Two-year-old male Fisher 344 rat (Rattus rattus).This male rat had been on test for 634 days as part of a 2-year carcinogenesis study; was found dead and reported as a natural death animal.

Gross Description:  

On gross examination, the abdomen was markedly distended with copious amounts of reddish-brown fluid (ascites).  In addition, numerous, pale tan, firm, raised nodules were diffusely lining peritoneal viscera; the testes and epididymis were swollen and discolored containing multiple, discrete, pale tan nodules.

Histopathologic Description:

Testis and epididymis: An unencapsulated, exophytic neoplasm composed of arborizing papillary projections is diffusely infiltrating the vaginal tunic and lining the serosal surfaces of the testis and epididymis, often extending into the adjacent adipose tissue.  The neoplasm is comprised of cuboidal epithelial cells, piling up to 4 cell-layers thick and supported by thick, fibrovascular stroma.  Neoplastic cells have indistinct cell borders, with small amounts of finely granular, eosinophilic cytoplasm and a single, central, round to oval nucleus with 1-3 variably distinct nucleoli and finely stippled chromatin.  Mitotic figures are rare.  Anisocytosis and anisokaryosis are mild.  Moderate numbers of mast cells with fewer hemosiderin-laden macrophages are scattered throughout the neoplasm.  Small caliber vessels occasionally contain aggregates of neoplastic cells admixed with variable amounts of eosinophilic flocculent material (fibrin) and necrotic debris (vascular invasion).  Multifocally within the adjacent adipose tissue, vascular lumina are partially occluded by adherent casts composed of fibrin and a mixture of inflammatory cells (vascular thrombosis).  The epididymal ducts are devoid of spermatids (hypospermia) and contain numerous sloughed germ cells within their lumina, admixed with necrotic debris (germ cell exfoliation).  Aggregates of basophilic, granular material are occasionally found within ducts or the vacuolated epithelial lining (mineralization and degeneration).

A second approximately 15 x 10mm, discrete, unencapsulated and expansile, multinodular neoplasm is diffusely effacing and replacing the testicular architecture.  The neoplasm is composed of polygonal cells arranged in nests and cords supported by small amounts of fine, fibrovascular stroma.  Neoplastic cells have variably distinct cell borders with abundant, finely granular, eosinophilic cytoplasm and a single, round to oval, eccentric to centrally located nucleus containing 1-2 distinct nucleoli and stippled chromatin.  Mitotic figures are rare.  Anisocytosis and anisokaryosis are mild.  Large numbers of lymphocytes are surrounding and infiltrating the neoplasm.  The majority of seminiferous tubules are lost; those remaining are shrunken and lined by vacuolated epithelial cells with pyknotic nuclei (tubular degeneration).  There are multifocal areas of hemorrhage and mineralization throughout the testis.  The contralateral testis is similar in appearance with two, discrete, neoplasms ranging from 2 x 2mm to 3 x 5mm and marked degeneration and atrophy of the seminiferous tubules.

Morphologic Diagnosis:  

1. Testis, tunica vaginalis: Malignant mesothelioma with vascular invasion and thrombosis.

2. Epididymis: 1) Malignant mesothelioma 2) Diffuse, subacute, severe hypospermia and degeneration with germ cell exfoliation and mineralization.

3. Testis, bilateral: 1) Interstitial cell adenoma, multiple 2) Diffuse, chronic, severe, tubular degeneration and atrophy with unilateral, marked lymphocytic infiltrate and multifocal mineralization.

Lab Results:  



Mesothelioma and interstitial cell tumor

Contributor Comment:  

Spontaneous mesothelioma is a rare neoplasm in rats with an incidence of 0.2–5%, the highest frequencies of which are reported in the Fischer 344 rat strain.  Rat mesotheliomas typically occur in aged male rats and originate from the tunica vaginalis with the potential to spread into the peritoneal cavity via transcoelomic extension or seeding.1-9  To date, 17 studies from the National Toxicology Program have reported xenobiotic-induced mesotheliomas in F344 rats; the F344 rat is particularly sensitive to developing this tumor type and is the only rodent strain reported to develop mesothelioma following xenobiotic exposure by a route other than peritoneal injection.6,7  Histologically, mesotheliomas may be difficult to distinguish from mesothelial hyperplasia as they can range from a single layer of hyperplastic mesothelial cells lining a thin fibrovascular stroma to papillary projections lined by multiple, irregular layers of cuboidal to polygonal cells that form a pavement or stratified pattern.1-9  Rat mesotheliomas have a classification scheme similar to humans with sarcomatous, epitheliomatous or mixed types that stain positively with Wilm’s tumor 1 (WT-1). Ultrastructural features of mesotheliomas include a distinct basal lamina, junctional complexes between mesothelial cells, microvilli, pinocytotic vesicles, abundant cytofilaments and dilated rough endoplasmic reticulum (RER).5-7 

