even though only few foci are present in some tumours. Variable degrees of inflammation (neutrophils, plasma cells, lymphocytes), necrosis, ulceration and desmoplasia are featured on regular basis. (Bilgic et al., 2015) Invasion in nearby connective tissue, skeletal muscle or bone, with possibility of osteolysis or bone resorption, is common. (Martin et al., 2011) Canine oral SCC has a similar histological appearance, but can further on be differentiated in various histological subtypes comparable with human oral SCC. (Nemec et al., 2012) Histological differentiation can be difficult between canine oral SCC and canine acanthomatous ameloblastoma which may result in inadequate treatment therapy. Using expression profiles of cytokeratins and calretinin can help to distinguish between these two epithelial-derived neoplasms. (Fulton et al., 2014)

5.2 Malignant melanoma Small foci of up to 20 heavily pigmented cells may be observed in the basal levels of the epithelium of adjacent mucosae. Intraepithelial cells and cells situated in this junctional change have a different appearance. Intraepithelial cells may vary in size and shape of both cytoplasma and nuclei, while cells of the second type have a uniformly round or polygonal appearance with uniformly round or oval central nuclei. Infiltration into submucosa is common with possible further migration to the epithelium. A malignant melanoma is divided into lobules, and the cells are supported by a minimum of collagenous stroma. In tumours and between tumours the mitotic index and melanin content can differ. Sections can be bleached with 1 percent potassium permanganate if the nucleus is masked by pigment granules. Poorly pigmented and amelanotic tumours can make diagnosis complicated, but respectively the detection of melanophages and the use of Masson Fontana silver stain may help in revealing their true nature. (Head et al., 2002) Other helpful tools in the visualization of these tumours are electron microscopy and monoclonal antibodies for melanoma. (Berrington et al., 1994; Carpenter et al., 1980; Turk and Leathers, 1981) A general agreement has been made that basically all canine oral melanomas are malignant, but benign forms have been reported as well. (Bostock, 1979) Feline oral melanoma has comparable histological features to dogs, but highly pigmented and pleomorphic tumours are uncommon. (Patnaik and Mooney, 1988)

5.3 Fibrosarcoma Fibrosarcomas show numerous uniform to pleomorphic spindle cells separated by small amounts of collagen or surrounded by reticulin fibers in silver stained sections. The tumour consists of interlacing

18

bundles, of which some are cut longitudinally forming elongated cells and others are cut tangentially or at right angles forming round cells. Highly malignant tumours are characterized with numerous mitotic figures, infiltrative borders, pleomorphic cells, and even multinucleate giant cells. Histopathological features of feline fibrosarcoma resemble the canine form comprising densely packed pleomorphic fibroblasts in interwoven fascicles with variable amounts of collagen. (Head et al., 2002) Feline melanoma has up to five mitoses per high power field, and in two studies including 43 cases 3 tumours revealed a few multinucleated cells. (Kemp et al., 1976; Stebbins et al., 1989)

5.4 Papilloma Papillomatosis is determined on histology by thickening of the epithelium due to the promotion of epithelial cell proliferation by papillomaviruses (PV). If a marked proliferation is present, it can result in folding of the thickened epithelium and a papilloma. Viral replication in papillomas can appear on histology as PV-induced changes with the possibility of enlarged cells with a shrunken nucleus surrounded by a clear cytoplasmic halo (koilocytes), cells with increased quantities of grey or blue fibrillary cytoplasm, cells with intracytoplasmic inclusions or cells with enlarged vesicular nuclei. Observation of intranuclear inclusions can be made, even though these inclusions can be difficult to differentiate from nucleoli due to its transient appearance. PV-induced lesions frequently include clumping of keratohyalin granules in the granular cell layer. In general, lesions with more marked epithelial proliferation, such as papillomas, are in favor of greater viral replication and are more likely to exhibit PV-induced cell changes. This contrasts lesions with more modest epithelial proliferation (such as a viral plaque) involve less viral replication and occasionally exhibit PV-induced cell changes. PV-induced cell changes present within a lesion do not support automatically PV infection as cause for the lesion. Nevertheless, the presence of PV-induced cell changes does prove viral replication in this lesion and consequently suggests the fact that PV may have influenced normal cell behavior. (Munday et al., 2017)

5.5 Adenocarcinoma In adenocarcinoma, the acinar aspect is structurally dominant, although papilliferous proliferations may also be seen. Cells have a round to polygonal appearance, with basophilic cytoplasm and small oval nuclei. Clear cells have been identified in small salivary glands tumours related to the dog tongue. A great variation in degree of cellular atypia, mitotic activity and infiltrative growth has been observed, even within the same tumor, but in particular from one case to another. (Head et al., 2003)

5.6 Mucoepidermoid carcinoma Mucoepidermoid carcinoma can be defined as a malignant tumour distinguished by the presence of mucous cells and the formation of cysts. The tumour is bordered by occasionally keratinized squamous cells and intermediate cells with one of the structures having a dominant presentation. Malignancy is revealed trough the grouping of mucoid cells into nests, numerous atypical mitoses, large polymorphic nuclei and infiltrative growth. The predominance of the epidermoid component is characterized by stratification of squamous cells and their arrangements in cords. These squamous cells are seen with large vesicular nuclei and prominent nucleoli, while the presence of keratin is easy to detect. The cell cytoplasm may reveal PAS-positive mucopolysaccharide drops and contain smaller cysts than low-grade tumours. Rupture of these cysts may be possible, provoking a granulomatous reaction with giant cells and cholesterol clefts. (Head et al., 2003)

5.7 Osteoma Osteoma consists of growing bone tissue, initially cancellous bone and progressively becoming compact. In soft tissue spaces between bony trabeculae one or more centrally located small caliber blood vessel, a sparse population of spindle cells, and a moderately fibrillary connective tissue matrix are present. Hematopoietic elements and adipose tissue may be present as well in this area. Actively growing osteomas are characterized with a border of connective tissue layer resembling the periosteum, while newly formed trabeculae are slender and oriented perpendicular to the surface of the osteoma. Older and less superficial trabeculae have a thicker appearance and may show no orientation. Usually osteomas are organized in an orderly zonal pattern. In an active growing osteoma,

19

the periosteum is well differentiated and consists of both fibrous and osteogenic layers. A layer of peripheral trabeculae is formed by a border of typical osteoblasts depositing woven bone. Due to normal remodeling activity woven bone is replaced partially or complete by finely fibered lamellar bone forming deeper trabeculae. Osteomas with minor organization may also be seen. This type of osteoma includes poorly differentiated periosteal covering, absence of osteoblasts in the periosteum, irregular shaped and disoriented trabeculae, and infrequent remodeling and replacement by lamellar bone. (Thompson and Pool, 2002)

5.8 Haemangioma On histological appearance haemangioma comprises proliferating, vasoformative mesenchymal tissue forming capillary and cavernous vessels and, less frequent, arterial and venous structures. Pericytes and fibroblasts were described to encircle endothelial cells. (Calonje and Fletcher, 2007; Gross et al., 2007) Haemangiomas may be divided histologically as capillary, cavernous, arteriovenous, lobular, spindle cell or epithelioid subtypes. (Calonje and Fletcher, 2007; Gross et al., 2007; Vos et al., 1986; Warren and Summers, 2007)

5.9 Anaplastic carcinoma Anaplastic carcinoma is primarily characterized by irregular masses of pleomorphic cells ranging from polyhedral to spindle-shaped and showing anisocytosis. Nuclei comprise anisokaryosis and are sometimes found hyperchromatic, while the prominent nucleoli exhibit many mitoses. Multinucleate giant cells may be exposed, and areas of necrosis and hemorrhage are common. (Head et al., 2003)

5.10 Fibromatous epulis of periodontal origin The main histological characteristic is the presence of a mesenchyme suggesting the periodontal ligament. This mesenchyme consists of a dense cellularity composed of small stellate to spindle fibroblast cells regularly positioned in a dense fibrillar collagen background. Localized deposition of collagen matrix is observed regularly, and the matrix consistency varies from bone to cementum and dentin. The stroma is filled with evenly spaced large empty blood vessels. A secondary feature is the presence of a frequently seen odontogenic epithelium. Long fronds can sometimes be found attaching to the surface epithelium; although the surface gingiva is not always attended meaning these epithelial cords can also originate from the cell rests of Malassez within the periodontal ligament. (Head et al., 2003)

5.11 Amyloid-producing odontogenic tumour The two basic histological features of an amyloid-producing odontogenic tumour (APOT) are irregularly shaped islands or strands of squamous epithelium within a fibrous connective tissue stroma and deposits of amyloid associated with the epithelium; with some of the amyloid calcified. In some areas of the epithelium palisading of the basal cells may be exposed. Occasionally stellate reticulum occurs in the centre. Next to the epithelium, a collagenous matrix with the appearance of dentine is present focally. (Gardner et al., 1994) APOT is often related in veterinary literature to calcifying epithelial odontogenic tumour (CEOT) (Abbott et al., 1986; Stebbins et al., 1989; Walsh et al., 1987), but has specific microscopical differences with the human CEOT (Pindborg tumour) of man. The main difference on histological aspect is the appearance of the epithelium, which in the CEOT comprises sheets of eosinophilic epithelial cells that often show considerable nuclear pleormorphism. Epithelial cells can vary from small and resembling reduced enamel epithelium to hyperchromatic and palisaded basal cells occasionally found in APOT, do not occur in CEOT. Another difference is the presence of focal areas of stellate reticulum in APOT, but not exhibited in CEOT. Amyloid can be found in both tumours, sometimes calcified. (Gardner et al., 1994)

5.12 Gingival hyperplasia Excessive amounts of nearly normal-looking gingival collagen and an intact epithelium are seen in gingival hyperplasia. Oedema, vascular dilation, and/or perivascular lymphocytes can be present. In the case of proliferative gingivitis, a profuse infiltrate of lymphocytes and plasma cells are observed.

20

Frequently, complex folds that extend both inward and outward are formed by the epithelium (pseudoepitheliomatous hyperplasia). (Head et al., 2003)

6. Therapy Surgery and radiation therapy are the most used methods in the local control of oral tumours. They are the only treatment options with the opportunity of achieving curative effects.

