Peliod hepatocellular carcinoma in a domesticated ferret (Mustela putorius furo)
Y. Jones, A. Wise, R. Maes, M. Kiupel
Abstract. Peliod hepatocellular carcinoma was diagnosed in a domesticated ferret (Mustela putorius furo). The diagnosis was made using immunohistochemical analysis, histologic examination, and the accepted classification schemes based on histomorphologic features. Bilateral, adrenocortical hyperplasia also was evident. Speculation about a possible association between the variant of hepatocellular neoplasia diagnosed in this animal and its adrenal pathologic changes was done.
Key words: Ferrets; hepatocellular carcinoma; liver; peliosis.
Neoplastic diseases in domesticated ferrets (Mustela putorius furo) are considered rare.5 Because the incidence of neoplasia increases with age, the scarcity of tumors reported in ferrets has been attributed to their short lifespan.17 With an increasing number of ferrets being kept in zoo facilities and as household pets, and being used in research, an increased number of reports are describing neoplastic conditions in this species.3-5,7,13,17 Primary neoplasms have been reported in almost every organ system of ferrets, except the respiratory tract. Pancreatic islet cell tumors, in particular, insulinomas, adrenocortical tumors (including adenomas and carcinomas), and lym- phomas, are the most commonly reported neoplasms in domesticated ferrets.17 We report a peliod hepatocellular carcinoma in a domesticated ferret. To our knowledge, this is the first report of such neoplasm in ferrets. The gross, microscopic, and immunohistochemical features of the neoplasm are described and are compared with those of similar neoplasms in human patients to speculate on the biological behavior of peliod hepatocellular carcinomas in domesticated ferrets. Additional tests that were performed to elucidate a possible infectious etiopathogenesis and speculation of the possible role of steroids in the carcinogenesis of this tumor, as suggested in human peliod hepatocellular carcinomas, are discussed.
An 8.5-year-old female ferret was presented to a local veterinarian with a history of listlessness, diarrhea, and anorexia. The ferret continued to deteriorate to the point of absence of defecation and loss of most of its body mass. The animal was euthanatized and submitted for necropsy to the Diagnostic Center for Population and Animal
Health at Michigan State University. At necropsy, the left medial liver lobe was markedly enlarged by a well circumscribed, tan mass that measured 12 cm in diameter. The mass had rubbery-to-soft consistency and had com- pletely effaced normal hepatic parenchyma (Fig. 1). On cut surface, the mass contained multifocal areas of blood-filled cystic spaces that ranged in size from 0.5 to 5.0 cm in diameter (Fig. 2). Gross lesions were not evident in any other tissues.
Samples of all major organs were selected for micro- scopic examination. Sections of selected tissues were fixed in neutral-buffered 10% formalin and embedded in paraffin wax, then 5-um-thick sections were cut in routine manner. After dewaxing, sections of tissue were stained with hematoxylin and eosin. Sections of liver, heart, brain, lung, kidney, pancreas, adrenal gland, spleen, multiple lymph nodes, and the digestive tract were examined microscopi- cally. The only relevant alterations were observed in the liver. On histologic examination, the grossly described liver mass consisted of sheets and trabeculae of neoplastic hepatocytes, commonly forming numerous cystic spaces, which contained variable amounts of red blood cells (Fig. 3). These blood-filled spaces were lined by neoplastic hepatocytes (Fig. 4) that were variably sized, with abundant, eosinophilic cytoplasm and distinct cell borders. They had round, vesiculate nuclei with prominent nucleoli and coarsely clumped chromatin. There was mild nuclear pleomorphism and minimal cellular atypia. Less than 1 mitotic figure per high-power field was observed. Within the mass was complete loss of lobular cord architecture with few recognizable portal vessels or centrilobular veins. The neoplasm compressed and multifocally invaded the surrounding hepatic parenchyma causing cellular degener- ation and lobular atrophy. The left and right adrenal glands had multifocal areas of nodular hyperplasia in the cortex. The hyperplastic cortical tissue was principally supracapsular and encompassed approximately 30-65% of each gland.
From the Department of Pathobiology and Diagnostic In- vestigation (Jones, Kiupel); the Diagnostic Center for Population and Animal Health (Jones, Wise, Maes, Kiupel); and the Department of Microbiology (Maes), Michigan State University, East Lansing, MI.
1Corresponding Author: Matti Kiupel.