In the male rat, mesotheliomas of the testis, epididymis, and peritoneum are considered to have originated in the tunica vaginalis; all tunica vaginalis mesotheliomas (TVM) are deemed malignant, even those confined to the scrotal sac and lacking features of malignancy, such as local invasiveness, cytological atypia and pleomorphism.  Proposed mechanisms of actions for both spontaneous and xenobiotic-induced mesotheliomas include endocrine disruption (leuteinizing hormone, prolactin and testosterone), mechanical stress (compression due to testicular tumors) or oxidative stress (asbestos-induced TVM).4,6,7

In the F344 rat, the most common proliferative disorders of the testes include interstitial cell (Leydig cell) hyperplasia and interstitial cell adenoma.  Because interstitial cell adenoma is considered a continuum of interstitial cell hyperplasia, it is sometimes difficult to distinguish hyperplasia from adenoma based solely on differences in cellular morphology.4,7 According to the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND), Leydig cell hyperplasia typically does not cause compression of the adjacent seminiferous tubules.  However, in instances where there is no appreciable compression, proliferative lesions greater than 3 seminiferous tubules in diameter are generally regarded as adenoma.3,4 Interstitial cell adenomas are common spontaneous and xenobiotic-induced neoplasms of rats and mice, particularly the F344 rat with an underlying pathogenesis that reflects endocrine disruption; a hormonal imbalance between the testicular levels of luteinizing hormone receptor and serum testosterone.

Five mechanisms by which hormone imbalance may contribute to Leydig cell tumor formation have been described and include: estrogen agonists (especially in mice), androgen receptor antagonists, gonadotropin releasing hormone (GnRH) and dopamine receptors, and 5α-reductase inhibitors.  Leydig (interstitial) cell adenomas are usually benign, un-encapsulated, discrete neoplasms composed of sheets of uniform polyhedral cells with abundant, finely granular, eosinophilic cytoplasm and a single, centrally located nucleus; compression atrophy of the adjacent seminiferous tubules is common.  Leydig cell adenomas are an age-related change, observed most frequently in F344 rats in their 2nd year of life, and appear to be inversely correlated with body weight, where increased body weights are associated with higher prolactin levels that reduce Leydig cell tumor incidence.4,7

JPC Diagnosis:  

1. Testis: Interstitial cell tumor, Fischer 344 rat, Rattus rattus.

2.  Testis, tunica vaginalis: Mesothelioma.

Conference Comment:  

The contributor provides an outstanding summary of the pathogenesis of spontaneous and xenobiotic-induced interstitial cell tumor (ICT) and mesothelioma in rats. ICTs (also known as Leydig cell tumor or interstitial adenona) are the most common spontaneous testicular tumor of rats with nearly 100% prevalence in 24-month-old Fisher 344 (F344) rats, as in this case.8 Additionally, ICTs are commonly multicentric and can occur in one or both testes.8

Leydig interstitial cells are normally found adjacent to the resident seminiferous tubules in the testis and produce testosterone under the stimulation of luteinizing hormone (LH). Hormonal imbalances as a result of disruption of the hypothalamic-pituitary-testicular axis can result in constitutive LH secretion, which is thought to be the main predisposing feature of ICT formation.1 ICTs are composed to two cell types readily recognized by conference participants: polygonal cells with granular vacuolated cytoplasm present within the central part of the neoplasm, in this case, and smaller cells with dense hyperchromatic nuclei and scant cytoplasm at the periphery.8 The conference moderator cautioned participants to not confuse the latter of these two cell types with lymphocytes. Ultrastructurally, interstitial cells contain lipid droplets, abundant smooth endoplasmic reticulum, and numerous mitochondria with tubulovesicular cristae, and desmosomes.3,8  This neoplasm typically exhibits benign biologic behavior; however, it may be hormonally active and has been associated with hypercalcemia and elevated plasma inhibin levels.1,3,8 It has a characteristic gross appearance of well-circumscribed yellow hemorrhagic nodules on the testes. Larger masses have areas of mineralization and necrosis.8