6.1 Surgery Surgical resection is the fastest, most economical and most curative treatment method. Due to the frequent adherence of tumour tissue on bone, bony margins should be included when determining surgical resection width. Maxillectomy (segmental), mandibulectomy (hemi or segmental) and orbitectomy are advised for most oral tumours, especially in tumours non-reactive to radiation therapy or with extended bone invasion. Malignant oral tumours such as fibrosarcomas, squamous cell carcinomas or malignant melanoma’s require margins of at least 2 cm. For oral squamous cell carcinoma in cats, margins greater than 2 cm are strongly indicated due to the high rate of local recurrence. This wide size of margins is usually not possible in domestic carnivores because of significant morbidity effects. Therefore, margins of 1 cm are encouraged by some clinicians if broad margins are technically not possible. (Lascelles et al., 2011) Logically, results of oral surgery will be affected by more narrow margins. Benign lesions and rostral SCC in dogs can be treated successfully with segmental or rostral resections. More aggressive tumours such as fibrosarcomas or tumours with pronounced caudal localisation demand large resections such as orbitectomy, hemimandubulectomy, hemimaxillectomy or radical maxillectomy. These procedures imply some morbidity, but are usually well accepted by cats and dogs. Most common peroperative complications are blood loss and hypotension and are mostly found during caudal mandibulectomy. (Lascelles et al., 2003) Owner satisfaction with the remaining life quality and cosmetic appearance of their pet can exceed 80%. (Lascelles et al., 2003; Northrup et al., 2006) Post-operative care has to be discussed with the owner because of possible temporary or permanent complications. Malocclusions and mandibular drift can cause trauma to the hard palate. Filling down of the damaging tooth in combination with root canal treatment is a possible resolution for this complication. In a situation of inability to eat or drink, feeding tubes have to be administered, but are usually not required after oral surgery in dogs. (Garrett et al., 2007) For tongue tumours, surgical resection is advised starting from the mobile rostral part. It is required to remove unilateral tumours not crossing the midline of the tongue with partial glossectomy of up to 60% of the tongue surface. Tumours located caudal to the midline or bilateral tumours necessitate larger resection, however this could interfere with basic functions of the tongue such as thermoregulations in dogs and grooming in cats. Case reports of resection or avulsion of the tongue varying from 50 - 100% in 5 dogs suggest more aggressive resections may be in favour of minimal postoperative complications and a decent life quality. (Dvorak et al., 2004) Short-term complications of glossectomy consist of ptyalism and dehiscence, while minor changes in drinking and eating habits are reported as long-term complications. (Lascelles et al., 2011) In patients with tonsillar SCC, bilateral tonsillectomy is advised due to the high frequency of bilateral disease. Cryosurgery can be used for lesions less than 2 cm in diameter with bony fixation or minimal invasion. However, serious side effects can be caused in more extensive lesions. Mandibular fractures or maxillary oronasal fistulas may be the result using too aggressive cryosurgery. In general, cryosurgery may not be used in tumours only involving soft tissues. (Liptak and Withrow, 2007)

6.2 Radiation therapy Radiation therapy can be used as primary treatment method or in addition to surgical resection. Sole therapy can be used for tumours that are radio responsive such as malignant melanoma, canine oral SCC and fibromatous epulis of periodontal ligament. In these tumours, tumour control is better with radiation therapy alone in the T1 and T2 stage. (Blackwood and Dobson, 1996; Theon et al., 1997a; Theon et al., 1997b) Adjunct radiation therapy is usually required for incomplete resected tumours or tumours with an aggressive local behaviour, since radiation therapy has specific locoregional

21

effectiveness. When treating resistant tumours, radiation therapy can be assisted by surgery, chemotherapy and radiation sensitizers such as gemcitabine and etanidazole in order to improve local tumour control and survival time (Evans et al., 1991; Hutson et al., 1992; Jones et al., 2003; LaRue et al., 1991; Ogilvie et al., 1993; Thrall et al., 1981) Fraction dose is in positive correlation with response rates, although one study didn't find any differences in survival time or local recurrence rate between two hypofractionated protocols and a conventional protocol. (Overgaard et al., 1986; Proulx et al., 2003) Tonsillar SCC can be controlled locally by regional radiation of the pharyngeal region and cervical lymph nodes in over 75% of cases, even though survival time of more than a year is only seen in 10% of the irradiated patients. (Brooks et al., 1988; Macmillan et al., 1982) A combination of radiation therapy and a variety of different chemotherapy drugs was reported to significantly improve the survival time and local tumour control of 22 dogs in one study. (Brooks et al., 1988) Acute effects can occur but are usually self-limiting. Depending on the irradiated region, side effects can consist of oral mucositis, dysphagia, alopecia, moist desquamation and ocular changes such as keraititis, uveitis, conjunctivitis and blepharitis. (Jamieson et al., 1991; Larue and Gillette, 2001; Roberts et al., 1987; Theon et al., 1997a; Theon et al., 1997b) Coarse fractionation results in less acute effects than full course protocols and generally disappears quickly. (Blackwood and Dobson, 1996) In general late complications are very uncommon (less than 5% of cases) but can include skin fibrosis; permanent alopecia; formation of oronasal fistula and bone necrosis; development of secondary malignancy within the radiation field and ocular changes such as keratoconjunctivitis sicca, ocular atrophy and cataract formation. (Theon et al., 1997a; Theon et al., 1997b; Thrall, 1984; Thrall et al., 1981)

6.3 Chemotherapy Although local tumour control is the biggest challenge in the treatment of oral tumours, some oral tumours such as oral melanoma or tonsillar SCC are linked with high metastatic potential. In these cases, chemotherapy could be a valuable tool in treatment management, but chemosensitivity is low in these tumours. Tonsillar SCC has been linked with significant increase in COX-2 expression but the use of nonsteroidal anti-inflammatory drugs has not proven any clinical results. For oral SCC in dogs, piroxicam may have some effect in combination with cisplatin, but cisplatin at standard dosage can cause renal toxicity. (Boria et al., 2004; de Vos et al., 2005) In cats, mitoxantrone in combination with radiation therapy has shown promising results against oral SCC in a small number of animals. (LaRue et al., 1991; Ogilvie et al., 1993) Although oral melanoma is considered chemoresistant, platinum drugs consisting of intralesional cisplatin and systemic carboplatin would achieve favourable but minor effects on treatment in dogs. (Rassnick et al., 2001) A promising pathway in the treatment of oral melanomas is the use of immunotherapy agents and biologic response modifiers. Preliminary studies including amongst others Corynebacterium parvum, liposome-muramyl tripeptide- phosphatidylethanolamine (L-MTP-PE), interleukin-2 (IL-2), tumour necrosis factor, pro-inflammatory cytokines, etc. have suggested improvements in survival time and local tumour control, but clinical trials on large scale are lacking. (Alexander et al., 2005; Bergman et al., 2004; Bergman et al., 2006; Bergman et al., 2003; Dow et al., 1998; Elmslie et al., 1994; Elmslie et al., 1995; Hogge et al., 1998; MacEwen et al., 1999; Macewen et al., 1986; Moore et al., 1991; QuintinColonna et al., 1996) This emerging approach is considered promising as a complementary method to surgery and radiation therapy.

7. Prognosis

Various case series covering a broad variety of oral tumour types, the majority surgically treated, have been published. In general, acanthomatous epulis and SCC have the best results in survival time and local tumour recurrence in dogs, while the highest rates are found in fibrosarcoma and malignant

22

melanoma. (Liptak and Withrow, 2007) Fibrosarcoma still has very high local recurrence rates; excessive resections and or the addition of adjuvant therapies are necessary. (Forrest et al., 2000) In melanoma's, high metastatic rate demands other adjuvant therapies, even though in 75% of cases the disease is controlled locally. (Liptak and Withrow, 2007) Oral SCC in cats has a poor prognosis mainly due to difficult achievement of complete surgical resection, high recurrence rates and unfavourable results using radiation therapy and/or chemotherapy. (Bilgic et al., 2015) For oral tumours in general, rostral localisation and histological complete resection are considered as favourable in terms of prognosis. Rostral located oral tumours are usually discovered earlier than caudal ones, which permits the increased probability of complete resection of the tumour in an earlier stage. Local recurrence rate has a positive correlation with incomplete resection (Schwarz et al., 1991a, b) and therefore a negative effect on survival time: consecutive treatments tend to be less successful and the effect on tumour response decreases. (Harvey et al., 1981; Overley et al., 2001) In two studies led by the same author, tumour related deaths were found multiple times more likely with malignant tumours, tumours located caudal to the canine teeth and incomplete resections. (Schwarz et al., 1991a, b) In dogs treated with megavoltage radiation the size of the tumour has an impact on local tumour control for malignant as well as benign tumours. Local recurrence as encountered in 30% of the cases is three and up to eight times more likely in respectively T2 (2-4 cm diameter) and T3 (>4 cm diameter) tumours compared to T1 tumours (< 2 cm diameter). Survival rates in dogs with malignant oral neoplasia are also influenced by tumour size with 3-year progression-free survival rates of 55%, 32% and 20% for T1, T2 and T3 tumours respectively. (Theon et al., 1997b) For tumours of the tongue, the prognosis depends on the type, grade and localisation of the tumour. (Carpenter 1993) Rostral localisation has better prognosis due to earlier tumour detection and easier resections widths. Cancer in the caudal tongue may have richer lymphatic and vascular vessels what is in favour of metastasis. (Liptak and Withrow, 2007)

23

Materials and methods

1. Survey outline

A questionnaire was drafted on www.enquetesmaken.be with the purpose of reaching as many veterinarians and veterinary pathologists dealing with wildlife carnivorans as possible. To reach this target audience, organisations or societies frequently dealing with these kinds of animals were contacted by email to send the questionnaire to their affiliated zoos or animal parks and veterinarians. In this questionnaire, wildlife facilities/veterinarians were being asked about characteristics of their encountered oral tumour(s). Questions of the survey were created to retrieve following data topics:  Species of the animal (Latin name)  Sex of the animal  Age of the animal at moment of diagnosis  Geographical region of tumour occurrence  Symptoms and clinical history of the animal  Presence of histopathological examination  (Histological) diagnosis of the tumour  Location of the tumour in the oral cavity  Shape of the tumour  Delineation of the tumour  Invasiveness of the tumour  Distribution  Cut surface appearance  Lymph node involvement  Presence of metastasis  Biopsy method  Treatment  Recurrence of the tumour after treatment  Survival time

Participants of this questionnaire were briefed about the possibility of the data being used in a peer reviewed paper later on and were asked for their permission to use this data before proceeding to further questioning. The survey can be found on the following internet link: https://www.enquetesmaken.com/s/84df898

2. Response

Thirty one survey participations were received until May 2018. In total, eleven additional cases were collected and considered as complete contributions to the study. These cases can be found under ‘Appendix B’.

24

Results

In total, 75 case reports of oral tumours in non-domesticated carnivorans with histopathological evidence were retrieved. 64 case reports were described in literature, while 11 useful case reports were gathered by sending out an online survey. 67 case reports (89%) were diagnosed as non- odontogenic oral tumours and 8 case reports (11%) as odontogenic oral tumours. In the group of non- odontogenic tumours, oral papilloma’s had the highest incidence with 37 described cases, followed by squamous cell carcinomas (17 cases) and melanomas (5 cases). Other less commonly retrieved non- odontogenic tumours were carcinoma (3 cases), muco-epidermoïd carcinoma, fibrosarcoma, osteoma and hemangioma (1 case each). Of the 8 reported odontogenic tumours, 4 were classified as fibromatous epulis of periodontal ligament, in some papers referred to as peripheral odontogenic fibroma (POF). Gingival hyperplasia was reported twice, while amyloid-producing odontogenic tumour (APOT) and calcifying epithelial odontogenic tumour (CEOT) both had one representative.

41 cases of oral tumours (54%) were reported in animals belonging to the suborder Feliformia and 34 cases (46%) were caniform species. In 42 out of 75 cases, the animal's sex was mentioned resulting in an equal representation of 21 male and 21 female species. Only 4 animals were neutered: one male and three females. Due to differences in species' physiology and life expectancy, it's very difficult to evaluate and compare the age of affected animals. A variety of different age groups was represented, from juvenile to (sub) adult and aged animals. In a group of 46 animals where age was mentioned or perceived, most of them were belonging to the group of adult to aged animals and only 2 out of 46 animals were categorized as non-adult animals. 30 cases of oral tumours were reported in free- ranging animals and all of them were diagnosed as oral papillomatosis.