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Figure 3. Photomicrograph of a section from the peliod hepatocellular carcinoma in the ferret. Microscopically, the neoplasm consisted of sheets and trabeculae of neoplastic hepatocytes commonly forming numerous cystic spaces that contained variable amounts of red blood cells, fibrin, and necrotic debris. HE. Bar = 200 um. Figure 4. Photomicrograph of a section from the peliod hepatocellular carcinoma in the ferret. The blood-filled spaces within the neoplastic mass are lined by neoplastic hepatocytes. HE. Bar = 40 um.
Figure 5. Photomicrograph of a section from the peliod hepatocellular carcinoma in the ferret. Cells lining blood-filled spaces within the neoplastic mass stained positive for pancytokeratin (arrows), confirming epithelial origin. Immunohistochemical analysis for pancytokeratin MNF116, with hematoxylin counterstain. Bar = 80 um.
For immunohistochemical analysis, sections of neoplas- tic hepatic tissue were dewaxed and rehydrated by use of routine methods.1º Briefly, antigen retrieval was accom- plished by incubation of slides in antigen-retrieval solutionª in a steamerb for 20 minutes. Endogenous peroxidase was blocked for 15 minutes by addition of 3% hydrogen peroxide. Nonspecific immunoglobulin binding was blocked by incubation of slides for 10 minutes with a protein-blocking agenta before application of the primary antibody. The latter was allowed to react for 30 minutes at room temperature. Sections were stained in a Dako autostainer apparatus.ª The slides were incubated with endothelial markers, factor VIIIª and CD31,ª and a pancy- tokeratin marker MNF116ª (dilution of 1:100). A strepta- vidin-immunoperoxidase staining procedureª was used for immunolabeling. The immunoreaction was “visualized” with 3,3’-diaminobenzidine substrate.ª Sections were coun- terstained with Mayer’s hematoxylin. Positive immunohis- tochemical controls included normal canine liver and a cutaneous hemangioma, to which the appropriate antisera were added. For negative controls, the primary antibodies were replaced with homologous nonimmune sera.
Most neoplastic cells stained positively with pancytokeratin (Fig. 5). The cells lining the blood-filled spaces did not stain with either of the endothelial markers (Fig. 6), but were positive for pancytokeratin (Fig. 5), confirming their epithelial origin. On the basis of the microscopic appearance and the immunohistochemical staining pattern, the neoplasm was subsequently classified as a peliod hepatocellular carcinoma, according to published classification schemes.9,12,13
The carcinogenesis of hepatocellular carcinomas has been extensively studied. In particular, an infectious etiopathogenesis in some cases has been well characterized. Hepadnaviruses, which may cause hepatitis B or C in humans, and woodchuck hepatitis virus in woodchucks, are known infectious causes of hepatic neoplasia. In the case presented here, universal primers for DNA that are capable of detecting all known Orthohepadnavirus species were used for polymerase chain reaction (PCR) analysis of affected sections of liver.14 There was no evidence of hepadnaviral DNA in this ferret. On the basis of negative PCR results, it was determined that the neoplasm was not associated with an Orthohepadnavirus.
Human hepatocellular neoplasms present a diagnostic and therapeutic challenge due to their geographic variabil- ity and association with chronic liver disease. Neoplastic transformation of hepatocytes secondary to infection and chronic inflammatory disease has been documented in multiple species. Helicobacter pylori infection is associated with primary gastric lymphoma and adenocarcinoma in humans.6,16 Hepatic disease in the ferret has been linked with physiologic status, diet, toxin, and infective agents,
including H. mustelae.8,17 There was no evidence of gastritis or Helicobacter infection in this ferret. In addition, sections of affected liver were treated with a Warthin-Starry silver stain and infective organisms were not identified.
