Signalment:  

Twelve-year-old, male, dwarf rabbit, (Oryctolagus cuniculus).Both testicles were submitted for histological examination because of increasing testicular size over time.

Gross Description:  

The formalin-fixed testicles measured 9 x 7 x 4 cm and 5 x 3 x 3 cm, respectively. The testicular parenchyma was almost completely replaced bilaterally by multilobulated, solid masses with a greyish cut surface. The mass was located on both sides within the testis and did not extend beyond the tunica albuginea.

Histopathologic Description:

The slide contains parts of the testicle, epididymis and tunica vaginalis. The majority of original testicular tissue is replaced by a multilobular, well demarcated, non-encapsulated, expansive, moderately cell-dense mass extending to the cut border on one side. Neoplastic cells are proliferated in solid fields supported by a small to moderate amount of fibrovascular stroma. The cells measure up to 70 µm in diameter and display a round to polygonal shape with an eccentrically located, 10 µm in diameter large, round to oval nucleus with finely stippled heterochromatin and one distinct, small, basophilic nucleolus. The abundant, eosinophilic, finely granular cytoplasm is surrounded by indistinct cell borders. Tumor cells show mild anisocytosis and anisokaryosis with a mitotic rate of 0-1 per high power field. In the cytoplasm of the tumor cells, a high number of PAS and PAS-diastase-resistant positive granules are present. These granules were also shown by Luxol Fast Blue staining. Remaining seminiferous tubules are compressed and lined by single Sertoli cells.
Multifocally within the tunica vaginalis there are few inflammatory cells, mainly consisting of plasma cells, lymphocytes, and fewer macrophages. In some slides eosinophilic, homogeneous, acellular material is present within the tunica vaginalis (edema). The epithelium of epididymal tubules is flattened and the diameter of the tubules is severely increased (dilatation). Within the epididymal tubules, no spermatozoa are present.

Morphologic Diagnosis:  

Testicle: Granular cell tumor with severe testicular atrophy, dilatation of epididymal tubules and mild, chronic, multifocal, lymphohistiocytic and plasmacellular infiltration of the tunica vaginalis with edema.

Lab Results:  

Immuno-histochemistry was applied using commercially available antibodies. Tumor cells showed a diffuse immunolabelling for Melan-A and a multifocal expression of neuron-specific enolase in about 40-50% of the tumor cells. About 20-30% of the tumor cells displayed an immunolabelling for S-100 protein and single tumor cells expressed vimentin.

Neoplastic cells were negative for cytokeratin, α-smooth muscle actin, glial fibrillary acidic protein, and myelin basic protein. Samples for transmission electron micro-scopy were processed by direct pop-off technique from the HE- stained slide. The cytoplasm of the tumor cells was filled with numerous round, membrane-bound structures measuring about 500 to 1000 nm in diameter. These structures contained several membrane-bound, moderately electron-dense, round structures, measuring about 50 to 150 nm in diameter.

Condition:  

Granular cell tumor

Contributor Comment:  

Testicular tumors in rabbits represent a rarely described entity. Mainly adult individuals are affected and interstitial cell tumors are most commonly reported.^6,7,19

Granular cell tumors occur rarely in domestic and pet animals and have been reported in different species including horses, dogs, cats, guinea pigs, and rats.^2,6 In ¹⁴ In horses, granular cell tumors represent the most common primary neoplasm of the lung.^2,9 In dogs and rarely in cats, they occur in the oral cavity.^2,11 In rats, a meningeal localization has been described.¹⁸ A variant of meningiomas, termed granular cell meningioma, has been reported in dogs located at the cerebral convexity, neurohypophysis, and spinal nerve roots.¹ Genital involvement of granular cell tumors has been reported in cats affecting the vulva.^5,6 Within the right atrium of a dog a granular cell tumor, also termed myo-blastoma, has been described exhibiting typical cytoplasmic granules.¹³ In guinea pigs, cutaneous granular cell tumors have been reported.¹⁷

The histogenetic origin of granular cell tumors remains unknown, but they are thought to derive from Schwann cells or related cells due to their histological, immunohistological and electron micro-scopic characteristics.^7,11 The immuno-reactivity of granular cells to antibodies specific for S-100 protein, Melan-A, and neuron-specific enolase supports the potential neuro-ectodermal origin.^6,7