Symptoms or a description of oral mass discovery were reported in 54 out of 75 cases. With 22 observations facial swellings were frequently reported. These swellings included swollen jawbones (6 observations), lingual masses (5 observations), swollen lips (6 observations), swollen gingiva (4 observations) and swollen skin under the eye (1 observation). Other more frequently mentioned symptoms were anorexia (10 observations), hypersalivation (4 observations), blood-tinged saliva (4 observations), presence of ulceration/hemorrhage/erythema in the region of lip or gingiva (4 observations), depression (3 observations) and weight loss (3 observations). Alopecia and staggering were both seen twice. Only one observation was reported for unilateral chewing, scratching, irregular adjacent mucosae, polydipsia, waning and waxing facial lesions, increased coughing and ataxia. In 3 papilloma cases in coyotes (Canis latrans) a good body condition despite the discovery of (severe) papilloma-like lesions was reported. In 22 cases, an oral mass was observed during a physical examination without a specific oral tumour check-up purpose. Oral tumours were thus discovered 12 times during translocation examination, 5 times during general physical examination, twice during dental treatment and once during behavioral/ecologic studies, surgical intervention and feeding time.

Treatment of an oral tumour was undertaken in 16 cases, divided in 7 odontogenic and 9 non- odontogenic cases. 6 out of 9 non-odontogenic cases were SCC, all of them appearing in Malayan sun bears (Helarctos malayanus). The 3 remaining non-odontogenic tumour treatments were applied in a malignant melanoma, fibrosarcoma and osteoma respectively. The odontogenic group consisted of 2 gingival hyperplasia cases, 1 calcifying epithelial odontogenic tumour and 4 fibromatous epulis of periodontal ligament. Treatment was considered successful in 5 out of 16 cases. Unsuccessful outcome was registered in 7 cases, while 4 cases were unclear about clearance or recurrence of the tumour. 3 of the 5 successful treatments were accomplished by local excision of the tumour: 2 gingival hyperplasia cases in cheetah’s (Acinonyx jubatus) and an osteoma in a female striped (Mephitis mephitis). The remaining 2 successfully treated tumours were the result of a multimodality therapy: malignant melanoma in a African lion (Panthera leo) and SCC in a Malayan (Helarctos malayanus). The African lion underwent a series of hypofractionated radiation therapies and immunotherapies followed by surgical excision of the tumour. In the case of the Malayan sun bear, bilateral rostral mandibulectomy and subsequent intra- and perilesional cisplatin injections were firstly performed, after

25

which radiation therapy and one additional cisplatin treatment were started due to histopathological incomplete resected margins. 5 of the 7 unsuccessful tumour treatments were cases of oral SCC, all of them appearing in adult to aged Malayan sun bears (Helarctos malayanus). 4 cases were found Gammaherpesvirus positive, while one case was Herpesvirus negative. Surgical excision of the oral tumour was undertaken in all of the 5 cases, but recurrence of an oral mass appeared in a time lapse of months to years depending from case to case. Another attempt of surgical resection was undertaken in 3 out of 5 cases, all of them herpesvirus positive. In one case, additional sub-lesional injections of carboplatin were administered following a second excision of the tumour. Another therapy that was started after tumour recurrence was the combination of intralesional injections of fluorouracil and cisplatin and oral administration of piroxicam and L-lysin. The 2 non-SCC that failed to achieve favorable results were fibrosarcoma in a (Chrysocyon brachyurus) and fibromatous epulis of periodontal ligament in an African lion (Panthera leo). Rostral maxillectomy was performed in the maned wolf, but recurrence and distant metastasis occurred after 7 months. In the case of the African lion, a histologically similar mass reappeared on the same location 6 months following excision. Of the 4 cases where tumour recurrence was unknown; 3 cases comprised fibromatous epulis of periodontal ligament, all appearing in cheetah’s (Acinonyx jubatus). The remaining case was CEOT in a Siberian tiger (Panthera leo altaica). All of these 4 tumours underwent local excision as only treatment method.

No curative treatment was attempted or treatment was unknown in 59 out of 75 cases (79%). In 22 cases, the oral mass was only observed at necropsy and obviously treatment couldn't be initiated. The affected animals and their diagnosed oral tumour can be found in Table 1. 24 cases were left untreated, all of them had papilloma. This papilloma group consisted of 2 coyotes (Canis latrans), 2 's (Felis concolor), 2 bobcats (Felis rufus), 4 Asiatic (Panthera leo persica), a snow leopard (Panthera uncia), a clouded leopard ( nebulosa), 11 Canadian lynx (Felis lynx canadensis) and one spotted (Crocuta crocuta). Euthanasia was performed in 9 cases, divided in 3 groups depending on the moment between histopathological diagnosis and euthanasia. A first group, consisting of 4 cases was euthanized shortly following the biopsy diagnosis: a tiger with gingival melanoma and both a (Suricata suricatta), a Californian sea lion (Zalophus californianus) and a coyote (Canis latrans) with SCC. In a second group animals diagnosed by histopathological proof were kept on palliative treatment until the decision was made to euthanize the patient because of a (severe) decrease in life quality. This palliative treatment was used in a bobcat (Felis rufus) and coyote (Canis latrans) both diagnosed with SCC. A third group of patients was euthanized before histopathological diagnosis of the oral mass was made and biopsy was therefore taken at necropsy. This group comprised three individuals: a lion (Panthera leo) with muco-epidermoïd carcinoma, a meerkat (Suricata suricatta) with anaplastic carcinoma and a wolf (Canis lupus) diagnosed with tonsillar SCC. In another case, a Bengal tiger (Panthera tigris tigris) was diagnosed with a mandibular Amyloid-producing Odontogenic Tumour (APOT) at necropsy after he was treated with antibiotics and was put on adapted nutrition (minced meat) for two months. Finally, 4 cases of diagnosed oral tumours were retrieved without further clarifications about possible attempted treatments. These comprise SCC in a red wolf (Canis rufus) and 2 papilloma cases in snow leopards (Panthera uncia).

Metastasis was mentioned in eight cases: 4 SCC, of which 2 were tonsillar SCC; one adenocarcinoma in a Syrian golden Jackal (Canis aureus syriacus), one muco-epidermoïd carcinoma in an African lion (Panthera leo), one malignant melanoma in a tiger (Panthera tigris), and one fibrosarcoma in a maned wolf (Chrysocyon brachyurus). The two tonsillar SCC were found in a polar wolf (Canis lupus arctos) and captive wolf (Canis lupus) while a mandibular SCC was reported in a meerkat (Suricata suricatta) and a lingual SCC in a Californian sea lion (Zalophus californianus). Nine times metastasis was found in the regional lymph nodes: Twice in a submandibular lymph node, both three times in a submandibular and retropharyngeal lymph node and once in a deep cervical lymph node. The lungs were reported metastasized six times. Other thoracic organs where metastasis was observed were mediastinum, heart and mediastinal lymph node with respectively two, one and one observation(s). Esophagus, diaphragm and kidney all were found metastasized each once. Only in the Syrian golden Jackall diagnosed with adenocarcinoma the metastasis localizations were not reported.

26

Species name Species name (Latin) Histopathological Localisation (English) diagnosis Lips, tongue and Coyote Canis latrans Papillomatosis adjacent tissue Wolves Canis lupus Papillomatosis Oral cavity Wolves Canis lupus Papillomatosis Oral cavity Lips, tongue and Coyote Canis latrans Papillomatosis adjacent tissue Coyote Canis latrans Papillomatosis Unknown Coyote Canis latrans Papillomatosis Oral cavity Coyote Canis latrans Papillomatosis Oral cavity Coyote Canis latrans Papillomatosis Oral cavity Coyote Canis latrans Papillomatosis Oral cavity Coyote Canis latrans Papillomatosis Oral cavity Coyote Canis latrans Papillomatosis Oral cavity Squamous cell California sea lion Zalophus californianus carcinoma Tongue Squamous cell Ocelot Leopardus pardalis carcinoma Tongue Syrian Golden Jackal C. aureus syriacus Adenocarcinoma Cheetah Acinonyx jubatus Hemangioma Tongue Black bear Ursus americanus Carcinoma Tongue Japanese brown bear Ursus arctos lasiotus Malignant melanoma Tongue Amercan Taxidea taxus Carcinoma Oral cavity Mexican Grey Wolf Canis lupus baileyi Amelanotic melanoma Gingiva mandibula left Buccal side left lower Mexican Grey Wolf Canis lupus baileyi Amelanotic melanoma lip Squamous cell Polar wolf Canis lupus arctos carcinoma Tonsilla Squamous cell Canadian lynx Lynx canadensis carcinoma Oral cavity Table 1: List of animals with discovered and diagnosed oral tumours at moment of necropsy Discussion

In this literature review, a large number of cases without curative treatment were reported: 59 cases or almost 8 cases out of 10. Under curative treatment, treatment methods with the purpose of improving the length of the patient's life are meant. For oral tumours, these methods can include surgical excision, radiation therapy, chemotherapy or more recently introduced immunologic approaches. It's clear that a curative treatment is not advised for every patient. This largely depends on the tumour type, stage, malignancy and presence of metastasis; all elements influencing the patient's prognosis. Other external factors also have to be taken into consideration. Firstly, financial responsibility of the treatment has to be taken, while medical curative cancer treatment comes with high costs. In companion animals, costs of tumour treatment are of less importance than it once was due to their newly perceived role as 'members of the household'. In wildlife species, this interacting bond with humans is less present, especially in wild-ranging animals. Captive animals are usually held in zoos or sanctuaries, while these facilities may be limited by available finances. Treatment of an individual wild- ranging animal is of less importance than the health of the entire group or even ecosystem. Secondly, technical equipment must be available. Some facilities may be located in rural areas with high distance to veterinary practices/hospitals or may not be equipped to provide proper intervention by external practitioners. The additional costs and clinical consequences of anesthesia necessary for

27

transportation and treatment of these non-domesticated patients neither may be neglected. It is recommended to trust an experienced team with the monitoring of anesthesia, considering the broad range of species variation in physiology and metabolism and of course guaranteed safety of the staff.

A large portion of the cases (24 out of 75 cases) were simply left untreated even after histopathological diagnosis. It has to be mentioned that all of these cases were diagnosed as papillomatosis which are considered to be self-limiting without further (major) clinical consequences. Papillomatosis was the most reported oral tumour in this study with an additional 13 cases diagnosed at necropsy. A total number of 37 case reports were classified as papillomatosis, but the actual number is estimated higher as a result of case restrictions in this study. For this literature review the amount of counted cases appearing in spotted (Nelson et al., 2013) was reduced to one report. The reason for this lays in the uncertainty in case numbers distinguishing oral and genital masses. The large representation of papillomatosis may be explainable through multiple reasons. Papillomatosis has a characteristic macroscopical appearance and thus is more easily to be suspected and examined further on. The tumour has been subject to large publications held by multidisciplinary, international scientific teams. In this study, the papilloma lesions were examined through advanced techniques such as immunohistochemistry, electron microscopy and molecular genomic sequencing in order to investigate the coevolution and migration of cat species and their pathogens. (Sundberg et al., 1996; Sundberg et al., 2000) Two other articles included each more than 10 individual case reports. (Samuel et al., 1978; Wolfe and Spraker, 2007) Oral tumours were frequently discovered at necropsy. Some necropsies revealed oral tumours which were discovered incidentally and were not considered as the cause of death. In the other cases, unfavorable clinical symptoms appeared before death of the animal without suspecting the presence of an oral mass. Unfortunately the exposed symptoms in these cases didn’t lead clinicians to examination of the oral cavity with consecutive diagnostic procedures and an eventual therapeutic treatment plan.

