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Vet Clin Exot Anim 7 (2004) 673–695
Vet Clin Exot Anim 7 (2004) 673–695

Amphibian oncology

Brian A. Stacy, DVM a, * , John M. Parker, DVM b

a Anatomic Pathology Service, Veterinary Medical Teaching Hospital, University of California–Davis, One Shields Avenue, Davis, CA 95616, USA b Office of Laboratory Animal Care, Northwest Animal Facility, University of California–Berkeley, Room 203C, Berkeley, CA 94720-7150, USA

The class Amphibia consists of three orders: Anura (frogs and toads), Urodela (a.k.a. Caudata) (salamanders), and Gymnophiona (caecilians). Captive and wild populations are important to many areas of scientific research, and are a focus of major global ecologic concerns, including pollution, climate changes, habitat destruction, and nonnative species trans- location. Such concerns have been identified as causes of decline in certain amphibian populations. Also, the number and diversity of wild and captive amphibians maintained and monitored by zoologic and teaching institutions, research facilities, and hobbyists continues to grow. Consequently, veter- inarians are consulted more frequently for information on health and disease. Reports of spontaneous neoplasia in wild amphibians are relatively rare and often limited to specific species or populations [1]. For example, viral- induced Lucke´ adenocarcinoma is documented only in northern leopard frogs (Rana pipiens), and a high incidence of neoplasia in barred tiger salamanders (Ambystoma mavortium) is limited to a population inhabiting a polluted pond in Texas. Research has shown that at least some anuran species possess inherent anticancer secretory products and cytoprotective devices, and are relatively resistant to some mammalian carcinogens [2–7]. Also, the remarkable regenerative capacity of urodeles is hypothesized to reduce tumor susceptibility [8,9]. To date, neoplasia has not been reported in Gymnophiona. Amphibians are comprised of the same basic tissue elements as other species, and any cell type can undergo neoplastic (malignant) transforma- tion. The biologic behavior of a given tumor type may vary among amphibian species because of natural species variation and etiologic differences, such as viral infection, exposure to pollutants, or inheritable

* Corresponding author. E-mail address: bastacydvm@glasselevator.net (B.A. Stacy).

1094-9194/04/$ - see front matter 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cvex.2004.04.001

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(genetic) predisposition [10–13]. Clinical management is facilitated by understanding documented features of tumors, which may provide insight into treatment options, identification of predisposing factors, and manage- ment of populations. Furthermore, definitive diagnosis is important because tumors with an infectious or acquired etiology and infectious ‘‘pseudoneo- plastic’’ conditions, such as mycobacteriosis, are relatively common, and may spread within the immediate group or population. Literature pertaining to amphibian neoplasia is scattered, often incom- plete, and difficult to interpret due to inconsistencies in diagnosis, nomencla- ture, and misdiagnosis of infectious and inflammatory conditions. Furthermore, clinically relevant information, such as long-term prognosis, biologic behavior, and therapy, is lacking, and many reports focus on aspects of tumor research rather than clinical management. Green and Harshbarger [14] provided a recent and thorough review of published cases and those submitted to the Registry of Tumors in Lower Animals (RTLA) (22,900 Shaw Road, Suite 107, Sterling, Virginia 20166-4311, USA). Such references, as well as materials maintained by the RTLA, are useful resources for veterinarians. This article focuses on relatively common types of neoplasia, methods for antemortem diagnosis, differential diagnoses, and techniques for surgical excision. Select cases from the large amphibian population maintained by the University of California, Berkeley (UCB) are used to exemplify some tumor types. Classically, the terms ‘‘tumor’’ and ‘‘neoplasia’’ have different definitions, and their use in the literature has led to some confusion. Most current references, however, use these terms interchangeably and regard them as synonyms. Thus, both terms are used interchangeably throughout this text. A variety of tumors have been induced experimentally, and those without clinical relevance are not considered here.

Tumors of the integument and soft tissues

Neoplastic diseases of the integument are documented in several anuran and urodele species, and constitute a large proportion of the literature on amphibian neoplasia [14,15]. Cases include neoplasia of the surface and glandular epithelium, dermal stroma, and melanophores. In some instances, tumors have unique features such as environmental and infectious etiology, temperature-dependent growth, and predictable regression under laboratory conditions. Many neoplasms are only reported in specific populations, and their occurrence and biologic behavior in other wild and captive populations is unknown.

Epidermal papillomas

Epidermal papillomas, also referred to as squamous papillomas, in- fectious warts, and epitheliomas, are described most frequently in urodele

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species, including free-ranging Japanese newts (Cynops pyrrhogaster), barred tiger salamanders (A mavortium) and Tohoku salamanders (Hyno- bius lichenatus) [11,16–19]. These papillomas are sessile, hypopigmented, and well demarcated with a folded or convoluted surface [16,17]. Histolog- ically, they consist of marked epidermal hyperplasia that forms papillary projections and occasionally involves the glandular epithelium and extends into the dermis [17,18]. Most are solitary lesions of little health concern, and are readily debulked or excised for diagnosis. In a few cases, however, papillomas were multicentric, and associated with poor body condition

[17,18].

