Conference 19 - 2016 Case: 4 20170215

Signalment:

25-year-old male California sea lion, (Zalophus californianus).This sea lion was born in captivity in a zoo, where it stayed its entire life. Two weeks prior to euthanasia the animal lost 40kg of weight, suffered from ascites and a heart murmur was heard.

Gross Description:

On post-mortem examination a yellow-white firm nodule was found near the trigone of the bladder, measuring 2 by 3 centimeters and 1 cm in thickness. Similar nodules were found extensively in the liver parenchyma and multifocally in the lung. The lung also contained large fibrotic interlobular septa. Several lymph nodes were severely enlarged, such as the mesenteric, pulmonary lymph nodes, and one lymph node close to the adrenal gland. The intestines were attached to one another and a large deposition of fibrin together with a large volume (700 ml) of serohemorrhagic fluid (fibrinoid exudative peritonitis).

Histopathologic Description:

Urinary bladder: Originating from the bladder epithelium and infiltrating in the lamina propria and the muscular layer (m. detrusor vesicae), a well-demarcated, densely cellular, multilobular neoplasm is present. In between these lobules lays a dense cellular fibrovascular stroma with large infiltrates of lymphocytes. On some places in the epithelium, multiple groups of disorganized layers of dysplastic cells are visible, which do not breach the basal membrane (carcinoma in situ). The neoplastic cells are organized in closely packed smaller lobules and larger nests. The cells have a polygonal appearance, range from 20-40 µm in diameter, contain a moderate amount of light basophilic granular to foamy cytoplasm and have a round to oval, central nucleus with clumped chromatin along the nuclear envelope and 1-2 basophilic nucleoli. Only a few cells have large cytoplasmic vacuoles which pushes the nucleus to the cell periphery which gives the appearance of so-called 'signet cells'. Some cells are multinucleated and there is marked anisokaryosis, anisocytosis, as well as an augmented ratio of nucleus:cytoplasm. Two to five, sometimes bizarre, mitotic figures per HPF are seen. In many lobules of neoplastic cells, central necrosis is present, along with a moderate number of inflammatory cells. Within widened lymphatic vessels and blood vessels, large clumps of neoplastic cells are present showing central necrosis (tumor emboli). Using immunohistochemistry, these neo-plastic cells stained positive for low molecular weight cytokeratin and uroplakin and negative for high molecular weight cytokeratin. A PAS-stain on the vacuoles within the 'signet cells' was negative.

Various metastases of this neoplasm were found in lymph nodes, liver, lung, adrenal gland, and eye. These metastases stained positive for uroplakin.

Morphologic Diagnosis:

Nonpapillary and infiltrating transitional cell carcinoma with multiple metastases to various lymph nodes, liver, lung, adrenal gland, and eye.

Lab Results:

Odocoileus adenovirus PCR of lung: Positive

Bovine viral diarrhea I&II PCR of lymph node: Negative

Aerobic culture of lung: Pasteurella multocida, heavy growth

Condition:

Bronchointerstitial pneumonia/Influenza virus

Contributor Comment:

Transitional cell carcinoma (TCC) is the most common tumor of the urinary tract in domestic animals.⁹ It is also a common neoplastic lesion in the urogenital tract of the Californian sea lion (Zalophus Californianus). In the Californian sea lion, it is a common cause of mortality.⁵

Most TCCs are found in the trigone of the bladder⁸ and have a papillary, polypoid, or sessile morphology.⁹Various patterns of TCCs have been described, with the papillary and infiltrating TCC as the most common type and the nonpapillary, infiltrating type as the second most common.⁹ Some TCCs may originate in the ureter or urethra and might be more difficult to diagnose.⁵

Histologically, nonpapillary infiltrating TCCs show a thickened bladder wall⁹ due to neoplastic growth of the urinary epithelium. Neoplastic cells are organized in small nests and form a multilobular pattern.⁵ Cyst-like structures may be visible, containing necrotic or keratinized debris may be seen.⁵ Neoplastic cells may show cystic degeneration and contain large cytoplasmic vacuoles, pushing the nucleus to the periphery creating a characteristic signet ring morphology. The large polygonal to round cells have distinct cell borders¹⁰, pale eosinophilic to amphophilic cytoplasm⁵and contain enlarged pleomorphic to polygonal and vesicular nuclei, with prominent nucleoli⁹ and clumped to reticular chromatin.³ Syncytial cells, atypical nuclei, mitotic figures are abundant.⁹ There might be variable desmoplasia and various amount of lymphoid inflammation. Vascular and lymphatic vessel invasion is often seen.⁹

The cause of TCC remains undetermined⁶, but is likely multifactorial.³ Environmental contaminants, such as polycyclic aromatic hydrocarbons like benzopyrine have been suggested to play a role.⁵ Lipscombet al. found intranuclear inclusion bodies in genital carcinoma and isolated a herpes virus (rhadinovirus) and an Epstein-Barr virus using PCR.^6,7 Polychlorinated biphenyls could also contribute to the occurrence this neoplasm.¹⁰

TCCs metastasize in about 50% of the cases in domestic animals; first affecting regional lymph nodes before spreading to other organs.⁹Metastases of TCC in Californian sea lions is also common, as in the current case³. The nonpapillary infiltrating type is the most likely to metastasize.⁹Bilateral hydronephrosis due to obstruction by the neoplasm in either ureter or urethra is a possible associated lesion.⁹

JPC Diagnosis:

Urinary bladder: Carcinoma, metastatic, California sea lion, Zalophus californianus.