Nine cases resulted in euthanasia, consisting of 3 cases where the patient was euthanized before histopathological examination confirmed an oral tumour. (Dadone et al., 2014; Dorso et al., 2008; Ryan and Nielsen, 1979) Specific reasons for not taking biopsies of the mass were not précised. In all the three cases, variable (non-specific) symptoms developed for a couple of weeks before further action was undertaken. Two animals went through oral examination under general anesthesia when in both cases a sublingual mass was discovered. Instead of performing biopsies, other diagnostic approaches were preferred (respectively cytological aspiration and radiographic imaging) to estimate the nature of the mass. In a meerkat, an odontogenic tumour was suspected and due to the age of the animal, presence of severe arthritis, and the stress associated with daily treatments further curative treatment was not attempted and palliative treatment was initiated. At later necropsy, the sublingual mass was diagnosed as a highly malignant anaplastic carcinoma. Waiting for the cytological results of the sublingual mass of the lion, NSAIDS were given for two days before the animal showed signs of dramatic dyspnea. Therefore, the lion was humanely euthanized and at necropsy the mass turned out to be a histological mucoepidermoid carcinoma. For the third case there were no specifications about attempted examinations or treatments but histopathological diagnosis of the tonsillar SCC was made after the captive wolf was euthanized. Although it would result in just a marginal benefit in these cases, histopathology is indispensable in the identification of oral tumours as this results in facilitating the choice of the most appropriate treatment therapy.

Six other patients were euthanized, all of them after histological examination of the mass. Two of them firstly had palliative treatment. The most common reason to perform euthanasia was because of poor prognosis and aggressive nature of the tumour. The impossibility of wide surgical resection and complications resulting in interference with eating were also mentioned. The six cases comprised 5 SCCs, all of them were non-tonsillar and occurred in 3 feliform and 2 caniform species, and one malignant melanoma in a tiger. Although the prognosis for dogs with oral SCC is considered good, two dog-like species (coyote and Californian sea lion) were amongst euthanized patients. Due to the reported malignancy of SCC (Californian sea lion) and wide anatomic extent (coyote), euthanasia was considered the most appropriate decision. Rostral tumour location is prognostic favorable, but wasn't

28

the case for these two cases: they both appeared on the gingiva of the mandibular molar region. In domestic dogs, local recurrence rate and survival time achieve better results with mandibulectomy than maxillectomy. (Kosovsky et al., 1991) For radiation therapy, smaller lesions, young age and maxillary location were mentioned as favorable prognostic factors. (Evans and Shofer, 1988; Theon et al., 1997b) In the coyote and sea lion, none of them were present. Results of chemotherapy in dogs with oral SCC are regarded as unsuccessful. Renal toxicity as a mentioned side-effect for the use of cisplatin and piroxicam could form an additional problem in a 19-year old coyote and 30-year old sea lion.

Curative treatment was only applied in a minority of cases (16 cases out of 75), with only 5 cases considered to result in a successful outcome. The specific description of a successful treatment is hard to define. No recurrence or visual detection of the tumour during a variable time lapse was mentioned in all of the cases. Two cases reported a time period of 4 months. One osteoma case in a skunk was only recently subjected to surgical intervention and until survey participation no recurrence occurred; while a cheetah with gingival hyperplasia had to be euthanized 4 months after surgical excision due to an unrelated neutrophilic cholecystitis. In the remaining three treatments, no visible detection of the tumour varied from 6 months to 6,5 years. Besides visual detection criteria such as blood and urine analysis, signs of metastasis on thoracic radiographs, signs of bone lysis on mandibular radiographs, survival time and lymph node cytology were used to assess successful outcome. Sole therapy consisting of surgical resection was performed in considerably benign neoplasia: gingival hyperplasia and osteoma. Malignant melanoma in a lion and SCC in a sun bear were treated by use of a multimodality therapy. Due to external-beam radiation and administration of an immunotherapeutic melanoma vaccine, the tumour size decreased by 50% which made surgical excision of the mandibular melanoma possible. The unsuccessfully treated tumours were primarily consisting of five oral SCC cases appearing in Malayan sun bears. Four of these bears tested positive for the presence of gammaherpesvirus, even though its pathogenicity and relationship to squamous cell carcinoma is unknown. In the same article, periodontal disease and traumatic stereotype behaviour were reported as other suggested causes of recurrence, but warranted further investigation. (Lam et al., 2013) Recurrence of the tumour could also be explained by the difficulty of complete resection due to large transition between normal tissue and malignant cells. The use of computer tomography in order to precisely determine tumour margins may contribute to avoid incomplete resections, but comes with additional financial costs. In the successfuly executed multimodality therapy, additional chemo- and radiation therapy was initiated because of incomplete surgical excision. Some bears repeatedly underwent surgical resection of a new growing mass, in two individuals intra- or sublesional administration of chemotherapy was performed after recurrence. Radiation therapy was never used in the unsuccessfull treatments. It is tempting to conclude that multimodality therapy use in tumours with associated malignancy is favorable in exotic carnivorans, even though the number of reported treatments is insignificantly low. Keeping in mind that the patients we are dealing with are of non-domesticated nature, the same considerations as being mentioned in the start of this discussion have to be made.

Similar to domesticated carnivores, oral tumors are frequently diagnosed in a more advanced stage, leading to a poorer prognosis. Contrary to their domestic relatives, examination of the oral cavity in exotic carnivorans is only possible under general anesthesia or in circumstances where the animal is trained to open the mouth. Even though exposure of the oral cavity is possible, it is still challenging to perform a decent examination in safe conditions. The appearances of particular symptoms are thus a paramount auxiliary method in the detection of oral neoplasia. Zookeepers or affiliated staff members hold a key role in the visual detection of minor signs that could imply oral pathology. Careful communication between these professionals and health practitioners are of extreme importance if diagnosis in an early stage wants to be achieved. Continuous education of caretakers and even veterinarians concerning oral discomfort signs is recommended as well in order to anticipate oral diseases and neoplasia. A great variety in signs suggestive for oral pathologies are described, but may include a decrease in appetite, external facial swelling, draining tracts, bleeding from the mouth, excessive salivation, nasal discharge, increased or reduced activity, aggression, and indications of pain or discomfort. More subtle signs mostly appearing in early oral disease comprise modification in

29

eating or chewing pattern, alterations from normal behaviour activity, a gradual weight loss, and periodic upper respiratory–type infections. (Wiggs and Bloom, 2003) Additional symptoms as seen in literature include irregular shaped mucosae and lips, alopecia, staggering, polydipsia, waning and waxing facial lesions, increased coughing and ataxia.

The importance of general check-ups was accentuated by the number of tumours discovered during such examinations: almost one third of the reported cases had almost incidental findings of an oral mass. Even if the intention of a procedure isn't necessarily oral, it's always worth paying attention to the oral cavity. If general anesthesia is undertaken for whatever reason, the opportunity has to be seized to perform a solid oral examination and further approaches such as (dental) radiography, dental scaling and other assessments or treatments can also be initiated if necessary. Conclusion

Oral tumours have been described in a variation of carnivoran species, but the number of reported cases is rather limited. All of the oral tumours reported in non-domesticated carnivorans have been described in domestic carnivores. A majority of these cases were of non-odontogenic origin, while papillomatosis, squamous cell carcinoma and melanoma represented the most frequently described tumour types. Regarding the odontogenic tumour group fibromatous epulis of periodontal origin was the most common tumour type.

Only a minority of animals diagnosed with an oral tumour were subjected to curative treatment. This treatment therapy comprised in all of the cases surgical resection. Clinical outcome of tumour excision was variable and tumour recurrence appeared in a slight minority of treated cases. Multimodality treatment was attempted in two cases diagnosed with a malignant tumour type and resulted in satisfying clinical outcome. Although highly dependent on tumour characteristics and external factors, multimodality treatment may be regarded as a promising approach in the resolution of oral tumours with distinct malignancy in non-domesticated carnivorans.

In a large number of cases, no cancer treatment approach was initiated. Frequently the presence of oral tumours was only confirmed at necropsy, while particular animals suffered from oral symptoms prior to death. In these cases, the presence of signs didn't lead to the suspicion of an oral neoplasia and further workup. Diagnosis of an oral tumour led to euthanasia in several cases. Poor prognosis and wide tumoral extent were commonly mentioned as the reason for this decision.

Due to various difficulties in treatment therapy of exotic carnivorans, special attention has to be paid on the early recognition of symptoms or discrete signs caused by oral tumours. The role of caretakers dealing with exotic carnivorans on daily basis is fundamental and it is advisable that animal keepers as well as affiliated veterinarians are retrained in the detection of suggestive signs associated with the presence of oral tumours. A second step in earlier detection of oral masses lays in more frequent examination of the oral cavity. Any situation of an anesthetized animal forms an excellent opportunity to achieve this goal.

This comprehensive enumeration can be considered not exhaustive in time and reality. Further submission of case reports in both captive and free-ranging animals future needs to be undertaken in order to evaluate tumour detection, diagnostic approach and treatment therapy; or in the case of free- ranging carnivorans, the impact of oral tumours on population dynamics.