Spontaneously regressing skin papillomas in wild C pyrrhogaster occur seasonally. The highest prevalence is in autumn, with affected newts comprising up to 5.4% of the animals examined [20]. In a case series of 10, seven regressed over 4 to 12 months and three progressed to large, multicentric lesions [18]. In another series, tumors in 44 newts regressed over a period of months when placed under laboratory conditions [17]. Studies have shown that temperature strongly influences tumor growth and regression. Tumors of newts housed at 4(C and 25(C regress in size and even sloughed off in some cases, and tumors increase in size at midrange temperatures of 10(C to 13(C [21]. Also, experimental exposure to ultraviolet radiation caused regression in one report [22]. A viral etiology is suspected based on the detection of herpesvirus-like particles in some tumors [20,23]. This suspicion was not pursued further, and tumor trans- mission could not be induced experimentally [17]. Regressing papillomas are also documented in a neotenic population of A mavortium that inhabits a polluted sewage pond [11]. These tumors are presumably caused by an environmental stimulus, having regressed after 4 months under laboratory conditions in a controlled study [11]. Whether papillomas will eventually regress spontaneously in most other species is not known. In anurans, reports of papillomas are limited to a single case in a black spotted frog (Rana nigromaculata) and a case series in R pipiens [14,24,25]. In addition, two long-term captive Surinam toads (Pipa pipa) housed at the UCB, developed large, insidious, pedunculated papillomas on the distal extremities (Fig. 1), both of which were excised successfully. This species is known for having ‘‘warts,’’ and an underlying viral etiology is suspected. Despite prolonged cohabitation, however, evidence for high infectivity was not observed in these cases [25; JMP, personal observation].

Other epithelial tumors

Other epithelial tumors reported in amphibians include squamous cell carcinomas and dermal gland tumors. Van der Steen et al [24] examined a series of cutaneous tumors in R pipiens that included 10 squamous cell carcinomas. These tumors were characterized grossly as hypopigmented, raised, sessile, or

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676 B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 Fig. 1. A

Fig. 1. A pedunculated papilloma extends from the dorsal aspect of the distal digit of a Surinam toad (Pipa pipa). The tumor was easily excised by ligation of the fibrovascular stalk.

pedunculated, 0.3 to 1.5 cm diameter masses. Histologically, tumors were comprised of infiltrative nests of nonkeratinizing epithelium. Most tumors were locally infiltrative but did not invade deep to the dermis, except for one case involving the deep skeletal muscle and bone. Adenomas and adeno- carcinomas of the dermal glands are documented as single case reports, as well as a larger series of 78 neoplasms in seven grass frogs (Rana temporaria) and 16 pond frogs (Rana ridibunda) [15]. Representing the majority of the literature on dermal gland tumors, this series described cystadenomas and cystadenocarcinomas as comprised of dilated glands lined by papillary projections of neoplastic epithelium. The diagnosis of cystadenocarcinoma was based on increased cellular pleomorphism and atypia. Tumors were confined to the dermis, and metastasis occurred in a single case. Other tumors of dermal glands include rarely reported mixed epithelial and myoepithelial (contractile glandular tissue) tumors and a presumed myoe- pithelial adenoma [14]. Based on these reports, surgical removal of squamous cell carcinomas and dermal gland tumors is advised and both may exhibit local infiltration and metastasis.

Melanophoromas

Melanophoromas, also referred to as melanoma, melanocytoma, and melanosarcoma, are described in multiple species of urodeles and anurans. Neoplasms vary greatly in behavior and gross morphology, ranging from solitary to multicentric, hypopigmented to pigmented, nonpalpable to nodular, and regressive to invasive masses. Histologically, as with melano- cytic tumors in mammals, they can exhibit several cell types, including dendritic, epithelioid, and spindle-shaped, but often are classified by the dominant cell type [14]. In some cases, melanophoromas were limited to the dermis and did not invade the underlying lymph sac, making them amenable

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to surgical excision [12,26]. Some of these benign forms are analogous to melanocytoma or melanocytic nevi in mammals. A few highly invasive or malignant melanophoromas are documented, and include darkly pigmented and hypopigmented variants [14]. An important differential diagnosis for melanophoroma is melanophore hyperplasia secondary to skin diseases (Fig. 2A). Some melanophoromas initially present as flat, darkly pigmented foci and grossly are indiscernible from hyperplastic lesions; therefore, biopsy is required for definitive diagnosis [26,27]. Melanophoromas have unique features in some species, such as hereditary predisposition or possible viral etiology [13,26]. In studies with transplant- able melanophoromas in crested newts (Triturus cristatus carnifex), tumor

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B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 677 to surgical excision

Fig. 2. (A) An unidentified Xenopus species with focal melanophore hyperplasia (arrow) associated with dermatitis. The inflammation in this case was due to nematodiasis of the dermal glands and required histopathology for diagnosis. Early melanomas may begin as grossly similar lesions. (B) The dorsum of a barred tiger salamander (Ambystoma mavortium) with a large melanophoroma. Melanophoromas in this population regressed under laboratory conditions and were presumed to have an environmental stimulus. (Courtesy of F.L. Rose, PhD, Texas State University.)

