JPC SYSTEMIC PATHOLOGY

DIGESTIVE SYSTEM

September 2024

D-N03

 

SIGNALMENT (JPC #1930834): Adult woodchuck (Marmota monax)

 

HISTORY: None

 

HISTOPATHOLOGIC DESCRIPTION: Liver: Infiltrating and replacing approximately 70% of the normal hepatic architecture is an unencapsulated, multilobulated, densely cellular neoplasm composed of polygonal cells arranged in disorganized, 5 to 6-cell- layer-thick trabeculae and fewer acini on a moderate fibrovascular stroma. Neoplastic cells have distinct cell borders, abundant eosinophilic granular to vacuolated cytoplasm, and round nuclei with finely stippled chromatin and one to two distinct magenta nucleoli. Anisocytosis and anisokaryosis are moderate and there are low to moderate numbers of cytomegalic, multinucleated neoplastic cells with occasional bizarre nuclei. The mitotic count averages 1 per 10 HPF (2.37mm²). Multifocally, neoplastic cells contain discrete, clear cytoplasmic vacuoles (vacuolar change, lipid type). Within the neoplasm, there are multifocal areas of lytic necrosis with loss of architecture and replacement by eosinophilic cellular and karyorrhectic debris, hemorrhage, fibrin, edema, and fibrosis. Also within the neoplasm, there are multifocal cystic spaces up to 500 micrometers in diameter that contain eosinophilic proteinaceous fluid and blood. Within the adjacent compressed hepatic parenchyma, portal areas are expanded by numerous lymphocytes and plasma cells and fewer neutrophils. This inflammatory infiltrate is admixed with variable amounts of fibrosis and increased small biliary duct profiles (ductular reaction) that occasionally bridge portal areas. Frequently, the cytoplasm of non-neoplastic hepatocytes is expanded by lacy, microvaculated to occasionally granular material (vacuolar change, glycogen type).  

MORPHOLOGIC DIAGNOSES: 

1. Liver: Hepatocellular carcinoma, woodchuck (Marmota monax), rodent.

2. Liver: Hepatitis, portal, lymphoplasmacytic and neutrophilic, chronic, diffuse, moderate, with biliary hyperplasia, bridging fibrosis, and hepatocellular degeneration and necrosis.

 

ETIOLOGY: Woodchuck hepatitis virus (WHV)

 

ETIOLOGIC DIAGNOSIS: Hepadnaviral hepatitis and hepatocellular carcinoma

 

GENERAL DISCUSSION: 

• Family Hepadnaviridae – Associated with hepatocellular carcinomas in humans, other mammals, birds; partially dsDNA virus; hepatotropic family of viruses
  • Genus Orthohepadnavirus (infects mammals): 
    • Ground squirrel hepatitis virus
    • Hepatitis B virus (man, chimpanzees)
    • Woodchuck hepatitis virus (wild American woodchucks)
    • Woolly monkey hepatitis B virus
  • Genus Avihepadnavirus (infects birds): 
    • Duck hepatitis B virus
    • Heron hepatitis B virus
• The woodchuck (groundhog) is a valuable animal model for studying the pathogenesis of viral hepatitis and viral hepatocarcinogenicity

PATHOGENESIS:

• WHV:
  • WHV causes the majority of hepatocellular damage through immunologic mechanisms, particularly through cytotoxic CD8+ T lymphocytes
  • Viral DNA is integrated into the host cell genome at the c-myc or n-myc2 locus
  • Normal growth control of infected hepatocytes is disrupted by transcriptional activation of host proto-oncogenes
• Hepatocellular carcinoma:
  • Venous invasion is typical of hepatocellular carcinomas
  • Morbidity more commonly due to expansive growth, local compression, necrosis, and/or hemorrhage of the mass (rather than metastasis)
  • Canine metastatic rate 25-61%, metastasis slightly more common in cats

TYPICAL CLINICAL FINDINGS:

• Nonspecific: Anorexia, vomiting, ascites, lethargy, and weakness; less commonly jaundice, diarrhea, weight loss
• Hepatomegaly with possible palpable cranial abdominal mass
• Chemistry: Nonspecific, increase in ALT, ALP, AST, GGT, increased fasting bile acids
• Infrequent: Seizures in dogs due to hypoglycemia or hepatoencephalopathy
  1. Hypoglycemia; cause is unknown; theories include tumoral secretion of an insulin-like substance or excess glucose consumption
• Infrequent: alopecia in cats

TYPICAL GROSS FINDINGS: 

• Massive (most common), nodular, or diffuse (rare) forms
• Variable color and consistency (both between different tumors and within a single tumor) due to variable areas of lipidosis, hemorrhage, necrosis; often friable and soft
• Hemoperitoneum common
• Intravascular invasion not commonly seen macroscopically but indicates malignancy

TYPICAL LIGHT MICROSCOPIC FINDINGS:

Hepatocellular carcinoma: 

• Classifications of hepatocellular carcinoma
  • Trabecular: Most common form
    • More differentiated (closely resembles normal liver)
    • Thickened trabeculae are a frequent feature though thickness is not uniform (unlike an adenoma)
  • Pseudoglandular (adenoid)
    • Neoplastic hepatocytes have a lobular arrangement within a scant connective tissue stroma
    • Crude acini formed by neoplastic hepatocytes
  • Solid
    • Solid sheets of poorly differentiated, pleomorphic neoplasic hepatocytes which do not form sinusoids
  • Scirrhous: Rare
    • Multiple foci of ductular formation and associated extracellular matrix (the ducts stain immunohistochemically with CD 7 and 19) 
• Histologic features:
  • Disorganized, thick hepatic cords (often >4 hepatocytes thick)
  • Necrosis, hemorrhage, dilated sinusoids, cystic cavities with blood or serum 
  • Atypical or bizarre cells and/or neoplastic giant cells with large, multilobed, or multiple nuclei
  • Mitotic figures are more frequent in carcinomas than adenomas
  • Focal to widespread lipidosis or glycogen accumulation within neoplastic hepatocytes

Hepatitis:

• Acute hepatitis: Inflammatory infiltrate confined by the limiting plate, with little or no hepatocellular necrosis and minimal fibroplasia and biliary ductular reaction 
• Chronic hepatitis: Numerous inflammatory cells expanding portal areas and extending beyond the limiting plate into the parenchyma, with proliferation of bile ducts, fibroplasia, and hepatocellular necrosis.

ULTRASTRUCTURE: 

• Electron microscopy to demonstrate the virus is definitive
• Woodchuck hepatitis virion is 42-48 nm, spherical, double-layered, with an electron-dense, 27-30 nm, slightly hexagonal or icosahedral core (nucleocapsid) surrounded by a closely adhered outer capsid or surface envelope

ADDITIONAL DIAGNOSTIC TESTS: 

• Cytology:
  • Unless cells have overt features of malignancy, distinguishing normal hepatocytes from hyperplasia, regeneration, adenoma, or well-differentiated carcinoma is difficult
  • Features of malignancy include significant anisocytosis, anisokaryosis, multinucleation, nucleolar atypia, and numerous mitotic figures (3+), which support cytologic diagnosis of HCC
  • In well-differentiated HCC, more subtle features such as cellular dissociation, naked nuclei, palisading arrangement, or admixture of capillaries with hepatocytes help support the diagnosis
• Immunohistochemistry for hepatocellular carcinoma (canine/feline): 
  • HepPar1: Binds a hepatocyte specific antigen, all but least-differentiated HCC are immunoreactive
  • CK19: Marker of hepatic progenitor cells, immature hepatocytes, and biliary epithelial cells
  • Arginase 1: Found in normal and diseased hepatocytes; an enzyme that catalzyes the hydrolysis of arginine to ornithine and urea during amino acid metabolism.