30

References

Abbott, D.P., Walsh, K., Diters, R.W., 1986. Calcifying Epithelial Odontogenic-Tumors in 3 Cats and a Dog. J Comp Pathol 96, 131-136. Alexander, A.N., K., H.M., Mitzey, M., 2005. Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a Phase II clinical trial in canine patients with malignant melanoma Vet Cancer Soc Proc 18, 1-10. Altamura, G., Corteggio, A., Pacini, L., Conte, A., Pierantoni, G.M., Tommasino, M., Accardi, R., Borzacchiello, G., 2016. Transforming properties of Felis catus papillomavirus type 2 E6 and E7 putative oncogenes in vitro and their transcriptional activity in feline squamous cell carcinoma in vivo. Virology 496, 1-8. Baba, A.I., Catoi, C., 2007. Bone and joint tumors, In: Comparative Oncology. The Publishing House of the Romanian Academy, Bucharest, pp. 87-407. Barnes, L., Eveson, J.W., Reichart, P., Sidransky, D., 2005. World Health Organization Classification of Tumours, Pathology & Genetics, Head and Neck Tumours. IARC Press, Lyon. Bergman, P.J., Camps-Palau, M.A., McKnight, J., 2004. Phase I & IB trials of murine tyrosinase +- human GM-CSF DNA vaccination in dogs with advanced malignant melanoma. Vet Cancer Soc Proc 24, 55. Bergman, P.J., Camps-Palau, M.A., McKnight, J.A., Leibman, N.F., Craft, D.M., Leung, C., Liao, J., Riviere, I., Sadelain, M., Hohenhaus, A.E., Gregor, P., Houghton, A.N., Perales, M.A., Wolchok, J.D., 2006. Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center. Vaccine 24, 4582-4585. Bergman, P.J., McKnight, J., Novosad, A., Charney, S., Farrelly, J., Craft, D., Wulderk, M., Jeffers, Y., Sadelain, M., 2003. Long-term survival of dogs with advanced malignant melanoma after DNA vaccination with xenogeneic human tyrosinase: A phase I trial. Clin Cancer Res 9, 1284-1290. Bernstein, K.S., Schelling, S.H., 1999. Oral squamous cell carcinoma in a coyote (Canis latrans). J Zoo Wildl Med 30, 305-307. Berrington, A.J., Jimbow, K., Haines, D.M., 1994. Immunohistochemical detection of melanoma- associated antigens on formalin-fixed, paraffin-embedded canine tumors. Vet Pathol 31, 455-461. Bertone, E.R., Snyder, L.A., Moore, A.S., 2003. Environmental and lifestyle risk factors for oral squamous cell carcinoma in domestic cats. J Vet Intern Med 17, 557-562. Bilgic, O., Duda, L., Sanchez, M.D., Lewis, J.R., 2015. Feline Oral Squamous Cell Carcinoma: Clinical Manifestations and Literature Review. J Vet Dent 32, 30-40. Blackwood, L., Dobson, J.M., 1996. Radiotherapy of oral malignant melanomas in dogs. J Am Vet Med Assoc 209, 98-102. Boehm, B., Breuer, W., Hermanns, W., 2011. [Odontogenic tumours in the dog and cat]. Tierarztl Prax Ausg K Kleintiere Heimtiere 39, 305-312. Bonfanti, U., Bertazzolo, W., Gracis, M., Roccabianca, P., Romanelli, G., Palermo, G., Zini, E., 2015. Diagnostic value of cytological analysis of tumours and tumour-like lesions of the oral cavity in dogs and cats: a prospective study on 114 cases. Veterinary journal (London, England : 1997) 205, 322- 327. Boria, P.A., Murry, D.J., Bennett, P.F., Glickman, N.W., Snyder, P.W., Merkel, B.L., Schlittler, D.L., Mutsaers, A.J., Thomas, R.M., Knapp, D.W., 2004. Evaluation of cisplatin combined with piroxicam for the treatment of oral malignant melanoma and oral squamous cell carcinoma in dogs. Javma-J Am Vet Med A 224, 388-394. Bossart, G.D., 1990. Invasive Gingival Squamous-Cell Carcinoma in a California Sea Lion (Zalophus- Californianus). J Zoo Wildlife Med 21, 92-94. Bostock, D.E., 1979. Prognosis after Surgical Excision of Canine Melanomas. Vet Pathol 16, 32-40. Brooks, M.B., Matus, R.E., Leifer, C.E., Alfieri, A.A., Patnaik, A.K., 1988. Chemotherapy Versus Chemotherapy Plus Radiotherapy in the Treatment of Tonsillar Squamous-Cell Carcinoma in the Dog. J Vet Intern Med 2, 206-211. Broughton, E., Graesser, F.E., Carbyn, L.N., Choquette, L.P., 1970. Oral papillomatosis in the coyote in Western Canada. J Wildlife Dis 6, 180-181.

31

Calonje, E., Fletcher, C.D.M., 2007. Vascular tumors, In: Diagnostic Histopathology of Tumors, 3rd ed. Churchill Livingstone, New York, pp. 41-81. Carpenter, J.W., Novilla, M.N., Griffing, W.J., 1980. Metastasis of a Malignant, Amelanotic Lingual Melanoma in a Dog. J Am Anim Hosp Assoc 16, 685-689. Ciekot, P.A., Powers, B.E., Withrow, S.J., Straw, R.C., Ogilvie, G.K., Larue, S.M., 1994. Histologically Low-Grade, yet Biologically High-Grade, Fibrosarcomas of the Mandible and Maxilla in Dogs - 25 Cases (1982-1991). J Am Vet Med Assoc 204, 610-615. Curry, S.S., Brown, D.R., Gaskin, J.M., Jacobson, E.R., Ehrhart, L.M., Blahak, S., Herbst, L.H., Klein, P.A., 2000. Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater. J Wildlife Dis 36, 792-797. Dadone, L.I., Garner, M.M., Klaphake, E., Johnston, M.S., Han, S.S., 2014. Anaplastic Mandibular Carcinoma in a Meerkat (Suricata Suricatta). J Zoo Wildlife Med 45, 413-416. Dalambiras, S., Boutsioukis, C., Tilaveridis, I., 2005. Peripheral osteoma of the maxilla: report of an unusual case. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 100, e19-24. de Vos, J.P., Burm, A.G., Focker, A.P., Boschloo, H., Karsijns, M., van der Waal, I., 2005. Piroxicam and carboplatin as a combination treatment of canine oral non-tonsillar squamous cell carcinoma: a pilot study and a literature review of a canine model of human head and neck squamous cell carcinoma. Vet Comp Oncol 3, 16-24. Dorso, L., Risi, E., Triau, S., Labrut, S., Nguyen, F., Guigand, L., Wyers, M., Abadie, J., 2008. High- grade mucoepidermoid carcinoma of the mandibular salivary gland in a lion (Panthera leo). Vet Pathol 45, 104-108. Dow, S.W., Elmslie, R.E., Willson, A.P., Roche, L., Gorman, C., Potter, T.A., 1998. In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma. Journal of Clinical Investigation 101, 2406-2414. Dubielzig, R.R., 2002. Odontogenic tumors and cysts, In: Tumors in domestic animals, 4th ed. Iowa State University Press, Ames, Iowa, pp. 402-410. Dubielzig, R.R., Goldschmidt, M.H., Brodey, R.S., 1979. Nomenclature of Periodontal Epulides in Dogs. Vet Pathol 16, 209-214. Dvorak, L.D., Beaver, D.P., Ellison, G.W., Bellah, J.R., Mann, F.A., Henry, C.J., 2004. Major glossectomy in dogs: A case series and proposed classification system. J Am Anim Hosp Assoc 40, 331-337. Effron, M., Griner, L., Benirschke, K., 1977. Nature and Rate of Neoplasia Found in Captive Wild , Birds, and Reptiles at Necropsy. J Natl Cancer I 59, 185-198. Elmslie, R.E., Dow, S.W., Potter, T.A., 1994. Genetic immunotherapy of canine oral melanoma. Vet Cancer Soc Proc 14, 111. Elmslie, R.E., Potter, T.A., Dow, S.W., 1995. Direct DNA injection for the treatment of malignant melanoma. Vet Cancer Soc Proc 15, 52. Evans, S.M., Lacreta, F., Helfand, S., Vanwinkle, T., Curran, W.J., Brown, D.Q., Hanks, G., 1991. Technique, Pharmacokinetics, Toxicity, and Efficacy of Intratumoral Etanidazole and Radiotherapy for Treatment of Spontaneous Feline Oral Squamous-Cell Carcinoma. Int J Radiat Oncol 20, 703-708. Evans, S.M., Shofer, F., 1988. Canine Oral Nontonsillar Squamous-Cell Carcinoma - Prognostic Factors for Recurrence and Survival Following Orthovoltage Radiation-Therapy. Vet Radiology 29, 133-137. Fagan, D.A., Oosterhuis, J.E., Kirkman, J.E., 1998. A review of the expanding field of exotic animal oral health care--veterinary dentistry. J Vet Dent 15, 117-128. Farrelly, J., Denman, D.L., Hohenhaus, A.E., Patnaik, A.K., Bergman, P.J., 2004. Hypofractionated radiation therapy of oral melanoma in five cats. Veterinary radiology & ultrasound : the official journal of the American College of Veterinary Radiology and the International Veterinary Radiology Association 45, 91-93. Featherstone, J.D., 2004. The continuum of dental caries--evidence for a dynamic disease process. J Dent Res 83 Spec No C, C39-42. Fecchio, R.S., Gomes, M.D.S., Xavier, J.G., Kunze, P.E., Gioso, M.A., 2015. Maxillary Calcifying Epithelial Odontogenic Tumor in a Siberian Tiger (Panthera tigris altaica). J Vet Dent 32, 120-121. Feldhamer, G.A., 2007. Mammalogy : adaptation, diversity, ecology, 3rd ed. Johns Hopkins University Press, Baltimore.

32

Fiani, N., Arzi, B., Johnson, E.G., Murphy, B., Verstraete, F.J., 2011a. Osteoma of the oral and maxillofacial regions in cats: 7 cases (1999-2009). J Am Vet Med Assoc 238, 1470-1475. Fiani, N., Verstraete, F.J.M., Kass, P.H., Cox, D.P., 2011b. Clinicopathologic characterization of odontogenic tumors and focal fibrous hyperplasia in dogs: 152 cases (1995-2005). Javma-J Am Vet Med A 238, 495-500. Forrest, L.J., Chun, R., Adams, W.M., Cooley, A.J., Vail, D.M., 2000. Postoperative radiotherapy for canine soft tissue sarcoma. J Vet Intern Med 14, 578-582. Frew, D.G., Dobson, J.M., 1992. Radiological Assessment of 50 Cases of Incisive or Maxillary Neoplasia in the Dog. J Small Anim Pract 33, 11-18. Fulton, A., Arzi, B., Murphy, B., Naydan, D.K., Verstraete, F.J., 2014. The expression of calretinin and cytokeratins in canine acanthomatous ameloblastoma and oral squamous cell carcinoma. Vet Comp Oncol 12, 258-265. Gardner, D.G., 1982. The Peripheral Odontogenic Fibroma - an Attempt at Clarification. Oral Surg Oral Med O 54, 40-48. Gardner, D.G., 1992. An Orderly Approach to the Study of Odontogenic-Tumors in Animals. J Comp Pathol 107, 427-438. Gardner, D.G., 1996. Epulides in the dog: A review. Journal of Oral Pathology & Medicine 25, 32-37. Gardner, D.G., Dubielzig, R.R., Mcgee, E.V., 1994. The So-Called Calcifying Epithelial Odontogenic- Tumor in Dogs and Cats (Amyloid-Producing Odontogenic-Tumor). J Comp Pathol 111, 221-230. Garrett, L., Marretta, S.M., Marretta, J.J., 2007. Feline oral squamous cell carcinoma: An overview. http://veterinarymedicine.dvm360.com/feline-oral-squamous-cell-carcinoma- overview?id=&pageID=1&sk=&date= (accessed 13/04/17. Gassel, A.D., Huber, M.L., 2002. What is your diagnosis? A 2.0 x 1.75-cm well-circumscribed ovoid mineralized opacity over the left mandible. J Am Vet Med Assoc 220, 1151-1152. Goldschmidt, M.H., Thrall, D.E., 1985. Benign bone tumors in the dog, In: Textbook of small animal orthopaedics. Lippincott, Philadelphia (Pa.), pp. 899 - 907. Gorlin, R.J., Chaudhry, A.P., Pindborg, J.J., 1961. Odontogenic tumors. Classification, histopathology, and clinical behavior in man and domesticated animals. Cancer 14, 73-101. Goudar, G., Ravi Kumar, R., Manjunath, G.A., Mahadesh, J., 2011. Osteoma of the mandible. J Dent Sci Res 2, 116-120. Gross, T.L., Ihrke, P.J., Walder, E.J., Affolter, V.K., 2007. Vascular tumors, In: Skin Disease of the Dog and Cat. Clinical and Histopathological Diagnosis, 2nd ed. Blackwell Science, pp. 735-758. Gulland, F.M.D., Trupkiewicz, J.G., Spraker, T.R., Lowenstine, L.J., 1996. Metastatic carcinoma of probable transitional cell origin in 66 free-living California sea lions (Zalophus californianus), 1979 to 1994 (vol 32, pg 250, 1996). J Wildlife Dis 32, 564-564. Hahn, K.A., DeNicola, D.B., Richardson, R.C., 1994. Canine oral malignant melanoma: Prognostic utility of an alternative staging system. J Small Anim Pract 35, 251. Harvey, C.E., Orr, H.S., British Small Animal Veterinary Association., 1990. Manual of small animal dentistry. British Small Animal Veterinary Association, Cheltenham. Harvey, H.J., Macewen, E.G., Braun, D., Patnaik, A.K., Withrow, S.J., Jongeward, S., 1981. Prognostic Criteria for Dogs with Oral Melanoma. J Am Vet Med Assoc 178, 580-582. Hawkins, C.E., Baars, C., Hesterman, H., Hocking, G.J., Jones, M.E., Lazenby, B., Mann, D., Mooney, N., Pemberton, D., Pyecroft, S., Restani, M., Wiersma, J., 2006. Emerging disease and population decline of an island endemic, the Tasmanian devil Sarcophilus harrisii. Biol Conserv 131, 307-324. Head, K.W., Cullen, J.M., Dubielzig, R.R., Else, R., Misdorp, W., Patnaik, A.K., Tateyama, S., van der Meer, I., Goodgame, R.W., van der Gaag, N.A., 2003. Histological Classification of Tumors of the Alimentary System of Domestic Animals. Armed Forces Institute of Pathology & World Health Organization, Washington DC. Head, K.W., Else, R.W., Dubielzig, R.R., 2002. Tumors of the alimentary tract, In: Tumors in domestic animals, 4th ed. Iowa State University Press, Ames, Iowa, p. 428. Hennet, P., Boutoille, F., 2013. Les dents et leur tissus d'attache, In: Guide pratique de stomatologie et de dentisterie vétérinaire. MED'COM, Paris, pp. 23-28. Herring, E.S., Smith, M.M., Robertson, J.L., 2002. Lymph node staging of oral and maxillofacial neoplasms in 31 dogs and cats. J Vet Dent 19, 122-126. Hillson, S., 2005. Teeth, 2nd ed. Cambridge University Press, Cambridge.