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growth rate and metastasis are temperature dependent. Namely, tumors were relatively static at 4(C, but grew rapidly and metastasized at 30(C [28]. Temperature-dependent growth is shared with Lucke´ renal adenocarcino- mas, epidermal papillomas of Japanese newts, and possibly other amphibian tumors [21,28,29]. Caution must be exercised, however, in extrapolating information about biologic behavior between different species, as different etiologies and biologic variations could result in different behavior. Several cases of melanophoroma are reported in Mexican axolotls (Ambystoma mexicanum), but little information on age and survivability is provided [15,26]. In cases reported by Khudoley and Mizgireuv [15], melanophoromas in both pigmented and albino A mexicanum consistently seemed to progress from a flat proliferation of melanophores to a nodular growth. For example, a male axolotl developed a tumor at 2.5 months of age and developed additional masses 16 months later. In a case series presented by Sheremetiva-Brunst, and Brunst [26], hereditary predisposition was evidenced by the high prevalence of tumors in offspring from two individuals with melanophoromas. The second generation developed tumors at approximately 1 year of age, and these grew faster and larger than those of the parent gen- eration. Histologically, neoplasms were confined to the dermis and comprised predominantly of dendritic and heavily pigmented cells with anisokaryosis, anisocytosis, and multinucleated cell formation. In both reports, authors describe two types of tumor growth—those with and those without deep tissue invasion [15,26]. Metastasis was not reported in any case. In summary, based on these few cases, melanomas in pigmented and albino axolotls may develop at a relatively early age, may be locally invasive and multicentric, but are not known to metastasize, and recurrence may follow surgical excision. In a population of A mavortium that inhabited a sewage pond, melano- phoromas were associated with other hyperplastic and neoplastic lesions of the integument (Fig. 2B) [30]. The gross appearance and potentially locally aggressive nature of these tumors were similar to those described in A mexicanum, but regression of some melanophoromas, including infiltrative tumors, followed the removal of animals from the polluted environment and metamorphosis (FL Rose, personal communication). Another series of melanophoromas was reported in the African clawed frog (Xenopus laevis) [13]. Eight frogs out of 40,000 developed single melanophoromas on the dorsum, ventrum, and limbs. These tumors differed from many of those observed in A mexicanum and A mavortium, because they were hypopigmented and highly invasive. Herpesvirus-like viral particles were observed within tumors; however, it was not determined if these tumors actually were caused by an oncogenic virus.

Mesenchymal skin tumors

The classification scheme and terminology of dermal mesenchymal tumors in amphibians is unclear and in need of further characterization.

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The terms dermal fibroma, polypoid dermal fibroma, and fibropapilloma have been used to describe a benign nodular proliferation of dermal fibroblasts covered by normal or hyperplastic epidermis arising anywhere on the skin [14]. Dermal mesenchymal tumors are reported in the same population of A mavortium, which is mentioned throughout this text as well as in a few geriatric albino A mexicanum at UCB ([30]; B.A. Stacy and J.M. Parker, personal observation). Grossly, masses are smooth and hypopig- mented (Fig. 3A). Histologically, they can be poorly demarcated, and are comprised of poorly organized streams of neoplastic fibroblasts with potentially marked anisokaryosis and generally rare to absent mitoses (Fig. 3B). The majority of dermal mesenchymal tumors are benign and exhibit slow growth, and likely do not represent a significant health threat.

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B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 679 The terms dermal

Fig. 3. (A) Geriatric albino Mexican axolotl (Ambystoma mexicanum) with a fibropapilloma of the rostral maxilla. This lesion exhibited slow growth and was an incidental finding. (B) Photomicrograph of the fibropapilloma in (A). The dermis is expanded by a nodular, well- circumscribed mass of haphazardly arranged streams of neoplastic fibroblasts. Some neoplastic cells have a large, bizarre nucleus (arrow). Mitotic figures are not observed. Hematoxylin and eosin; 20 (inset 400 ).

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Fibrosarcomas, which may be locally aggressive, are reported in A mavortium and a few other species [14,31].

Neuroepithelial tumors

Tumors of neuroepithelium are reported in A mexicanum and an alpine newt (Triturus alpestris) [32,33]. These tumors presumably originate from olfactory tissue to form space-occupying masses in the maxilla. Histolog- ically, lobules of elongated or bipolar neoplastic epithelium palisade around cystic spaces containing coagulated material. There is no mention of long-term survivability. In one case, the tumor was transplanted to other adult A mexicanum, and one was alive 3 years after successful transplantation, which may suggest a prolonged clinical course [32]. Tumor location, however, can interfere with feeding and thus present a significant health threat. Surgical excision has not been attempted. Palliative care is recommended until quality of life declines below accept- able standards.