DIFFERENTIAL DIAGNOSIS: 

• Nodular hyperplasia: Usually multiple nodules less than 3 cm diameter with retention of normal liver architecture including central vein and portal areas; well-differentiated cords 1-2 cells thick; rare in non-canines
• Regenerative nodules: Lack normal lobular architecture and can be difficult to differentiate on needle biopsies; hepatic plates no more than 2 cells thick; usually contain a single portal tract; occur on a background of significant hepatic injury and fibrosis 
• Hepatocellular adenoma: Typically solitary; up to 12 cm; monomorphic population with trabeculae 2-3 cells thick (thinner, more uniform than trabeculae in hepatocellular carcinoma); symmetric, well-delineated growth (vs. hepatocellular carcinoma that has invasive growth pattern); hemorrhage and necrosis uncommon; no metastasis
• Hepatoblastoma: Rare, benign neoplasm; reported in sheep, dogs, horses and a bull; cells similar to fetal hepatocytes; no or partial HepPar-1 immunoreactivity; alpha-fetoprotein immunoreactive, and some are immunoreactive for neuroendocrine markers (S100, chromogranin A, and NSE)
• Hepatic carcinoids: Rare, aggressive neuroendocrine cell tumor; extra- or intra-hepatic; can metastasize to lymph nodes and peritoneum; forms nests and packets with frequent mitotic figures on a fine fibrovascular stroma 
  • Neuroendocrine cell markers: Chromogranin A, NSE, or neurosecretory products such as glucagon or serotonin
  • Cytoplasmic neurosecretory granules can be seen on EM or silver stains (e.g. Churukian-Schenk)
• Cholangiocellular tumors: CCTs have acinar/tubular composition, cuboidal to columnar lining epithelium; pseudoglandular HCTs can form rudimentary acini but they lack mucus produced by biliary epithelium
  • HCC immunoreactive for: Alpha-fetoprotein; HepPar-1
  • CCT immunoreactive for: Cytokeratin 7, epithelial membrane antigen/mucus 1, carcinoembryonic antigen
• Mixed hepatocellular and cholangiocellular carcinoma: Rare; IHCs as described above can confirm hepatocytic nature
• Metastatic neoplasia: Often multiple neoplastic foci; certain metastatic carcinomas may require immunohistochemistry to differentiate from hepatic or cholangiocellular origin

COMPARATIVE PATHOLOGY:

• Hepatocellular carcinoma occurs in all veterinary species and humans 
• Chronic inflammation and hepatitis virus are associated with hepatocellular carcinoma in many species, including humans, prairie dogs, and woodchucks (and other similar rodents e.g. squirrels, prairie dogs)
• Viral or chemical (aflatoxin, nitrosamines, permethrin) etiologies are suggested in laboratory and domestic animals
• Dogs: More common than in other species, left lateral liver lobe often affected
• Cats: Cholangiocellular neoplasia is more common than HCC
• Bears and fennec foxes: Hepatocellular carcinoma is common; etiology may be related to chronic inflammation, carcinogens, or possibly viral
• Nonhuman Primates: Although monkeys can be infected with human hepatitis B viruses, development of HCC or cirrhosis does not seem to follow acute hepatitis 
  1. Experimental induction of HCC with carcinogens (nitrosamine), hepatitis C, hepatitis B in multiple species
  2. Natural infection with Schistosoma mansoni caused HCC secondary to liver granulomas in chimpanzees
• Prosimians: Hepatocellular tumors are the most common neoplasms, of which hepatocellular carcinoma is the most frequent; no known cause and iron, aflatoxin, viruses are not associated
• Bats: HCC and cholangiocarcinoma are common neoplasms of Egyptian fruit bats due to dietary iron excess
• Ducks: Aflatoxin and duck hepatitis virus B virus are both inducers of HCC
• Snakes: Hepatic and biliary tumors are common, especially in colubrids
• Fish: Development of HCC may be a consequence of carcinogen entry into waterways (e.g. creosote)
• Sheep: Case report of a clear cell HCC (Hughes, Vet Path, 2023).
• Mice (CD-1): Chronic dietary exposure to permethrin increases incidence of hepatocellular adenoma (not carcinoma) (Quist, Tox Pathol. 2019)
• Mice (A strain): Helicobacter hepaticus infection is associated with increased incidence and earlier onset of hepatitis and hepatocellular tumors

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