Classifications of hepatocellular carcinoma on the basis of gross features have been proposed; however, diagnosis and classification methods that are based on histomorpho- logic features and tumor interaction with the surrounding liver are more widely accepted, descriptive, and accurate. Peliod heptocellular carcinomas are rarely diagnosed in humans and other mammalian species. To our knowledge, this is the first report of a peliod hepatocellular carcinoma in a ferret. In contrast, intrahepatic vascular neoplasms have been described in a colony of ferrets.4 These neoplasms were histologically consistent with hemangioma and hemangiosarcoma. The hemangioma was described as cavernous, consisting of large lakes of blood, bounded by a single layer of flattened endothelium. Histologically, hemangiosarcomas often lacked the large lakes of blood, but were composed of neoplastic endothelial cells that formed small clefts, many of which contained erythrocytes. The neoplastic endothelial cells also formed solid sheets that invaded and disrupted the adjacent hepatic architec- ture.4 To differentiate hepatocellular carcinomas forming blood-filled cavernous spaces from vascular neoplasms infiltrating the liver, use of immunohistochemical analysis is commonly necessary to identify the cells lining blood- filled spaces in such liver lesions. Immunohistochemical staining for pancytokeratin and endothelial cell markers (CD31 and factor VIII) confirmed a diagnosis of peliod hepatocellular carcinoma in the ferret of this report.
Peliosis hepatis (or peliosis) also must be differentiated from peliod hepatocellular carcinoma. Peliosis is a rare disorder that is characterized by the presence of blood-filled spaces. Affected individuals often succumb to hemoper- itoneum or liver failure. Microscopically, peliosis hepatis is composed of blood-filled spaces, most of which are lined by endothelial cells that communicate with the surrounding hepatic sinusoids, which often are dilated. The blood is often liquid, but may be clotted; in a few instances, the contents may be partially organized. Two types of peliosis hepatis have been reported on the basis of histomorpho- logic features. In the parenchymal variant, cystic spaces do not have an endothelial lining and are attributable to hemorrhagic parenchymal necrosis.2,9,15 In the phlebotic variant, the prominent vascular cavities consist of dilated sinusoids, and thus, have an endothelial lining.2,9,15 In cases of peliod hepatocellular carcinoma, masses in the liver have the typical features of malignancy associated with hepato- cellular carcinomas; however, blood-filled lakes will de- velop in the tumor, mimicking the parenchymal variant of peliosis hepatis, as described in this ferret.9
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Figure 6. Photomicrograph of a section from the peliod hepatocellular carcinoma in the ferret. Cells lining blood-filled spaces within the neoplastic mass were negative for von Willebrand factor (arrows), excluding an endothelial origin. Notice clumped positive staining within the lumen of blood-filled spaces (asterisk). Immunohistochemical analysis for von Willebrand factor (factor VIII), with hematoxylin counterstaining. Bar = 80 um.
Besides the association of peliosis hepatis with hepato- cellular neoplasms, both carcinoma and adenoma, peliosis hepatis more commonly results from complications asso- ciated with anabolic steroid therapy.1,9 In human patients, peliosis hepatis has been associated with long-term treatment with oral contraceptives, estrogens, and immu- noglobulin therapy.1,9 In addition, androgenic and estro- genic steroids have been implicated in the development and course of several liver diseases in humans including hepatocellular carcinoma.2,11 Estrogens are well known as hyperplastic agents in the liver, and have been implicated in cases of hepatic adenoma and focal nodular hyperplasia in males and females. It has been suggested that estrogen and estrogen receptor are involved in the early stages of hepatocarcinogenesis. It has also been reported that treatment of rodents and humans with natural and synthetic estrogens results in neoplasia.2,11
Clear association among peliosis hepatis, hepatocellular hyperplasia, or neoplasia and estrogen therapy has been established for human patients.11 Interestingly, adrenocor- tical tumors and/or nodular adrenocortical hyperplasia commonly cause increased plasma estradiol concentration in ferrets. Variable degrees of estradiol reactivity in such hyperplastic, benign, and malignant adrenocortical lesions in ferrets have been confirmed by immunohistochemical evaluation.13 It is unclear whether the described hyperplas- tic lesions in the adrenal cortex of the ferret of this report were associated with increased plasma estradiol concentra- tion, as antemortem estradiol values were not available. Considering that the case reported here represents the first peliod hepatocellular carcinoma in a ferret, it seems unlikely that the increased plasma estradiol concentration, which is commonly observed in this species, is associated with proliferative hepatocellular lesions. It also is uncertain whether development of the peliod component in this hepatocellular carcinoma was related to hyperplasia of the adrenal cortex. We contend that, if a ferret presents with a proliferative lesion of the adrenal cortex, it is worthwhile to closely examine the liver and, in particular, to search for possible vascular abnormalities within that organ.
Sources and manufacturers
a. DAKO Cytomation Inc., Carpinteria, CA.
b. Black & Decker, Hampstead, MD.
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