Mast cell tumors

Reports of mastocytomas, also refered to as mast cell tumors, are limited to urodeles, including A mexicanum, neotenic A mavortium, and red-spotted newts (Notophthalmus viridescens) [14,33]. A mexicanum were 10 to 17 years old, and all of A mavortium originated from a polluted sewage pond. Tumors developed within the dermis or subcutaneous tissues anywhere on the body, and appeared grossly as domed, firm, gray-white or red, ulcerated, less than 3.0-cm diameter masses. Histologically, neoplastic mast cells were polygonal to spindle-shaped with marked anisocytosis and anisokaryosis in some tumors. These tumors were locally invasive and regional edema in some tumors suggested the possible release of histamine. Systemic in- volvement, rate of tumor growth, and longevity following diagnosis were not addressed in these studies. Giemsa stain revealed metachromatic granules in 5% to 25% of the neoplastic cells [14]. Initially, many of these tumors were misidentified as mesenchymal or neural tumors and later were reclassified after examination with a Giemsa stain [14]. Thus, a Giemsa stain should be considered when examining similar dermal tumors. Mast cells should exfoliate well on fine-needle aspirates or impression smears, which may aid in antemortem diagnosis. It is not known whether amphibians develop poorly differentiated, agranulocytic tumors, which could present a diagnostic challenge. Surgical treatment depends on the location of the mass. Tumors restricted to the limbs or tail may be cured by amputation of affected appendage. Tumors on the trunk of urodeles are technically challenging because skin defects in these animals are not readily amenable to closure and healing occurs by contraction and epithelialization.

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Hyperplastic skin conditions

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As in mammals, hyperplasia is a nonspecific response of the amphibian integument to injury and typically subsides after resolution of the un- derlying cause. Cutaneous hyperplasia may present as distinct skin mass(es), and is caused by a variety of diseases, including parasitic, fungal, and viral infections or imbalances in water quality [34,35]. In some cases, hyperplastic lesions are associated with intraepithelial keratin-filled cysts, and have been documented in association with various skin tumors [24]. The previously described population of A mavortium inhabiting a polluted sewage pond in Texas had papillomas and melanophoromas as well as epithelial hyperpla- sia, which was proposed as a preneoplastic change [30]. Both the hyperplasia and neoplasia regressed when the animals were removed from the environment, suggesting an environmental cause.

Tumors of the hemolymphatic system

The anatomy of hematopoietic tissues in amphibians differs from that in other groups of animals more commonly encountered by veterinarians. Even within amphibians, there is significant anatomic variation among orders and genera, as well as different life stages. To those unfamiliar with amphibian pathology, the character, abundance, and distribution of hemopoietic tissue and proliferation in response to a variety of stimuli is a potential source of confusion and misinterpretation. The best example of this difficulty is the large body of literature pertaining to the misdiagnosis of mycobacteriosis as lymphoma, often referred to as ‘‘transmissible lympho- sarcomata’’ [36].

Lymphoma

The literature on lymphoma in amphibians often is confusing and contradictory. Much of this confusion results from the misdiagnosis of mycobacteriosis as lymphoma; therefore, older papers on lymphoprolifer- ative disease in amphibians should be scrutinized carefully. For reference, proven cases of lymphoma are characterized best in X laevis and A mexicanum. A series of T-cell lymphomas was reported in X laevis [37,38]. These cases primarily involved the thymus, presenting initially as a unilateral swelling of the caudal cephalic region (Fig. 4A). Histologically, monomorphic neo- plastic lymphocytes form densely cellular sheets and may have a high mitotic rate. Lymphoma may become generalized to involve multiple organs and progress to leukemia (Fig. 4B). Thus, a diagnosis may be obtained by complete blood cell count and examination of a blood smear. Lymphoma in axolotls is the subject of transplantation and histocom- patibility research [39]. For spontaneous lymphoma, such features as

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682 B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 Fig. 4. (
682 B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 Fig. 4. (

Fig. 4. (A) African clawed frog (Xenopus laevis) with thymic lymphoma resulting in a large mass in the lateral cervical region. The tumor was soft and white on section and obliterated the associated skeletal muscle and lymph sac. (Courtesy of Greg Trimmel, UCB) (B) Photomicrograph of a transverse process of a cervical vertebrae from the X laevis in (A). The tumor had progressed to leukemia, as evidenced by effacement of the bone marrow by a monomorphic population of neoplastic lumphocytes. Neoplastic lymphocytes fill the vasculature of the medullary cavity (asterisks). Hematoxylin and eosin; 100 (inset 1000 ).

location and progression of disease are not well documented. Lymphoma in transplant recipients, however, is noted to progress rapidly to leukemia.

Other round cell tumors

The spontaneous occurrence of other types of round cell tumors is limited to two cases of granulocytic leukemia in a toad (Bufo sp.) [14]. No additional information is available on these animals.