33

Hogge, G.S., Burkholder, J.K., Culp, J., Albertini, M.R., Dubielzig, R.R., Keller, E.T., Yang, N.S., MacEwen, E.G., 1998. Development of human granulocyte-macrophage colony-stimulating factor- transfected tumor cell vaccines for the treatment of spontaneous canine cancer. Hum Gene Ther 9, 1851-1861. Hollings, T., McCallum, H., Kreger, K., Mooney, N., Jones, M., 2015. Relaxation of risk-sensitive behaviour of prey following disease-induced decline of an apex predator, the Tasmanian devil. P Roy Soc B-Biol Sci 282. Hutson, C.A., Willauer, C.C., Walder, E.J., Stone, J.L., Klein, M.K., 1992. Treatment of Mandibular Squamous-Cell Carcinoma in Cats by Use of Mandibulectomy and Radiotherapy - 7 Cases (1987- 1989). Journal of the American Veterinary Medical Association 201, 777-781. ITIS, 2018. Carnivora. https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=180539#null (accessed 11/03/2018. Jamieson, V.E., Davidson, M.G., Nasisse, M.P., English, R.V., 1991. Ocular Complications Following Cobalt 60 Radiotherapy of Neoplasms in the Canine Head Region. J Am Anim Hosp Assoc 27, 51-55. Jones, P.D., de Lorimier, L.P., Kitchell, B.E., Losonsky, J.M., 2003. Gemcitabine as a radiosensitizer for nonresectable feline oral squamous cell carcinoma. J Am Anim Hosp Assoc 39, 463-467. Joslin, J.A., Garner, M., Collins, D., Kamaka, E., Sinabaldi, K., Meleo, K., Montali, R.J., Sundberg, J.P., Jenson, A.B., Ghim, S., Davidow, B., Hargis, A.M., West, K., Clark, T., Haines, D., 2000. Viral papilloma and squamous cell carcinomas in snow leopards (Uncia uncia), In: Proceedings of the American Association of Zoo Veterinarians and International Association for Aquatic Animal Medicine Joint Conference, New Orleans, Louisiana, USA, pp. 155-158. Kang, M.S., Park, M.S., Kwon, S.W., Ma, S.A., Cho, D.Y., Kim, D.Y., Kim, Y., 2006. Amyloid- producing odontogenic tumour (calcifying epithelial odontogenic tumour) in the mandible of a Bengal tiger (Panthera tigris tigris). J Comp Pathol 134, 236-240. Kemp, W.B., Abbey, L.M., Taylor, L.A., 1976. Pseudosarcomatous Fasciitis of Upper Lip of a Cat. Vet Med Sm Anim Clin 71, 923-925. Kosovsky, J.K., Matthiesen, D.T., Marretta, S.M., Patnaik, A.K., 1991. Results of partial mandibulectomy for the treatment of oral tumors in 142 dogs. Veterinary surgery : VS 20, 397-401. Kudnig, S.T., Ehrhart, L.M., Withrow, S.J., 2003. Survival analysis of oral melanoma in dogs. Vet Cancer Soc Proc 23, 39. Lachish, S., Jones, M., Mccallum, H., 2007. The impact of disease on the survival and population growth rate of the Tasmanian devil. J Anim Ecol 76, 926-936. Lam, L., Garner, M.M., Miller, C.L., Milne, V.E., Cook, K.A., Riggs, G., Grillo, J.F., Childress, A.L., Wellehan, J.F.X., 2013. A novel gammaherpesvirus found in oral squamous cell carcinomas in sun bears (Helarctos malayanus). J Vet Diagn Invest 25, 99-106. Larue, S.M., Gillette, E.L., 2001. Radiation therapy, In: Small animal clinical oncology. Saunders, Philadelphia. LaRue, S.M., Vail, D.M., Ogilvie, G.K., 1991. Shrinking-field radiation therapy in combination with mitoxantrone chemotherapy for the treatment of oral squamous cell carcinoma in the cat. Proc Vet Cancer Society, 99. Lascelles, B.D., Thomson, M.J., Dernell, W.S., Straw, R.C., Lafferty, M., Withrow, S.J., 2003. Combined dorsolateral and intraoral approach for the resection of tumors of the maxilla in the dog. J Am Anim Hosp Assoc 39, 294-305. Lascelles, B.D.X., Dobson, J.M., British Small Animal Veterinary Association., 2011. BSAVA manual of canine and feline oncology, 3rd ed. British Small Animal Veterinary Association, Quedgeley, Gloucester. Liptak, J.M., Withrow, S.J., 2007. Oral tumors, In: Withrow & MacEwen's small animal clinical oncology, 4th ed. Saunders Elsevier, St. Louis, Mo., pp. 455-475. Loh, R., Bergfeld, J., Hayes, D., O'Hara, A., Pyecroft, S., Raidal, S., Sharpe, R., 2006. The pathology of devil facial tumor disease (DFTD) in Tasmanian devils (Sarcophilus harrisii). Vet Pathol 43, 890- 895. Lombard, L.S., Witte, E.J., 1959. Frequency and Types of Tumors in Mammals and Birds of the Philadelphia-Zoological-Garden. Cancer Res 19, 127-141.

34

Luff, J., Rowland, P., Mader, M., Orr, C., Yuan, H., 2016. Two Canine Papillomaviruses Associated With Metastatic Squamous Cell Carcinoma in Two Related Basenji Dogs. Vet Pathol 53, 1160-1163. Maccubbin, A.E., Black, P., Trzeciak, L., Black, J.J., 1985. Evidence for Polynuclear Aromatic- Hydrocarbons in the Diet of Bottom-Feeding Fish. B Environ Contam Tox 34, 876-882. MacEwen, E.G., Kurzman, I.D., Vail, D.M., Dubielzig, R.R., Everlith, K., Madewell, B.R., Rodriguez, C.O., Phillips, B., Zwahlen, C.H., Obradovich, J., Rosenthal, R.C., , L.E., Rosenberg, M., Henry, C., Fidel, J., 1999. Adjuvant therapy for melanoma in dogs: Results of randomized clinical trials using surgery, liposome-encapsulated muramyl tripeptide, and granulocyte macrophage colony-stimulating factor. Clin Cancer Res 5, 4249-4258. Macewen, E.G., Patnaik, A.K., Harvey, H.J., Hayes, A.A., Matus, R., 1986. Canine Oral Melanoma - Comparison of Surgery Versus Surgery Plus Corynebacterium-Parvum. Cancer Invest 4, 397-402. Macmillan, R., Withrow, S.J., Gillette, E.L., 1982. Surgery and Regional Irradiation for Treatment of Canine Tonsillar Squamous-Cell Carcinoma - Retrospective Review of 8 Cases. J Am Anim Hosp Assoc 18, 311-314. Martin, C.K., Tannehill-Gregg, S.H., Wolfe, T.D., Rosol, T.J., 2011. Bone-invasive oral squamous cell carcinoma in cats: pathology and expression of parathyroid hormone-related protein. Vet Pathol 48, 302-312. Martineau, D., Lemberger, K., Dallaire, A., Labelle, P., Lipscomb, T.P., Michel, P., Mikaelian, I., 2002. Cancer in wildlife, a case study: Beluga from the St. Lawrence estuary, Quebec,Canada. Environ Health Persp 110, 285-292. Matthews, J., Preston, P., 2004. Welcome to the order carnivora. http://www.nhc.ed.ac.uk/index.php?page=4 (accessed 11/08/18. McAloose, D., Munson, L., Naydan, D.K., 2007. Histologic features of mammary carcinomas in zoo felids treated with melengestrol acetate (MGA) contraceptives. Vet Pathol 44, 320-326. McAloose, D., Newton, A.L., 2009. Wildlife cancer: a conservation perspective. Nat Rev Cancer 9, 517-526. McCallum, H., Tompkins, D.M., Jones, M., Lachish, S., Marvanek, S., Lazenby, B., Hocking, G., Wiersma, J., Hawkins, C.E., 2007. Distribution and impacts of Tasmanian devil facial tumor disease. Ecohealth 4, 318-325. McNulty, E.E., Gilson, S.D., Houser, B.S., Ouse, A., 2000. Treatment of fibrosarcoma in a maned wolf (Chrysocyon brachyurus) by rostral maxillectomy. J Zoo Wildlife Med 31, 394-399. Misdorp, W., Cotchin, E., Hampe, J.F., Jabara, A.G., Sandersl.Jv, 1973. Canine Malignant Mammary- Tumors .3. Special Types of Carcinomas Malignant Mixed Tumors. Vet Pathol 10, 241-256. Misdorp, W., Vanderheul, R.O., 1976. Tumors of Bones and Joints. B World Health Organ 53, 265- 282. Moore, A.S., Theilen, G.H., Newell, A.D., Madewell, B.R., Rudolf, A.R., 1991. Preclinical Study of Sequential Tumor-Necrosis-Factor and Interleukin-2 in the Treatment of Spontaneous Canine Neoplasms. Cancer Res 51, 233-238. Munday, J.S., 2014. Papillomaviruses in felids. Vet J 199, 340-347. Munday, J.S., Kiupel, M., 2010. Papillomavirus-Associated Cutaneous Neoplasia in Mammals. Vet Pathol 47, 254-264. Munday, J.S., O'Connor, K.I., Smits, B., 2011. Development of multiple pigmented viral plaques and squamous cell carcinomas in a dog infected by a novel papillomavirus. Vet Dermatol 22, 104-110. Munday, J.S., Thomson, N.A., Luff, J.A., 2017. Papillomaviruses in dogs and cats. Vet J 225, 23-31. Mylniczenko, N.D., Manharth, A.L., Clayton, L.A., Feinmehl, R., Robbins, M., 2005. Successful treatment of mandibular squamous cell carcinoma in a Malayan sun bear (Helarctos malayanus). J Zoo Wildlife Med 36, 346-348. Nanci, A., Ten Cate, A.R., 2008. Ten Cate's oral histology : development, structure, and function, 7th ed. Mosby Elsevier, St. Louis, Mo. NCI, 2018. NCI Dictionary of Cancer Terms. https://www.cancer.gov/publications/dictionaries/cancer- terms/def/oral-cavity (accessed 02/04/18. Nelson, K.G., Engh, A.L., McKnight, C.A., Klupel, M., Wise, A.G., Maes, R.K., Stevens, H., Heylen, E., De Keyser, K., Rector, A., Van Ranst, M., Flies, A.S., Holekamp, K.E., 2013. Papillomavirus- associated Cutaneous Papillomas in a Population of Wild Spotted Hyenas (Crocuta crocuta). J Wildlife Dis 49, 627-631.

