Two-year-old male B6C3F1 mouse (Mus musculus).This mouse was in the control group of a two-year study investigating the carcinogenicity of kava kava extract. It was sacrificed on Test Day 668, due to moribund condition. Clinical observations included ruffled fur, lethargy and thin body condition.

Gross Description:  

Gross lesions described at necropsy included: a single tan mass, approximately 27x17x12 mm, on the left lateral liver lobe; and several white nodules, up to 5 mm diameter, on the visceral pleura, lungs, and mediastinum.

Histopathologic Description:

Most of this section of liver lobe is replaced by a large mass extending to the capsular surface. The mass is composed of irregular acini and variably-sized cystic spaces interspersed with more densely cellular areas and areas of fibrosis or necrosis. Acini are lined by columnar epithelium with prominent hyper-chromatic nuclei and scant cytoplasm. More densely cellular areas are comprised of hepatocytes with a central vesicular nucleus containing 1 or 2 prominent nucleoli, and abundant cytoplasm. These cells are arranged in packets, in trabeculae more than 3 cells thick, or around cystic spaces. The cystic spaces are lined by one or more layers of flattened to plump hepatocytes, with occasional papillary projections extending into the lumens; lumens often contain sloughed epithelial cells, lightly stained fibrillar eosinophilic material, and occasional extravasated erythrocytes. In other areas, densely-packed, small round to elongated cells with hyperchromatic nuclei form a sarcomatous pattern, often extending into areas of fibrosis. Occasional endothelial-lined spaces contain erythrocytes, small clusters of white blood cells (mainly neutrophils) and single or clusters of neoplastic cells.

Morphologic Diagnosis:  

Liver – Hepatocholangiocarcinoma. Tissue not included: Lung – Hepatocholangiocarcinoma, metasta-tic
Lymph node, mediastinal – Hepatocholan-giocarcinoma, metastatic
Thymus – Hepatocholangiocarcinoma, metastatic
Heart, pericardium – Hepatocholangio-carcinoma, metastatic

Lab Results:  



Hepatocholangicarcinoma, mouse

Contributor Comment:  

This hepato-cholangiocarcinoma (HCCC) typically contains both malignant hepatocellular and malignant biliary components, as well as foci of markedly undifferentiated sarcoma-like cells, as described in a poster presented by the National Institute of Environmental Health Sciences (NIEHS) and Experimental Pathology Laboratories, Inc. (EPL).4 HCCC is rarely reported in mice and rats. However, it has been induced by some chemicals tested in the National Toxicology Program (NTP) studies.2,4  It is an aggressive neoplasm that metastasizes readily.

JPC Diagnosis:  

Liver: Hepato-cholangiocarcinoma, B6C3F1 mouse, Mus musculus.

Conference Comment:  

Hepato-cholangiocarcinoma (HCCC) is a rare spontaneous neoplasm with an incidence of less than 0.5% in B6C3F1 mice, according to the National Toxicology Program (NTP) database.4,6  HCCC is a primary liver neoplasm comprised of both neoplastic hepatocytes and neoplastic bile duct epithelial cells.4,6 The biliary component forms tubules and acini or small nests, while the hepatocellular component forms trabeculae, glands, or solidly cellular areas of neoplastic cells. Both neoplastic hepatocytes and bile duct epithelial cells have many characteristics of malignancy, such as frequent mitoses, nuclear pleo-morphism, local invasion, and widespread metastasis.4,6,7 HCCC often contains large cystic or necrotic areas within the central portions of the neoplasm, as demonstrated in this case. Additionally, there are occasional ducts within the neoplasm, lined by both hepatocytic and biliary epithelial cells, in conjunction with multifocal areas of poorly differentiated mesenchymal spindloid cells arranged in short interlacing streams and bundles. Proliferation of sarcomatous cells is a frequently reported feature of this malignant neoplasm.6,7 In previously studies, 84% of mice with HCCC had metastasis to multiple tissues, similar to what is reported by the contributor here.6 The histo-morphologic variability of this neoplasm and widespread metastasis may pose a diagnostic challenge in determining the site of origin. Metastatic lesions may contain only one of the three neoplastic populations mentioned above, further confounding the diagnosis.6

As mentioned previously, spontaneous occurrence of HCCC is exceedingly rare in mice; however, malignant transformation has been experimentally associated with chemical carcinogens, such as trimethylolpropane tricrylate, benzidine dihydrochloride, and N-2-acetylaminofluorene.4 The B6C3F1mouse is a hybrid strain that is the result of a cross between a male C3H and a female C57BL/6 mouse. This relatively hardy mouse strain has been used by the NTP for decades as part of carcinogenesis, toxicity, and transplant studies.2


1.      Adams ET, Auerbach S, Blackshear PE et al. Proceeding of the 2010 National Toxicology Program satellite symposium. Toxicol Pathol. 2010; 39:240-246.

2.      Gad SC, Clifford C, Goodman D. The mouse. In Gad SC ed. Animal Models in Toxicology. 3rd ed. Boca Raton, FL: CRC Press; 2016:76-78.

3.      Hailey JR, Nold JB, Brown RH et al. Biliary proliferative lesions in the Sprague-Dawley rat: Adverse/non-adverse. Toxicol Pathol. 2014; 42:844-854.

4.Harada T, Enomoto A, Boorman GA, Maronpot RR. Liver and gallbladder. In: Maronpot RR, Boorman GA, Gaul BW eds. Pathology of the Mouse. Vienna, IL: Cache River Press; 1999:153.

5.      Maronpot RR. Rodent Liver - Neoplastic. 2015.

6.      Moore R,Willson G, Miller R, Malarkey D, Kissling et al. Hepatocholangiocarcinomas in B6C3F1 Mice in National Toxicology Program (NTP) Studies.

7.      Thoolen B, Maronpot RR, Harada T et al. Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system. Toxicol Pathol. 2011; 38:5S-81S.

Click the slide to view.

4-1. Liver, B6C3F1 mouse.

4-2. Liver, B6C3F1 mouse.

4-3. Liver, B6C3F1 mouse.

4-4. Lung, B6C3F1 mouse.

4-5. Mediastinum, B6C3F1 mouse.

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