Tumors of the hepatobiliary system

Neoplasia of the hepatobiliary system is rare in amphibians. As in other species, accurate diagnosis of liver tumors and the distinction between

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benign and malignant tumors and tumors verses hyperplastic or regenera- tive nodules may present a diagnostic challenge in some cases. Studies are needed to characterize the liver’s natural response to insults and regener- ative capability. Some reports suggest similarities between hepatic lesions and regenerative capability of mammals and amphibians [14]. For example, hepatic cirrhosis was documented in wild A mavortium [30]. In other species and perhaps in amphibians, features that help distinguish nodular re- generation from benign hepatocellular adenoma or hepatoma are the multifocal distribution of the former and absence of normal portal structures in the latter. The absence of portal structures was described in an adenoma in the barking tree frog (Hyla gratiosa) [31]. All reported cases of hepatocellular carcinoma in amphibians were induced experimentally or associated with environmental carcinogens [14]. Features of malignancy, including local invasion, poorly differentiated neoplastic hepatocytes, and metastasis, may be present; however, in well-differentiated carcinomas, the distinction between benign and malignant can be difficult. Spontaneous tumors of the biliary system in amphibians are also rare. One case was reported in a group of Asian pond frogs (Rana spp.) with a high prevalence of pancreatic carcinoma [40]. This hepatic tumor was diagnosed as a biliary adenoma, but was not well described. The most common differential diagnosis for hepatic masses is mycobac- terial granulomas. Fine-needle aspirates or impression smears examined with acid-fast stains are instrumental in making a diagnosis. Additional differentials include other causes of granulomas, biliary cysts, and meso- thelial hyperplasia, the latter of which may occur in response to chronic effusion or coelomitis.

Tumors of the gastrointestinal tract

Neoplasia of the gastrointestinal tract is poorly documented and limited to a few reported cases, most of which were malignant and diagnosed at necropsy. Elkan [41] described a gastric adenocarcinoma with metastasis in X laevis. A small number of intestinal adenocarcinomas are described in various anuran species, although it is often unclear whether the tumors arose from the enteric mucosa, pancreas, or another organ [14]. Green and Harshbarger [14] described an intestinal adenocarcinoma in a marine toad (Bufo marinus) from the RTLA (Herron, RTLA #1921). It was character- ized by a prominent scirrhous or desmoplastic response, which is a common feature of gastroenteric adenocarcinomas in mammals. A group of R pipiens that were infected experimentally with Mycobacterium marinum developed intestinal adenomas and adenocarcinomas that resulted in obstruction, ascites, and in some cases death [42]. The definitive tissue of origin and true neoplastic nature are uncertain because these tumors were poorly described. A few cloacal tumors, including an adenocarcinoma in a Mexican axolotl

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and a squamous cell carcinoma of the colorectal junction in an aged Oriental fire-belly toad (Bombina sp.) are documented in the RTLA [14]. A major differential diagnosis for masses in the alimentary tract includes granulomas, especially of mycobacterial and parasitic origin, which can be serosal, intramural, or submucosal. Furthermore, it is recommended that all suspected stromal tumors of the alimentary tract are examined with an acid- fast stain.

Tumors of the urogenital system

Lucke´ renal adenocarcinoma

Lucke´ renal adenocarcinoma of R pipiens is the most well-known amphibian neoplasm. This tumor is caused by a herpesvirus known as Ranid herpesvirus-1 or Lucke´ ’s herpesvirus, which is considered endemic in wild northern leopard frog populations [29]. The Lucke´ tumor has been the focus of much research since it was first described in 1934, and early accounts date back as far as 1905 [43]. Confirmed cases of spontaneous nonviral associated renal neoplasia has not been reported in R pipiens, and only a few cases have been reported in other species. The pathogenesis of viral infection and tumorigenesis has several interesting features. Viral transmission is hypothesized to occur from infected urine in breeding ponds when frogs emerge from winter hibernation [43]. Two forms of the tumor are recognized. In the winter or algid phase, tumors are relatively static in size, and viral replication is active. In the summer or calid phase, tumor growth and metastasis occur, and detectable viral replication is minimal or absent. Tumor growth and metastasis are profoundly greater at higher temperatures. Experimental studies demon- strated that large tumors developed at 28(C, whereas colder temperatures (7(C) inhibited tumor growth, but supported viral replication [45]. The prevalence of tumors in wild populations fluctuates by year, season, region, and method of detection. In some populations, greater than 90% prevalence was reported when evaluated for microscopic tumors by histologic examination [44]. Leopard frogs are a common model used for research and education. Recently, their taxonomy was reclassified, which facilitated understanding of the regional distribution of this neoplasm. Any true R pipiens should be considered potentially infected with this oncogenic virus. Thus, a significant number of wild-caught R pipiens may develop tumors under laboratory conditions where they are maintained at high ambient temperatures. This can be disruptive to research as large or metastatic tumors will result in death or necessitate euthanasia. Frogs develop carcinomas only if exposed to the virus as eggs or young embryos [46–49]. Furthermore, infections in other anuran species have occurred only under experimental conditions.