Furthermore, ultrastructurally numerous round cytoplasmic structures are found surrounded by a membrane with a diameter of about 500 to 1000 nm containing round electron-dense structures, measuring about 50 to 150 nm in diameter. These findings are similar to reports about the ultrastructure of equine pulmonary granular cell tumors.^12,15 The membrane-bound structures tend to contain fragments of mitochondria or other organelles and are interpreted as secondary lysosomes.
The majority of granular cell tumors are reported to be benign, except one case in a cat, showing recurrence after excision and a high degree of pleomorphism with a high mitotic rate.¹¹ Granular cell tumors in cats generally tend to be more anaplastic.¹¹
As a differential diagnosis interstitial cell or Leydig cell tumor has to be considered because it shares histologic features with the granular cell tumor, e. g. eosinophilic granules within the cytoplasm.⁶ In hematoxylin-eosin stained slides they are almost indistinguishable. However, testicular interstitial cell tumors lack PAS-positive and diastase-resistant cytoplasmic granules as well as Luxol Fast Blue stainable granules that are indicative for a granular cell tumor.^7,11 Furthermore, granular cell tumors express neuron-specific enolase, S-100 protein, and vimentin to a variable extent^6,7,19 as was shown in the present case. Additionally, transmission electron microscopy is a suitable tool to demonstrate the typical membrane-bound granules reported for granular cell tumors, leading to their name.^6,12<

JPC Diagnosis:  

Testis: Granular cell tumor, dwarf rabbit, (Oryctolagus cuniculus).

Conference Comment:  

The contributor provides an excellent summary of the major features of granular cell tumors (GCT). GCTs are histologically characterized by proliferating uniform polygonal neoplastic cells that contain abundant eosinophilic granules in their cytoplasm and a round eccentrically placed nucleus.² Conference participants discussed differential diagnoses for neoplasms with abundant granular eosinophilic cytoplasm to include rhabdomyomas, oncocytomas, balloon cell melanoma, and interstitial cell tumors. In addition to the histochemical and immunohistochemical stains mentioned by the contributor, transmission electron microscopy remains the best way to differentiate these histologically similar neoplasms. Ultrastructurally, oncocytomas and rhabdomyomas both contain large numbers of mitochondria which explain the abundant acidophilic granular appearance histologically.^2,3,6 In balloon cell melanoma, electron microscopically reveals numerous heterogeneous melanosomes within the cytoplasm. Interstitial cell tumors (also known as Leydig cell tumors) have an abundance of smooth endoplasmic reticulum and well-developed mitochondria with tubular and vesicular cristae. In GCT, the granules are thought to be composed of numerous membrane-bound secondary lysosomes.^2,3,6

In a 2015 Veterinary Pathology article, Suzuki et al. demonstrates that in canine lingual GCT, the cytoplasmic granules are positive for LC3, p62, NBR1, and ubiquitin. LC3 is localized on the membranes of autophagosomes and is a potent marker of autophagy. In addition, LC3, p62, and NBR1 are all required for production of the autophagosome.¹⁴ This suggests that the cytoplasmic granules found in canine lingual GCT cells are autophagolysosomes which are a subset of secondary lysosomes that result from the fusion of a primary lysosome with a phagosome containing cytoplasmic cellular constituents that are to be digested.¹⁴
Recently, it has been suggested that autophagy might play a suppressive role in the initiation stages of a neoplasm by maintaining genomic stability and inducing cell senescence and autophagic death; but conversely plays a maintaining role in tumor growth in the later stages of tumorigenesis by supplying metabolic substrate, limiting oxidative stress, and maintaining cancer stem cell population.⁸

Due to near diffuse effacement and compression of the normal testicular architecture and distortion from diffuse ductular ectasia in the adjacent epididymis, conference participants had some trouble identifying the tissue of origin in this section. The key to identification of the tissue is dependent on a close investigation of the epididymis. The epididymis is a tightly coiled mass of thin tubules that carries sperm from the testes to the ductus deferens in the male reproductive system. In this case, the epididymal tubules are markedly dilated, lined by a single layer of attenuated cuboidal epithelium and contain an abundant amount of amphophilic inspissated proteinaceous material. Some participants noted that scattered throughout the lumen are very small numbers of degenerate spermatozoa thus identifying the tissue as epididymis with adjacent testis and tunica vaginalis.

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