The female Wistar rat from the control group of a carcinogenicity study had to be killed in a moribund condition after 6 months on test. At necropsy, the animal was skinny. In the thoracic cavity, a nodulated covering was recorded and in the thymus, a brownish-red nodule with a diameter of 4 cm was found.

Click on the images below for a larger view.
Fig. 1: H&E, x1	Fig. 2: H&E, x7
Fig. 3: H&E, x23	Fig. 4: H&E, x40

Histopathologically, the tumor (low magnification shown in fig. 1) consisted mainly of nests, isolated cells (fig. 2) or cords of large and pleomorphic cells, with often bizarre nuclei (fig. 4, fig. 8) and frequent mitotic figures and necrosis. They are embedded in a hyaline matrix which is positive with the Periodic Acid Schiff (PAS) reaction (fig.5). PAS-positive material was also visible in tumor cells (fig. 5). Immunohistochemically, the cells are distinctly positive for cytokeratin (CK) (fig 6). In some regions of the tumor, the cells were densely packed, small, partly spindle-shaped and extracellular material was not discernible in the H&E stain. Fig. 3 shows an area with tumor cells embedded in a huge amount of eosinophilic extracellular matrix. At the bottom of fig.3, spindle-shaped cells and epithelial cells associated with a low amount of extracellular material are visible. At the surface of the tumor in the upper part of fig. 3, a small papilla is present. CK- immunohistochemistry shows that in this area positive large cells are intermingled smaller CK-negative cells (fig. 7). With the PAS-reaction, positive material is seen in the cells and in an extracellular position (fig. 8). Staining of the tumor for vimentin showed in most regions a negative result whilst the stroma was distinctly positive (fig. 9). In some areas, tumor cells exhibited however a positive reaction for vimentin (fig. 10). An attempt was made to demonstrate alpha-fetoprotein (AFP) with a polyclonal antibody from rabbit against human, rat, and mouse AFP. The tumor cells were positive (fig. 11) including those associated with eosinophilic matrix. The result was confounded by cross-reactivity with other tissue components in the same section and in control sections from other tissues, thereby giving an inconclusive result due to lack of specificity.

Click on the images below for a larger view.
Fig. 5: PAS, x40	Fig. 6: cytokeratin, x20
Fig. 7: cytokeratin, x10	Fig. 8: PAS, x20
Fig. 9: vimentin, x20	Fig. 10: vimentin, x20
Fig. 11: alpha-fetoprotein, x20

For the case, 14 responses were received. One of the senders proposed two diagnoses.

Six of the contributors proposed thymoma or more specifically malignant thymoma. Benign and malignant thymomas are the most frequent tumor entities observed in the mediastinum of rats.They are derived from the epithelial cells of the thymus and morphology may vary considerably. Most thymomas display an admixture of epithelial cells and lymphocytes with a prevailing of lymphocytes. In epithelial thymomas lymphocytes are scarce or even be absent so that only this type of thymoma has to be taken into account for differential diagnosis. Epithelial cells have often a squamous differentiation whereas extracellular hyaline material is not a feature of thymoma. Immunohistochemically, thymomas are positive for CK.

Four contributors proposed chondrosarcoma. This is an appropriate differential diagnosis based on the H&E-stained slides. Chondrosarcomas are characterized by large basophilic cells, which can be very large and pleomorphic. They lie individually in lacunae and are surrounded by hyaline matrix. The matrix normally stains bluish due to proteoglycan content, in particular in the proximity of chondrocytes similar to matrix of normal hyaline cartilage. In contrast to chondrosarcoma, the cells of the present tumor form nests and are not arranged similar as chondrons in lacunae. The matrix shows no basophilia. A case of an extraskeletal myxoid chondrosarcoma has been observed in an aged F344 rat (Nakamura et al. 2007) which was positive for vimentin and S-100 but not for CK.

Other diagnoses, which were given by one contributor each were lymphoblastoma, biphasic mesothelioma, osteosarcoma and metastatic germ cell tumor (choriocarcinoma).

Pleural mesothelioma is a rare spontaneous tumor in rats. It can show papillary or glandular growth patterns, and in the biphasic type epitheloid and sarcomatoid parts made up of spindle-shaped cells are present side by side. Immunohistochemically, the tumor is positive for cytokeratin. Although PAS-positive material may be present in the tumor, it is an unusual feature and extensive formation of PAS-positive extracellular matrix has not been described in rats. The high proportion of extracellular hyaline material in the present tumor would also preclude lymphoblastoma. In lymphoblastic lymphoma the tumor cells resemble lymphoblasts (monomorphic population of lymphoid cells and no extracellular material) and can be either of B- or T-cell origin.

The reason for the proposal of osteosarcoma was likely the presence eosinophilic extracellular matrix which resembles decalcified osteoid, particularly in fig. 2.

The occurrence of a metastatic germ cell tumor has also to be taken into account. In the animal, a full spectrum of organs including gonads was investigated grossly and histopathologically. No tumor or tissue which could indicate a primary tumor or even regressive tumor remnants could be detected elsewhere in the animal.

The choriocarcinoma is characterized by small basophilic cells and giant cells with large nuclei and prominent nucleoli. Typical for choriocarcinoma is extensive necrosis and hemorrhage whereas PAS-positive extracellular matrix is not typical.

Yolk sac carcinoma (syn. endodermal sinus tumor) was proposed by one sender. This type of germ cell tumor often mimicks the parietal and visceral part of the fetal membranes but may also consist of one of the components only:

• The parietal cells produce an abundant PAS-positive matrix (yolk) around nests and cords of tumor cells. The extracellular matrix seen in the present tumor is typical for yolk sac tumor and PAS-positive granules could also be detected in the tumor cells. Growth patterns include rosettes, cords, papillary structures or cysts.
• The visceral cells produce alpha-Fetoprotein, have a cylindrical shape and may grow papillary around a central capillary which are called Schiller-Duval bodies (Sobis 1987). These structures, originally found in rodents, were the reason to name this tumor endodermal sinus tumor despite fundamental interspecies differences in placenta and yolk sac (Nogales 2012).

In the present case, there is clear evidence of the parietal component with its huge amount of extracellular material and PAS-positive inclusions in the tumor cells. In a small part of the tumor, this material is not detected in the H&E stain but present to a low extent in the PAS-stain where it is also seen intracellularly in some of the cells. There is no clear evidence of a visceral part in the sections investigated. For clarification, alpha-fetoprotein (AFP) is of interest since this protein is known to be produced by the visceral cells. In a case of metastatic ovarian yolk sac carcinoma in a rat, AFP could however be demonstrated in visceral as well as in parietal cells (Sakamoto et al. 2011). In the present tumor, the antibody used regrettably gave inconclusive results.

Spontaneous occurrence of yolk sac tumors seems to be very rare, in particular in male rats. A few case reports refer to female rats and only one testicular yolk sac carcinoma was reported in a male Sprague-Dawley rat (Nakazawa et al. 1998).

The extragenital development of the present tumor is inferred from the absence of any tumor tissue in the testes or elsewhere except in the mediastinum where the tumor was observed grossly. Mediastinal yolk sac tumors are known to occur in humans, where they have been observed to date for unknown reasons in the male sex only (Moran and Suster 1997). The origin of extragenital germ cell tumors is speculative, and may originate from misrouted germ cells or germ cells physiologically distributed to extragonadal tissues during embryogenesis (Bokemeyer et al. 2002).

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