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There are currently no major efforts underway to establish virus-free R pipiens colonies. Clinically, R pipiens with Lucke´ ’s tumor may present with coelomic distension, palpable intracoelomic masses, atrophy of thigh musculature, pelvic limb paralysis, lethargy, and sudden death. Often, clinical signs are not apparent until tumors are quite large or impinge on nerves of the pelvic limbs. Smaller tumors may be detected by coelomic palpation. On exploratory celiotomy or postmortem examination, tumors may be single or multiple and typically are white, slightly firm, and smooth or multinodular (Fig. 5A). Larger tumors often will have central necrosis and regional visceral adhesions. Histologically, Lucke´ tumors typically are well demarcated, and comprised of neoplastic tubules that may be ectatic or lined by papillary projections. Intranuclear inclusion bodies are numerous in the algid phase and rare in the calid phase [43,49]. Also, calid phase tumors can have a high mitotic rate, consistent with rapid growth [14]. Tumors may arise anywhere within the kidneys and affect both kidneys similarly. A slight predisposition for the right kidney is reported [50]. Metastasis can occur to any organ, especially the liver [14]. The reproductive tract may be intimately associated with or compressed by the tumor, requiring careful dissection. Diagnosis is based on the species and presence of renal tumor(s). Differential diagnoses include other causes of coelomic distension, such as effusion, coelomitis, parasitism, and egg production. Renal granulomas, such as from mycobacteriosis, should be considered with smaller masses (Fig. 5B). A fine-needle aspirate or touch preparations may facilitate ante- mortem diagnosis. Neoplastic epithelial cells exfoliate well and often are easily identifiable.

Other renal tumors

Other types of renal tumors are rare and include single case reports of nephroblastoma and renal carcinoma in various species of anurans and urodeles [14,51,52]. One exception is a high prevalence of polycystic kidneys in a population of hybrid Japanese toads (Bufo japonicus) and Chinese toads (Bufo raddei). The disease appears to follow a progression from hyperplasia of the tubular epithelium to tubular or papillary renal adenocarcinomas [53]. These tumors are believed to have a genetic basis, and in some cases, metastasized to the lungs, liver, and kidneys.

Testicular tumors

Amphibian testes are internal and undergo seasonal variation in spermatogenetic activity. Reports of neoplasia are limited to Sertoli cell tumors in both anurans and urodeles. These tumors are considered an incidental finding at necropsy. A series of testicular tumors reported in 16 A

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Fig. 5. (A) A Northern leopard frog (Rana pipiens) with a large Lucke´ adenocarcinoma of the right kidney that presented with marked coelomic distention. This is a large calid phase tumor and metastases to the liver and lungs was noted on histologic examination. Lucke´ tumors were subsequently diagnosed in five out of 10 animals in this group. (B) Coelomic viscera from a R pipiens (left) and Tropical clawed frog (Xenopus (Silurana) tropicalis) (right) demonstrating the similar gross appearance of neoplasia and granulomatous inflammation. The R pipien’s kidneys are expanded by multiple Lucke’s adenocarcinomas. One cranial pole of the X tropicalis kidney is effaced by granulomatous inflammation due to mycobacteriosis.

mexicanum by Humphrey [54] were classified as Sertoli cell tumors in the RTLA [14,54]. All of these cases occurred in a single colony, and 14 of the animals were closely related, which suggests a hereditary predisposition in this group. Many of these tumors were pedunculated and white with deep fissures and a ‘‘pebbled’’ appearance. Neoplastic cells formed elongated, branched tubules. Atrophy of the preexisting testicular tissue was noted in some cases. All of the tumors were benign and nonfatal. Sertoli cell tumors also are described in a Hellbender (Cryptobranchus alleganiensis) and a R pipiens R palustris hybrid [14,55]. Seminomas and interstitial (Leydig) cell tumors are not reported.

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Ovarian tumors

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Ovarian tumors are poorly described in amphibians. Many are identified simply as epithelial neoplasia or carcinomas. Use of nonspecific classifica- tion schemes makes it difficult to draw firm conclusions regarding specific tissue of origin, biologic behavior, and similarities with primary ovarian tumors in other species. In other species, the term ‘‘epithelial’’ usually implies an origin from the surface epithelium of the ovary. Canine ovarian cystadenomas and cystadenocarcinomas are relatively common examples. One cystadenocarcinoma was reported in a R pipiens [56]. Green and Harshbarger [14] report a single case of a granulosa cell tumor in an ornate horn frog, and Streett [57] reports an ovarian teratoma in R pipiens. Dysgerminomas are not documented in amphibians.

Endocrine organs

Neoplasia of endocrine organs is reported rarely. With the exception of the Asian frog population described below, endocrine tumors are limited to a presumed adenoma of the interrenal gland in R pipiens and suspect pancreatic carcinoma in X laevis [14].

