Peribiliary Cysts Associated with Severe Liver Disease: A Previously Unrecognized Tumor in a Lion ( Panthera Leo )

Abstract

A 13-year-old male lion (Panthera leo) from Dae Jeon Zoo, Republic of Korea, presented with clinical signs of lethargy and anorexia. Despite treatment with antibiotics and fluid therapy, the lion died 6 days after initial presentation. Postmortem examination revealed multiple masses measuring 5–10 cm in diameter and cysts throughout the liver. A diagnosis of spontaneous peribiliary cysts was made on the basis of microscopic lesions as well as special staining and immunohistochemical characteristics. Histologically, the neoplasm was surrounded and composed of compact collagenous tissue. The inner cystic single layer resembled biliary mucosa and was composed of cuboidal or flattened epithelial lining that was strongly immunopositive for cytokeratin AE1/AE3. This layer was surrounded by fibrous tissue that stained blue by Masson's trichrome staining. Given the presence of multiple organized cysts in the liver, the lesion was consistent with peribiliary cysts. To the authors' knowledge, this is the first report of peribiliary cysts in an animal.

Peribiliary cysts are characterized by multiple cysts along the portal radicle. They result from the cystic dilatation of intrahepatic extramural peribiliary glands around the intrahepatic large bile ducts. 9 To date, peribiliary cysts have been reported only in cases of advanced liver disease in humans. 2,5,9 , The tumor was first reported as an incidental finding in 7 postmortem livers and initially called multiple hilar cysts of the liver. 5 Peribiliary cysts are similar to hepatobiliary cystadenoma, an uncommon benign tumor that has been reported in some animals, including cats, dogs, horses, sheep, and swine. 1,2,6 It is characterized by multiple cystic masses and consists of thin-walled cysts that contain clear, watery mucin-like fluids. The tumor is often raised above the capsular surface and involves more than 1 liver lobe. 5 The clinicopathologic features of hepatobiliary cysta-denoma are well described in cats and horses. 1,8 The present report describes for the first time a case of hepatic biliary tumor in a lion, exhibiting macroscopic and microscopic features similar to peribiliary cysts in humans.

Figure 1.

Gross liver microscopic lesion and special staining in liver of peribiliary cysts from lion. There are multiple cysts in the entire liver lobes.

Figure 2.

Gross liver microscopic lesion and special staining in liver of peribiliary cysts from lion. The cysts are lined by a single layer of cuboidal or flattened epithelial cells and located in the portal area. Hematoxylin and eosin. Bar = 100 μm. Inset: Detail of a single layer of flattened epithelial cells. Bar = 55 μm.

Figure 3.

Gross liver microscopic lesion and special staining in liver of peribiliary cysts from lion. The cysts are varied size and surrounded by mature collagen. Masson's trichrome stain. Bar = 210 μm. Inset: Detail of fibrous tissue around cysts. Bar = 85 μm.

Figure 4.

Gross liver microscopic lesion and special staining in liver of peribiliary cysts from lion. Lined cuboidal or flattened epitheliums were immunoreactive to cytokeratin AE1/AE3. Envision system-AP. Hematoxylin counterstain. Bar = 55 μm.

A 13-year-old lion, initially imported from Safari Zoo, Germany, in 2002, died at the Dae Jeon Zoo in January 2004. Seven days before death, the lion was referred to the attending veterinarian at Dae Jeon Zoo with signs of lethargy, anorexia, and weakness. The animal died despite supportive fluid therapy. Postmortem examination revealed 5–10-cm multiple cystic masses buldging on the surface of both the left and right lobes of the liver (Fig. 1). The cysts contained clear fluid and were located along the bile ducts in the large intrahepatic portal areas. There was no communication between the cysts and the lumen of the bile ducts. Other organs, including regional lymph nodes, were normal in appearance.

A liver specimen collected at necropsy was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin for evaluation by light microscopy. Immunohistochemical analysis of the liver sections was performed using the 2-step Envision system-AP® (alkaline phosphatase) a according to manufacturer's instructions. The primary antibody was a mouse anti-human cytokeratin AE1/AE3 monoclonal antibody. a In brief, tissue sections were first treated with 5% H₂O₂ in phosphate buffered saline (PBS) for 20 min at room temperature (RT) to quench endogenous peroxidase, followed by 3 PBS washes. The sections were then placed in Tris-EDTA (pH 9.0), and heat-induced epitope retrieval was performed by heating in a microwave oven b (high power) for 10 min and cooling to RT, followed by 3 washes in PBS. The primary antibody was applied to the slides at a dilution of 1:50 overnight at 4°C. After washing 3 times in PBS, sections were incubated with a ready-to-use polymer-alkaline phosphatase conjugate a for 40 min. Sections were then washed before incubation with substrate for AP systems for 5–10 min, and the color reaction was stopped by washing the slides in deionized water. All slides were counterstained with hematoxylin and covered with glasses cover slips. A section of the same liver treated with PBS in place of the primary antibody was used as the negative control. To confirm mucin-like fluid and fibrous tissue, liver sections were stained with alcian blue (pH 2.5) and Masson's trichrome stain according to standard histotechnology methods. 7