35

Nemec, A., Murphy, B., Kass, P.H., Verstraete, F.J., 2012. Histological subtypes of oral non-tonsillar squamous cell carcinoma in dogs. J Comp Pathol 147, 111-120. Northrup N .C., Selting K. A., M., R.K., 2006. Outcomes of cats with oral tumors treated with mandibulectomy: 42 cases. J Am Anim Hosp Assoc 42, 350-360. Northrup, N.C., Selting, K.A., Rassnick, K.M., Kristal, O., O'Brien, M.G., Dank, G., Dhaliwal, R.S., Jagannatha, S., Cornell, K.K., Gieger, T.L., 2006. Outcomes of cats with oral tumors treated with mandibulectomy: 42 cases. J Am Anim Hosp Assoc 42, 350-360. Ogbureke, K.U.E., Nashed, M.N., Ayoub, A.F., 2007. Huge peripheral osteoma of the mandible: A case report and review of the literature. Pathol Res Pract 203, 185-188. Ogilvie, G.K., Moore, A.S., Obradovich, J.E., Elmslie, R.E., Vail, D.M., Straw, R.C., Salmon, M.D., Klein, M.K., Atwater, S.W., Ciekot, P.E., Larue, S.M., Peaston, A., Withrow, S.J., 1993. Toxicoses and Efficacy Associated with Administration of Mitoxantrone to Cats with Malignant-Tumors. J Am Vet Med Assoc 202, 1839-1844. Overgaard, J., Overgaard, M., Hansen, P.V., Vondermaase, H., 1986. Some Factors of Importance in the Radiation Treatment of Malignant-Melanoma. Radiother Oncol 5, 183-192. Overley, B., Goldschmidt, M.H., Shofer, F.S., 2001. Canine oral melanoma: a retrospective study. Vet Cancer Soc Proc 21, 43. Owen, L.N., 1980. TNM classification of tumors in domestic animals. World Health Organization, Geneva. Owston, M.A., Ramsay, E.C., Rotstein, D.S., 2008. Neoplasia in Felids at the Knoxville Zoological Gardens, 1979-2003. J Zoo Wildlife Med 39, 608-613. Padgett, S.L., Tillson, D.M., Henry, C.J., Buss, M.S., 1997. Gingival vascular hamartoma with associated paraneoplastic hyperglycemia in a kitten. J Am Vet Med Assoc 210, 914-915. Patnaik, A.K., Mooney, S., 1988. Feline Melanoma - a Comparative-Study of Ocular, Oral, and Dermal Neoplasms. Vet Pathol 25, 105-112. Pessier, A.P., 1999. Soft tissue sarcomas associated with identification of microchip implants in two small zoo mammals, In: Proc. Am. Assoc. Zoo Vet. Pindborg, J.J., Kramer, I.R.H., Torloni, H., 1971. Histological typing of odontogenic tumours, jaw cysts and allied lesions. World Health Organisation, Geneva. Postorino Reeves, N.C., Turrel, J.M., Withrow, S.J., 1993. Oral squamous cell carcinoma in the cat. J Am Anim Hosp Assoc 29, 438-441. Poulet, F.M., Valentine, B.A., Summers, B.A., 1992. A survey of epithelial odontogenic tumors and cysts in dogs and cats. Vet Pathol 29, 369-380. Proulx, D.R., Ruslander, D.M., Dodge, R.K., Hauck, M.L., Williams, L.E., Horn, B., Price, G.S., Thrall, D.E., 2003. A retrospective analysis of 140 dogs with oral melanoma treated with external beam radiation. Vet Radiol Ultrasoun 44, 352-359. Pyecroft, S., 2007. Transmission trials: devil facial tumor disease. Devil Facial Tumour Diseases: Senior Scientist’s Scientific Forum 18. QuintinColonna, F., Devauchelle, P., Fradelizi, D., Mourot, B., Faure, T., Kourilsky, P., Roth, C., Mehtali, M., 1996. Gene therapy of spontaneous canine melanoma and feline fibrosarcoma by intratumoral administration of histoincompatible cells expressing human interleukin-2. Gene Ther 3, 1104-1112. Rassnick, K.M., Ruslander, D.M., Cotter, S.M., Al-Sarraf, R., Bruyette, D.S., Gamblin, R.M., Meleo, K.A., Moore, A.S., 2001. Use of carboplatin for treatment of dogs with malignant melanoma: 27 cases (1989-2000). J Am Vet Med Assoc 218, 1444-1448. Regalado, A., 2016. Malignant transformation of a canine viral papilloma to oral squamous cell carcinoma, In: Proceedings of the Veterinary Dental Forum, Minneapolis, Minnesota, USA. Roberts, S.M., Lavach, J.D., Severin, G.A., Withrow, S.J., Gillette, E.L., 1987. Ophthalmic complications following megavoltage irradiation of the nasal and paranasal cavities in dogs. J Am Vet Med Assoc 190, 43-47. Roden, R.B.S., Lowy, D.R., Schiller, J.T., 1997. Papillomavirus is resistant to desiccation. J Infect Dis 176, 1076-1079. Ryan, M.J., Nielsen, S.W., 1979. Tonsillar Carcinoma with Metastases in a Captive Wolf. J Wildlife Dis 15, 295-298.

36

Sacco, T., Van Valkenburgh, B., 2004. Ecomorphological indicators of feeding behaviour in the bears (Carnivora : Ursidae). J Zool 263, 41-54. Samuel, W.M., Chalmers, G.A., Gunson, J.R., 1978. Oral Papillomatosis in Coyotes (Canis-Latrans) and Wolves (Canis-Lupus) of Alberta. J Wildlife Dis 14, 165-169. Sato, T., Higuchi, T., Shibuya, H., Ohba, S., Nogami, S., Shirai, W., Watanabe, H., Honda, S., 2002. Lingual squamous cell carcinoma in a California sea lion (Zalophus californianus). J Zoo Wildlife Med 33, 367-370. Schmidt, J.M., North, S.M., Freeman, K.P., Ramiro-Ibanez, F., 2010. Canine paediatric oncology: retrospective assessment of 9522 tumours in dogs up to 12 months (1993-2008). Vet Comp Oncol 8, 283-292. Schoofs, S.H., 1997. Lingual hemangioma in a puppy: A case report and literature review. J Am Anim Hosp Assoc 33, 161-165. Schwarz, P.D., Withrow, S.J., Curtis, C.R., Powers, B.E., Straw, R.C., 1991a. Mandibular Resection as a Treatment for Oral-Cancer in 81 Dogs. J Am Anim Hosp Assoc 27, 601-610. Schwarz, P.D., Withrow, S.J., Curtis, C.R., Powers, B.E., Straw, R.C., 1991b. Partial Maxillary Resection as a Treatment for Oral-Cancer in 61 Dogs. J Am Anim Hosp Assoc 27, 617-624. Siegal-Willott, J., Heard, D., Sliess, N., Naydan, D., Roberts, J., 2007. Microchip-associated leiomyosarcoma in an Egyptian fruit bat (Rousettus aegyptiacus). J Zoo Wildl Med 38, 352-356. Sladakovic, I., Burnum, A., Blas-Machado, U., Kelly, L.S., Garner, B.C., Holmes, S.P., Divers, S.J., 2016. Mandibular Squamous Cell Carcinoma in a Bobcat (Lynx Rufus). J Zoo Wildlife Med 47, 370- 373. Smith, M.M., 1995. Surgical approach for lymph node staging of oral and maxillofacial neoplasms in dogs. J Am Anim Hosp Assoc 31, 514-518. Snyder, L.A., Bertone, E.R., Jakowski, R.M., Dooner, M.S., Jennings-Ritchie, J., Moore, A.S., 2004. p53 expression and environmental tobacco smoke exposure in feline oral squamous cell carcinoma. Vet Pathol 41, 209-214. Stains, H., 1984. Carnivores, In: Orders and Families of Recent Mammals of the World. John Wiley and Sons, New York, pp. 491-521. Stebbins, K.E., Morse, C.C., Goldschmidt, M.H., 1989. Feline Oral Neoplasia - a 10-Year Survey. Vet Pathol 26, 121-128. Sundberg, J.P., Montali, R.J., Bush, M., Phillips, L.G., OBrien, S.J., Jenson, A.B., Burk, R.D., VanRanst, M., 1996. Papillomavirus-associated focal oral hyperplasia in wild and captive Asian lions (Panthera leo persica). J Zoo Wildlife Med 27, 61-70. Sundberg, J.P., Van Ranst, M., Montali, R., Homer, B.L., Miller, W.H., Rowland, P.H., Scott, D.W., England, J.J., Dunstan, R.W., Mikaelian, I., Jenson, A.B., 2000. Feline papillomas and papillomaviruses. Vet Pathol 37, 1-10. Theodorou, D.J., Theodorou, S.J., Sartoris, D.J., 2003. Primary non-odontogenic tumors of the jawbones: an overview of essential radiographic findings. Clin Imaging 27, 59-70. Theon, A.P., Rodriguez, C., Griffey, S., Madewell, B.R., 1997a. Analysis of prognostic factors and patterns of failure in dogs with periodontal tumors treated with megavoltage irradiation. J Am Vet Med Assoc 210, 785-788. Theon, A.P., Rodriguez, C., Madewell, B.R., 1997b. Analysis of prognostic factors and patterns of failure in dogs with malignant oral tumors treated with megavoltage irradiation. J Am Vet Med Assoc 210, 778-784. Thoma, K.H., Goldman, H.M., 1946. Odontogenic Tumors: A Classification Based on Observations of the Epithelial, Mesenchymal, and Mixed Varieties. Am J Pathol 22, 433-471. Thompson, K.G., Pool, R.R., 2002. Tumors of bones, In: Tumors in domestic animals, 4th ed. Iowa State Press, Ames, Iowa, pp. 248-252. Thomson, N.A., Munday, J.S., Dittmer, K.E., 2016. Frequent detection of transcriptionally active Felis catus papillomavirus 2 in feline cutaneous squamous cell carcinomas. J Gen Virol 97, 1189-1197. Thrall, D.E., 1984. Orthovoltage radiotherapy of acanthomatous epulides in 39 dogs. J Am Vet Med Assoc 184, 826-829. Thrall, D.E., Goldschmidt, M.H., Biery, D.N., 1981. Malignant tumor formation at the site of previously irradiated acanthomatous epulides in four dogs. J Am Vet Med Assoc 178, 127-132.