Pancreatic carcinoma of Asian pond frogs

Masahito et al [40] described over 101 tumors mostly of pancreatic origin in a captive population that included multiple species of pond frogs originating from Japan (Rana nigromaculata and R porosa brevipoda), China (R nigromaculata and R plancyi plancyi), Korea (R nigromaculata), and Taiwan (R plancyi fukienensis). Tumors ranged from 0.5 to 3.7 cm in diameter, expanding the region of the pancreas. Most tumors originated from the endocrine (islet) cells with typical histomorphology of tightly packed groups of nests, rudimentary tubules, and acini. Areas of exocrine differentiation were noted in some tumors, which also is a feature of some islet tumors in mammalian species [58]. Immunohistochemical character- ization of a subset of cases revealed expression of insulin or somatostatin in many tumors. Metastases were observed in the liver, kidney, heart, lung, stomach, and ovary. Tumors were observed first in hybrids bred from the imported Chinese frogs, and eventually in other hybrid and nonhybrid frogs. Subsequent examination by transmission electron microscopy revealed type C retrovirus particles in the tumor cells. The epidemiology and presence of viral particles in these tumors suggest a viral etiology. Two other types of tumors were identified in this group including a rhabdomyosarcoma and a hepatic adenoma. It is not known whether the etiology of these tumors was associated with the pancreatic carcinomas.

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Pseudoneoplastic conditions

In general, the majority of masses of amphibians are nonneoplastic and caused by bacteria, fungi, parasites, or other infectious or inflammatory conditions. Here we review two commonly encountered pseudoneoplastic conditions, mycobacteriosis, and oral inflammatory lesions.

Mycobacteriosis

Mycobacteriosis is a common disease of captive anurans, and can cause a variety of clinical signs. Multiple mycobacterial species are demonstrated as causative agents. Currently, it is considered an incurable disease in amphibians and its eradication from a collection is problematic. Granulomatous inflammation caused by mycobacteria has been mis- identified on gross and histologic evaluation as various types of neoplasia, including lymphoma and mesenchymal tumors, and often requires histopa- thology for definitive diagnosis (Fig. 6). Misdiagnosis of mycobacterial lesions is the source of great confusion in the literature. Reports and experimental studies of lymphoma in X laevis and T pyrrhogaster ultimately were shown to be mycobacteriosis [59,60]. During the investigation of this disease, various hypotheses were proposed, such as secondary infection of tumors by mycobacteria and mycobacteria-induced neoplastic transforma- tion [59,61]. Eventually, most investigators disregarded these hypotheses [60,62]. Certain features of mycobacterial lesions contribute to misinterpre- tation, especially by individuals not familiar with amphibian pathophysiol- ogy and histology. Namely, macrophages and reactive fibroblasts of granulomas were misidentified as sarcomas or lymphomas. Also, myeloid hyperplasia of the hepatosplenic hematopoietic tissue is a cellular response in infectious processes, such as mycobacterial infections, and can be misinterpreted as lymphoma. Histologically, there are various degrees of proliferation of hematopoietic tissue in the periportal and subcapsular regions of the liver and spleen. The reactive cell population is pleocellular, with a progression of leukocyte development that may include a large number of blastic precursor cells (Fig. 7). This response is described in the literature and is observed commonly in Xenopus species and A mexicanum at UCB that are infected with mycobacteria and, less frequently, other bacteria species such as Chryseobacterium (Flavobacterium). Myeloid hyperplasia may occur in the absence of hepatic granulomas or other visceral involvement, such as in some cases of cutaneous mycobacteriosis. Mis- diagnosis can be avoided by careful histologic examination and prudent use of acid-fast stains.

Inflammatory lesions and ‘‘myxoid tumors’’

Veterinarians have encountered proliferative, mass-like lesions protrud- ing from the oral cavity, characterized by a prominent myxoid or mucinous

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B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 689 Fig. 6. Ovaries,

Fig. 6. Ovaries, oviducts, and kidneys (center) from an African clawed frog (Xenopus laevis) demonstrating the ‘‘pseudoneoplastic’’ appearance of granulomatous inflammation. The abnormal smooth tissue (arrows) expands and obliterates the normal ovary (asterisks), as well as the kidneys, and corresponds to areas of intense granulomatous inflammation. This is another example of mycobacteriosis and diagnosis required histological examination.

matrix. Anecdotal evidence indicates that these lesions occur with some frequency, especially in the maxilla or other regions of the oral cavity. A few such cases have been reported in the literature and accessioned into the RTLA [14]. Diagnoses such as myxoma, myxosarcoma, and chondromyx- oma are applied; however, neoplastic origin is not proven. Green and Harshbarger [14] comment on the uncertain etiology and consider differen- tial causes, such as chronic, exuberant inflammation, and callus formation. One case encountered at UCB responded well to surgical excision, and histologic examination revealed features consistent with a chronic, reactive lesion, including inflammation, fibroplasia, and periosteal response (Figs. 8A and 8B). More work is needed to characterize these conditions.