Macroscopically, the cysts were oval-shaped, variable in size, and concentrated around the hepatic hilus and large portal area at the section of liver (data not shown). Microscopically, the cysts were lined by a single layer of cuboidal or flattened epithelium surrounded by an abundant amount of fibrous tissue and mature collagen. The cysts were located in the portal area, which is composed of artery, vein, and bile ducts (Fig. 2). Cellular atypia, mitoses, necrosis, inflammatory infiltrates, or lymphatic invasion were not observed. Liver parenchyma outside the tumors was unremarkable. The cystic epithelial lining was negative by Alcian blue stain, indicating that the tumor is different from biliary cystadenoma in which mucinous fluids stain positive with Alcian blue. Based on Masson's trichrome stain, the cysts were surrounded by fibrous tissue and mature collagen in which were scattered peribiliary glands, some showing microcyst formation (Fig. 3).

Immunohistochemical staining showed that all the epithelial cells were strongly immunoreactive to cytokeratin AE1/AE3, thus confirming their biliary nature because this result is typical of developing and mature normal biliary ducts (Fig. 4). By contrast, no staining was observed in the absence of primary antibody (data not shown).

The macroscopic, histopathology, and immunohistochemical findings in this case led to a diagnosis of peribiliary cysts. Although this neoplasm is grossly and histologically similar to biliary cystadenoma of domestic animals, the authors prefer to describe it as peribiliary cysts because biliary cysadenoma cysts are located within the parenchyma, whereas peribiliary cysts are located in the connective tissue of hepatic hilus and also within the large intrahepatic portal tracts. 3 Also, the typical biliary cystadenoma of domestic animals has a smaller amount of stromal content, 8 in contrast to this case that exhibited abundant stromal component around the cysts. Another piece of unequivocal evidence that supports the classification of this tumor as peribiliary cysts is the failure to stain for mucin in the cytoplasm of the epithelial lining. This is in contrast to the cells of the multiple epithelial lining of biliary cystadenomas, which typically produce mucinous fluids. 4 In short, a comparative histological analysis between biliary cystadenoma and peribiliary cysts reveals significant differences: biliary cystadenoma is characterized by location of cysts in the parenchyma, mucin secreting epithelium, scant fibrous tissue, and multiple layers of epithelial cells, whereas peribiliary cysts are mainly located in the portal area, possess non-mucin-secreting epithelium, abundant fibrous tissue, and a single layer of epithelial cells. Another neoplasm to be distinguished from peribiliary cysts is biliary adenofibroma, a morphological variant of biliary cystadenoma that is basically a solid lesion with microcysts not exceeding 2 mm. 8 Peribiliary cysts are distinguishable from biliary adenofibroma by their gross appearance as a multilocular cystic mass with single cysts of variable size ranging from less than 10 mm to more than 38 mm. 4

Peribiliary cysts in humans were first reported by Nakanuma et al. in 1984. 5 These authors speculated that peribiliary cysts arose from cystic dilatation of the intrahepatic extramural peribiliary glands in the periductal connective tissue. A subsequent systematic study of postmortem livers indicated that the peribiliary cysts are quite common in humans; indeed, 202 livers (20.2%) of 1,000 consecutive postmortem livers were found to harbor the condition. 10 The degree of cystic dilatation was quite variable, and the majority were recognizable microscopically. In clinical practice, however, this disease is still poorly recognized. 4 The etiology of peribiliary cysts is obscure. It is known that genetic background, chemical carcinogens such as nitrosamine, and hepatobiliary diseases (including cirrhosis, portal hypertension, and extrahepatic portal obstruction) may play a role in developing peribiliary cysts. 9,11 One report suggested that the disturbance of the portal venous flow could be the precipitating factor in their pathogenesis. 5 Another report postulated that periductal inflammation, fibrosis, and portal venous thrombosis could obliterate the necks of the peribiliary glands and result in the formation of retention cysts. 11

This study provides the first evidence that peribiliary cysts, known to occur in humans, can also occur in lions. Cytokeratin AE1/AE3 immunoreactivity and staining with Alcian blue and Masson trichrome were used to identify the neoplastic hepatobiliary tumor. The immunohistochemical and histologic results confirmed for the first time the occurrence of peribiliary cysts in a lion. To the authors' knowledge, this neoplasm has never been described in domestic animals. It is possible that it may occur in domestic animals but might have been misdiagnosed as biliary cystadenoma. Therefore, the authors propose that peribiliary cysts should be included in the differential diagnosis of cystic hepatobiliary neoplasms in domestic animals.

Acknowledgements. The study was supported by the Ministry of Agriculture and Forestry (MAF) of Korea. K-T Kim contributed to this paper as the co-first author. The technical assistance of Ms. R-W Jang is highly appreciated.