Conference Comment:

This case generated spirited discussion among conference participants regarding the tissue of origin of the numerous small islands and nests of neoplastic cells in the muscular layers of this section of urinary bladder. Most participants agreed with the contributor and favored the diagnosis of urothelial carcinoma (also known as transitional cell carcinoma); however, others preferred the diagnosis of metastatic carcinoma not otherwise specified, because the neoplastic cells generally do not associate with the overlying transitional epithelium, rather forming distinctive intra-epithelial neoplasms (IEN) and distinct islands within the muscular layers of the urinary bladder. IEN has been previously associated with genital carcinoma of California sea lions (CSL) described in similar lesions in the lower urinary tract (vagina, cervix, uterus, prepuce, penis, and urethra).^3,6,7 Interestingly, lesions of the urinary bladder had not been described in previous cases of genital carcinomas in CSL.^6,7 Unfortunately, in this case, most of the hematoxylin and eosin (HE) stained tissue sections provided to participants contained little to no overlying epithelium, so it was difficult to accurately assess the origin of the neoplasm. We thank the contributor for proving excellent quality images of strong and highly specific membranous immunoreactivity of the neoplastic cells for uroplakin-III in both the urinary bladder and eye metastasis.¹¹ Prior to the conference, the Joint Pathology Center also ran uroplakin-III and pancytokeratin and confirmed strong immunoreactivity of the neoplastic cells for both stains.

The mammalian urothelium is composed of 610 layers of cells that includes superficial, intermediate, and basal layers.^2,12 The superficial layer, commonly known as the umbrella cell layer, contains a specialized plasma membrane that forms rigid plaques covering the apical surface of the urothelium. Uroplakin is one of the major protein components of the umbrella cells in the superficial layer and is highly conserved through most, if not all, mammalian species. The immunohistochemical stain, uroplakin-III, is marker for urothelial cells of the renal pelvis, ureter, bladder and urethra and has been used to detect transitional cell neoplasms in humans, dogs, cattle, and laboratory rodents. Loss of uroplakin-III expression in bladder cancers has been associated with malignant, invasive and anaplastic urothelial carcinomas in humans and dogs.^2,12 Uroplakin-III is generally considered to be specific for urothelial carcinomas; however, positive immuno-reactivity for uroplakin-III has also been reported in prostatic carcinoma in humans and dogs.^2,12 Positive immunostaining for uroplakin-III provides strong evidence for urothelial origin of the neoplasm in this case; however, further study may be needed to determine the specificity of uroplakin-III in the genital and urinary tract in California sea lions.

Invasive and metastatic genital and urinary tract carcinomas are commonly diagnosed in stranded California sea lions. As mentioned by the contributor, these neoplasms have been associated with a number of predisposing causes, including otariine gamma herpesvirus^6,7 infection and exposure to polychlorinated biphenyl water pollutants.^3,10 Additionally, there is a reported association between development of urogenital carcinoma in sea lions with genetic homogenicity at the Pv11 microsatellite locus for the heparanase 2 gene (HPSE2).¹ Homogenicity at this site is also strongly correlated with decreased overall fitness within the stranded California sea lion population and may be secondary to inbreeding.¹ Conference participants did not note eosinophilic intranuclear inclusion bodies characteristic of herpesvirus infection in this tissue section.

References:

1. Browning HM, Acevedo-Whitehouse K, et al. Evidence for a genetic basis of urogenital carcinoma in the wild California sea lion. Proc Biol Sci. 2014; 281(1796):20140240.

2. Charney VA, Miller MA, et al. Skeletal metastasis of canine urothelial carcinoma: Pathologic and computed tomographic features. Vet Pathol. 2016; pii:0300985816677152. [Epub ahead of print].

3. Colegrove KM, Gulland FMD, Naydan DK, et al. Tumor morphology and immuno-histochemical expression of estrogen receptor, progesterone receptor, p53, and Ki67 in urogenital carcinomas of California sea lions (Zalophus Californianus). Vet Pathol. 2009; 46:642-655.

4. Dierauf, L, Gulland FMD. CRC Handbook of Marine Mammal Medicine: Health, Disease, and Rehabilitation. CRC press; 2001.

5. Gulland, FMD, Trupkiewicz JG, Spraker TR, et al. Metastatic carcinoma of probable transitional cell origin in 66 free-living California sea lions (Zalophus Californianus), 1979 to 1994. J Wildl Dis.1996; 32:250258.

6. Lipscomb TP, Scott DP, Schulman FY. Primary site of sea lion carcinomas. Vet Pathol. 2010; 47:185.

7. Lipscomb TP, Scott DP, Garber RL, et al. Common metastatic carcinoma of California sea lions (Zalophus Californianus): Evidence of genital origin and association with novel gammaherpesvirus. Vet Pathol. 2000; 37:60917.

8. Martineau D, Lagace A, Masse R, et al. Transitional cell carcinoma of the urinary bladder in a beluga whale (Delphinapterus Leucas). Can Vet J. 1985; 26:297.

9. Meuten DJ, Everitt J, Inskeep W, et al. Histological Classification of Tumors of the Urinary System of Domestic Animals. 2^nd ed. Vol. XI. Second Series. Washington; DC: World Health Organization and Armed Forces Institute of Pathology, 2004.

10. Newman SJ, Smith SA. Marine mammal neoplasia: A review. Vet Pathol. 2006; 43:865880.

11. Van de Velde N, De Laender P, Chiers K. Transitional cell carcinoma metastasizing to different organs including the eye in a California sea lion (Zaliophus californianus). J Comp Pathol. 2017; 156(1):132.

12. Wilson CJ, Flake GP, et al. Immunohistochemical expression of cyclin D1, cytokeratin 20, and uroplakin III in proliferative urinary bladder lesions induced by o-nitroanisole in Fischer 344/N rats. Vet Pathol. 2016; 53(3):682-690.