Treatment

Treatment options for neoplasia are limited to removal of predisposing condition(s), supportive care, and surgical excision. To the best of the authors’ knowledge, no studies have examined the efficacy of chemotherapy in amphibians. As with many physiologic processes in ectotherms, the growth of some types of neoplasia is temperature dependent. Thus, ambient temperature can influence growth rate, treatment, and outcome, and may be manipulated to influence biologic behavior and tumor incidence. When treatment options fail or are not suitable, humane euthanasia may be elected. Many tumors of the integument in amphibians are limited to the dermis, and those affecting anurans are often amenable to surgical excision.

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690 B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 Fig. 7. Photomicrograph

Fig. 7. Photomicrograph of the liver of a Tropical clawed frog (Xenopus tropicalis) with mycobacteriosis. Hepatic cords (asterisks) are separated by proliferating myeloid cells (M) (myeloid hyperplasia), including mitotically active progenitor cells (arrow). This reactive change is commonly encountered in amphibians with mycobacteriosis and may be misinterpreted as lymphoproliferative disease. Hematoxylin and eosin; 400 .

Furthermore, amphibians are excellent surgical candidates due to their remarkable capacity for regeneration, which varies among species. Uro- deles, newts in particular, have remarkable regenerative abilities of the appendages and tail [63]. In the authors’ experience, there are marked differences in the degree of technical difficulty between surgical procedures performed on anurans and urodeles. Anurans have highly vascularized, pliable, loosely attached integument that is quite amenable to surgical manipulation. Conversely, the skin of urodeles is adhered firmly to the deep muscular layer, making wound closure more difficult. A complete review of amphibian anatomy is beyond the scope of this article; however, before performing any major surgery, a review of vital structures, such as lymph hearts and the midline coelomic vein, is recommended.

Basic surgical procedure

In amphibians, the most common anesthetic protocol for induction is immersion in a known concentration of tricaine (MS222). Benzocaine, clove oil, and volatile anesthetics may be suitable alternatives, and are imprecisely administered ‘‘to effect.’’ Gauze soaked in sterile water should be placed underneath the patient to maintain cutaneous moisture. Information on postoperative pain management is limited. A few studies suggest that opiods, a-adrenergic, and nonsteroidal antiinflammatory agents may pro- vide pain relief [64,65]. Surgical procedures on amphibians are considered ‘‘clean contaminated.’’ Sterile preparation of the surgical site is accomplished by placing gauze soaked in dilute chlorhexidine solution (0.75%) or benzalkonium solution (2 mg/L) on the surgical site for 10 minutes. Other disinfectants commonly

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B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 691 Fig. 8. (
B.A. Stacy, J.M. Parker / Vet Clin Exot Anim 7 (2004) 673–695 691 Fig. 8. (

Fig. 8. (A) Two-year-old Pacific tree frog (Hyla (Pseudacris) regilla) with a large, multinodular, fleshy mass originating from the ventral aspect of the rostral maxilla. The mass was successfully excised, and the defect was allowed to heal by contraction and epithelialization. (B) Photomicrograph of the maxillary mass from the Hyla regilla in (A). The mass is comprised of reactive fibroblasts and abundant mucinous stroma (asterisks). Inflammatory cells (arrows) diffusely infiltrate the tissue. These histologic features are consistent with a reactive inflammatory process. Hematoxylin and eosin; 20 (inset 400 ).

used for mammals, such as iodine compounds or alcohol-based solutions, should be avoided due to possible toxic affects. In anurans but not urodeles, the skin is tented easily to make a stab incision. Once a window is created, the deep surface of the skin can be inspected visually to avoid transection of major vessels. An excisional tumor margin of at least 0.2 to 1.0 cm is recommended, but will be limited by species, size, and location. Skin closure is performed using nonabsorbable, monofilament suture in an interrupted everting pattern. In several anuran species, extensive wound management is not required because of inherent antimicrobial peptides that are excreted from the skin [66–68]. Hygienic and optimal environmental conditions, however, are required in the postoperative interval to avoid opportunistic bacterial and fungal infections.

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Summary

This article is a review of the more common types of neoplasia in amphibians. When possible, clinically relevant features, such as biologic behavior, anatomy, associated etiologies, and major differential diagnoses, are included. It is important to recognize neoplasia caused by oncogenic viruses or predisposing conditions, and to distinguish neoplasia from infectious pseudoneoplastic conditions that can impact population and group health. Treatment options are limited to removal of identified predisposing condition(s), supportive care, controlling ambient temper- atures, surgical excision and, when necessary, humane euthanasia.

Acknowledgments

We would like to thank Drs. Hilde E. V. De Cock, Helen E. Diggs, Nina Hahn, John Harshbarger, Tyrone Hayes, Linda J. Lowenstine, Yuka Nakamura, Allen Pessier, Francis Rose, Gregory Timmel, and Tanja S. Zabka for their editorial support, consultation, and contribution of cases. We also thank Jerry Fields (UCD) for his assistance with photography; Stephen Friet, Sam Cunningham, Loren Johnson, Jeanine Hamel, Rachel Casamina, Aaron Vonk, and Magdalena Mleczko of the UC Berkeley, Office of Laboratory Animal Care; and Barry Puget, Asi Djan, and Diane Naydan of the UC Davis histology laboratory.

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