37

Todoroff, R.J., Brodey, R.S., 1979. Oral and pharyngeal neoplasia in the dog: a retrospective survey of 361 cases. J Am Vet Med Assoc 175, 567-571. Turk, J.R., Leathers, C.W., 1981. Light and Electron-Microscopic Study of the Large Pale Cell in a Canine Malignant-Melanoma. Vet Pathol 18, 829-832. Vaughan, T.A., Ryan, J.M., Czaplewski, N.J., 2000. Mammalogy, Fourth edition. ed. Thomson Learning, London. Verhaert, L., 2001. Oral Proliferative Lesions in Dogs and Cats, In: 26th World Congress of the WSAVA, Vancouver, pp. 218-220. Verstraete, F.J., Ligthelm, A.J., Weber, A., 1992. The histological nature of epulides in dogs. J Comp Pathol 106, 169-182. Vos, J.H., Vandergaag, I., Vandijk, J.E., Wouda, W., 1986. Lobular Capillary Hemangiomas in Young Horses. J Comp Pathol 96, 637-644. Wallace, J., Matthiesen, D.T., Patnaik, A.K., 1992. Hemimaxillectomy for the Treatment of Oral Tumors in 69 Dogs. Vet Surg 21, 337-341. Walsh, K.M., Denholm, L.J., Cooper, B.J., 1987. Epithelial Odontogenic-Tumors in Domestic-Animals. J Comp Pathol 97, 503-521. Warren, A.L., Summers, B.A., 2007. Epithelioid variant of hemangioma and hemangiosarcoma in the dog, horse, and cow. Vet Pathol 44, 15-24. Widmer, W.R., Carlton, W.W., 1990. Persistent Hematuria in a Dog with Renal Hemangioma. J Am Vet Med Assoc 197, 237-239. Wiggs, R.B., Bloom, B.C., 2003. Exotic placental carnivore dentistry. Vet Clin North Am Exot Anim Pract 6, 571-599, vi. Williams, L.E., Packer, R.A., 2003. Association between lymph node size and metastasis in dogs with oral malignant melanoma: 100 cases (1987-2001). J Am Vet Med Assoc 222, 1234-1236. Withrow, S.J., MacEwan, E.G., 2001. Cancer in the oral cavity, In: Small animal clinical oncology, 3rd ed. W. B. Saunders, Philadelphia, pp. xvii, 718 p. Woldenberg, Y., Nash, M., Bodner, L., 2005. Peripheral osteoma of the maxillofacial region. Diagnosis and management: a study of 14 cases. Medicina oral, patologia oral y cirugia bucal 10 Suppl 2, E139- 142. Wolfe, L.L., Spraker, T.R., 2007. Oral papillomatosis in Canada lynx (Lynx canadensis). J Wildlife Dis 43, 731-733. Yanai, T., Noda, A., Murata, K., Yasuda, S., Hama, N., Sakai, H., Masegi, T., 2003. Lingual squamous cell carcinoma in an ocelot (Felix pardalis). Vet Rec 152, 656-657. Yoshida, K., Yanai, T., Iwasaki, T., Sakai, H., Ohta, J., Kati, S., Minami, T., Lackner, A.A., Masegi, T., 1999. Clinicopathological study of canine oral epulides. J Vet Med Sci 61, 897-902.

38

Appendix A: Taxonomy chart of the Carnivora order (ITIS, 2018)

Eupleridae Pantherinae Felidae Felinae Herpestidae Crocuta Feliformia

Hyaenidae Hyaena

Nandiiidae Proteles

Viverridae

Carnivora Ailuridae

Canidae Martes

Mephitidae Mustela Lutrinae Mustelidae Meles Mustelinae Caniformia Odobenidae Mellivora

Otariidae Neovison

Phocidae Ailuropoda Taxidea

Procyonidae Helarctos

Ursidae Melursus

Tremarctos

Ursus

39

Appendix B: Reported cases of oral tumours in non- domesticated carnivorans described in literature, classified per tumour type

Abbreviations: M: Male intact F: Female intact MC: Male castrated FC: Female castrated

1. Odontogenic tumours 1.1 Amyloid-producing odontogenic tumour

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Panthera Mandibula M 13 Kang et al. Journal of Comparative 2006 tigris tigris (incisors) Pathology 1.2 Calcifying epithelial odontogenic tumour

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Panthera Gingiva at M Adult Fecchio et al. Journal of Veterinay 2015 tigris altaica right Dentistry maxillary canine tooth

1.3 Fibromatous epulis of periodontal origin

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Panthera leo Gingiva at M Aged Castro et al. Brazilian Journal of 2011 left maxillar Veterinary Pathology canine tooth

2. Non-odontogenic tumours 2.1 Papillomatosis

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Canis latrans Lips, tongue, F ? Broughton et al. Journal of wildlife 1970 adjacent diseases tissue Canis lupus unknown ? puppy Samuel et al. Journal of wildlife 1978 diseases

40

Canis lupus unknown ? puppy Samuel et al. Journal of wildlife 1978 diseases Canis latrans Lips, tongue, ? ? Samuel et al. Journal of wildlife 1978 adjacent diseases tissue Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Canis latrans unknown ? ? Samuel et al. Journal of wildlife 1978 diseases Panthera Sublingual ? ? Joslin et al. PROCEEDINGS AAZV AND 2000 uncia IAAAM JOINT CONFERENCE Panthera Sublingual ? ? Joslin et al. PROCEEDINGS AAZV AND 2001 uncia IAAAM JOINT CONFERENCE Felis Tongue ? Adult Sundberg et al. Veterinary Pathology 2000 Concolor Felis Tongue ? Adult Sundberg et al. Veterinary Pathology 2000 Concolor Felis rufus Tongue ? Adult Sundberg et al. Veterinary Pathology 2000 Felis rufus Tongue ? Adult Sundberg et al. Veterinary Pathology 2000 Panthera leo Tongue M Adult Sundberg et al. Journal of Zoo and 1996 Wildlife Medicine Panthera leo Tongue M Adult Sundberg et al. Journal of Zoo and 1996 Wildlife Medicine Panthera leo Tongue M Adult Sundberg et al. Journal of Zoo and 1996 Wildlife Medicine Panthera leo Tongue F Adult Sundberg et al. Journal of Zoo and 1996 Wildlife Medicine Panthera Tongue, ? 5-16 y Sundberg et al. Veterinary Pathology 2000 uncia buccal mucosae Neofelis Tongue ? Adult Sundberg et al. Veterinary Pathology 2000

41

nebulosa Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Felis lynx Tongue ? ? Wolfe and Journal of Wildlife 2007 Spraker diseases Crocuta Muzzle ? (Sub)- Nelson et al. Journal of wildlife 2013 crocuta adult diseases

2.2 Squamous cell carcinoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Lynx Rufus Right F 23 Sladakovic et al. Journal of Zoo and 2016 mandibula Wildlife Medicine Canis latrans Maxilla right FC 19 Bernstein et al. Journal of Zoo and 1999 Wildlife Medicine Zalophus Tongue F 28 Sato et al. Journal of Zoo and 2002 californianus Wildlife Medicine Leopardus Tongue F 19 Yanai et al. The Veterinary record 2003 pardalis Zalophus Left F 30 Bossart Journal of Zoo and 1990 calif Mandibula Wildlife Medicine ornianus Lynx unknown ? ? Effron et al. Journal of national cancer 1978 canadensis institute Helarctos Left lower lip F 19 Lam et al. Journal of Veterinary 2013 malayanus Diagnostic Investigation

42

Helarctos Right lower M 18 Lam et al. Journal of Veterinary 2013 malayanus lip Diagnostic Investigation

Helarctos Left lower lip M 8 Lam et al. Journal of Veterinary 2013 malayanus Diagnostic Investigation Helarctos Left lower lip F 17 Lam et al. Journal of Veterinary 2013 malayanus Diagnostic Investigation Helarctos Right lower M 18 Lam et al. Journal of Veterinary 2013 malayanus lip Diagnostic Investigation Helarctos Mandibula: F 6 Mylniczenko et Journal of Zoo and 2005 malayanus lingual al. Wildlife Medicine surface incisors Canis lupus Tonsilla M 11 Teifke et al. Journal of Zoo and 2005 arctos Wildlife Medicine Canis lupus Tonsilla M 13 Ryan et al. Journal of wildlife 1979 diseases Suricata Mandibular M 10 Sladky et al. Veterinary Pathology 2000 suricatta gingiva

2.3 Adenocarcinoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year C. aureus oral cavity M ? Effron et al. Journal of national cancer 1977 syriacus glandular institute epithelium

2.4 Mucoepidermoid carcinoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Panthera leo Left MC 13 Dorso et al. Veterinary Pathology 2008 mandibular salivary gland

2.5 Anaplastic carcinoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Suricata Tongue, F 8 Dadone et al. Journal of Zoo and 2014 suricatta rostral Wildlife Medicine mandibula

43

2.6 Secondary carcinoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Ursus Tongue M ? Lombard and Cancer research 1959 americanus Witte Taxidea Mouth M ? Lombard and Cancer research 1959 taxus Witte

2.7 Malignant melanoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Panthera leo Left F 13 Steeil et al. Journal of Zoo and 2013 maxillary lip Wildlife Medicine thv canine Ursus arctos Tongue F ? Lombard and Cancer research 1959 lasiotus Witte

2.8 Haemangioma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Acinonyx Tongue F ? Effron et al. Journal of national cancer 1978 jubatus institute

2.9 Fibrosarcoma

Species Oral Age name localisation (Latin) Sex (years) Author Journal Year Chrysocyon Journal of Zoo and brachyurus Left maxilla M 12 McNulty et al. Wildlife Medicine 2000

44

Appendix C: Reported cases of oral tumours in non- domesticated carnivorans retrieved through survey

Abbreviations: M: Male intact F: Female intact MC: Male castrated FC: Female castrated

Species name Histopathological diagnosis Age (Latin) Sex (years) Region Acinonyx jubatus Fibromatous epulis of periodontal ligament origin M 1,5 North America Acinonyx jubatus Fibromatous epulis of periodontal ligament origin M 1,5 North America Acinonyx jubatus Fibromatous epulis of periodontal ligament origin M 1,5 North America Acinonyx jubatus Gingival hyperplasia M 6,5 North America Acinonyx jubatus Chronic fibrosing gingival hyperplasia F 14 North America Canis latrans Squamous cell carcinoma FC 8 North America Canis rufus Squamous cell carcinoma FC 15 North America Panthera tigris Malignant melanoma F 12 North America Canis lupus baileyi Amelanotic melanoma F 12 North America Canis lupus baileyi Amelanotic melanoma F 14 North America Mephitis mephitis Osteoma F >4 North America

45