Mesotheliomas are the third most common spontaneous testicular tumor of rats and are also particularly common in the F344 strain and they commonly occur concurrently with ICT, as in this case.8 Mesothelial cells form a lining around the heart, pleural and peritoneal cavities, and the surface of most organs. In males, it extends into the scrotum and tunica vaginalis and wraps the testes and epididymis.1,2,3,5,8 In male rats, the tunica vaginalis is the most common primary location, with subsequent spread to the rest of the mesothelial-lined surfaces.8 Interestingly, mesothelalial cells have both an epithelial and mesenchymal component. Stains run by the Joint Pathology Center prior to the conference confirmed immuno-reactivity of neoplastic mesothelial cells to both pancytokeratin and vimentin. Other neoplasms of interest that typically express both vimentin and cytokeratin include: chordoma, synovial sarcoma, meningioma, renal cell carcinoma, adrenal carcinoma, and endometrial sarcoma.9 Grossly, mesotheliomas are multiple, well-circumscribed, firm sessile or pedunculated nodules, villous projections, or plaque-like with fibrous adhesions. Concurrent ascites and severe scrotal edema are not uncommon sequelae.8 Ultrastructurally, mesothelial cells have a microvillous cell membrane, junctional complexes between cells, pinocytotic vesicles, and a distinct basal lamina.3,5

As mentioned by the contributor, several studies have attempted to establish a connection between the high incidences of concurrent ICT with mesothelioma in Fisher 344 rats. Some hypotheses include ICT causing direct mechanical stress on mesothelial cells of the tunica vaginalis leading to neoplastic transformation.1,2 Another theory posits that LH and testosterone secretion by neoplastic ICT cells triggers local mesothelium proliferation. Others suggest that Fisher 344 strain-related sensitivity to chemical exposure causes the development of mesotheliomas and ICTs are an unrelated coincidental background lesion.1,2 A definitive connection has not yet been established.


1. Blackshear PE, Pandiri AR, et al. Gene expression of mesothelioma in vindylidene chloride-exposed F344/N rats reveals immune dysfunction, tissue damage, and inflammatory pathways. Toxicol Pathol. 2014; 43:171-185.
2. Blackshear PE, Pandiri AR, et al. Spontaneous mesotheliomas in F344/N rats are characterized by dysregulation of cellular growth and immune function pathways. Toxicol Pathol. 2013; 42:863-876.
3. Boorman GA, Chapin BE, Mitsumori K.  Testis and epididymis. In: Boorman GA, ed.  Pathology of the Fisher Rat.  San Diego, CA: Academic Press, Inc., 1990: 405-418.
4. Creasy D, Bube A, de Rijk E, Kandori H, Kuwahara M, Masson R, Nolte T, Reams R, Regan K, Rehm S, Rogerson P, Whitney K.  Proliferative and nonproliferative lesions of the rat and mouse male reproductive system.  Toxicol Pathol. 2012; 40: 40S-121S.
5. Hall W.  Peritoneum, retroperitoneum, mesentery and abdominal cavity.  In: Boorman GA, ed.  Pathology of the Fisher Rat.  San Diego, CA: Academic Press, Inc., 1990: 63-70.
6. Maronpot RR, Zeiger E, McConnell EE, Kolenda-Roberts H, Wall H, Friedman MA.  Induction of tunica vaginalis mesotheliomas in rats by xenobiotics. Crit Rev Toxicol. 2009; 39(6):512-537.
7. Maronpot RR, Abraham N, Foreman JE, Ramot Y.  The legacy of the F344 rat as a cancer bioassay model (a retrospective summary of three common F344 rat neoplasms).  Crit Rev Toxicol. 2016; 46(8):641-675.
8. Percy DH and Barthold SW. Rat: Neoplasms. In: Percy DH and Barthold SW, eds. Pathology of Laboratory Rodents and Rabbits. 4th ed. Ames, IA: Blackwell Publishing, 2016:169-170.
9. Percy DH and Barthold SW. Rat: Neoplasms. In: Percy DH and Barthold SW, eds. Pathology of Laboratory Rodents and Rabbits. 4th ed. Ames, IA: Blackwell Publishing, 2016:169-170.

Click the slide to view.

4-1. Testis, Fischer 344 rat.

4-2. Testis, Fischer 344 rat.

4-3. Testis, Fischer 344 rat.

4-4. Testis, Fischer 344 rat.

4-5. Testis, Fischer 344 rat.

4-6. Testis, Fischer 344 rat.

4-7. Testis, Fischer 344 rat.

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