Spontaneous Subcutaneous Sarcoma in a 50-week-old Male Wistar Hannover GALAS Rat.

A subcutaneous mass was noted in the abdomen of a 50-week-old male Wistar Hannover GALAS rat. Histologically, the tumor was composed of vimentin-positive small round cells with scant cytoplasm arranged in a trabecular, sheet or pericytoma-like pattern and spindle cells arranged in a bundle pattern and vimentin-negative round cells proliferating in an island-shaped pattern. Argentophilic thin fibers were observed to wrap up the individual cells, and some of the tumor cells showed coexpression of vimentin and cytokeratin that formed juxtanuclear globes in the cytoplasm by double immunohistostaining. Transmission electron microscopy did not reveal any characteristic features suggesting cellular differention toward a specific cell type. Based on these findings, it was difficult to specify the origin, and the tumor was diagnosed as a poorly differentiated mesenchymal tumor and classified as a sarcoma, NOS (not otherwise specified).

There are few reports available concerning tumor incidence in Wistar Hannover GALAS rats. According to the Historical control data of Wistar Hannover GALAS rats (from RCC Ltd.), the incidence of cutaneous nonepithelial neoplasm in male Wistar Hannover GALAS rats was approximately 2.4%, and cutaneous nonepithelial malignancies with higher incidences were malignant neurinoma (0.76%); sarcoma, NOS (0.54%); hemangiosarcoma (0.49%); fibrosarcoma (0.27%); and liposarcoma (0.22%). Although cutaneous sarcoma, NOS, was diagnosed in a relatively large number of rats, no case report was found. Thus, we herein report a case of mesenchymal tumor that was ultimately diagnosed as a sarcoma, NOS, in a male GALAS rat.

The Wistar Hannover GALAS rat was obtained from CLEA Japan, Inc. (Shizuoka, Japan) at 5 weeks old and was allocated to the intact group in a toxicity study. The animal was fed a pelleted basal diet sterilized by γ-ray irradiation at 10 kGy (CE-2: CLEA Japan, Inc., Tokyo, Japan) for 45 weeks ad libitum and was maintained in a room with a barrier-sustained system under the following conditions: temperature of 22.1–24.3 °C, relative humidity of 39.3–73.2%, ventilation frequency of 10–25 times/h and a 12-h light/dark cycle. The animal was housed individually in a stainless steel cage and given tap water ad libitum. At 48 weeks of age, a hard palpable subcutaneous mass was observed in the abdomen region, and the animal was euthanized by exsanguination under isoflurane anesthesia at 50 weeks of age. The animal experiment complied with the Guide for Care and Use of Experimental Animals of the Toxicology Research Laboratory, Kissei Pharmaceutical Co., Ltd.

At necropsy, a massive subcutaneous mass approximately 5 × 4 × 3 cm in size was found in the lower abdomen. The mass had a flesh color appearance, was surrounded by thin connective tissue and was well demarcated from the skeletal muscle but poorly demarcated from the skin. The cut surface of the mass was orangish white, and the central part was necrotic. No other findings were noted in other organs grossly.

The mass was fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin-eosin (HE) for microscopic examination. Additional histochemical stains including Watanabe’s silver impregnation, Masson’s trichrome staining, Grimelius reaction and periodic acid-Schiff stain (PAS) were performed. Immunostaining was also performed by the peroxidase-anti-peroxidase complex method using antibodies against CD31, CD99, chromogranin A, cytokeratin (clones AE1/AE3, MNF116), cytokeratin 20, leukocyte common antigen (LCA), melanoma PNL2, neurofilament, neuron-specific enolase (NSE), P63, proliferating cell nuclear antigen (PCNA), synaptophysin, S-100 protein, vimentin, von Willebrand factor and α-smooth muscle actin (α-SMA). To examine coexpression of cytokeratin and vimentin in the neoplastic cells, double immunostaining was also performed with vimentin and cytokeratin (clone AE1/AE3). For electron microscopic examination, pieces of the formalin-fixed tissue were immersed in a half-strength Karnofsky solution for 2 hours and fixed in 1% osmium tetraoxide. The tissues were then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, evaporated with carbon and then observed under a transmission electron microscope (JEM-1200EX; JEOL, Tokyo, Japan).

Histologically, the tumor was encapsulated by thin fibrous connective tissue, and some tumor cells had invaded the surrounding tissue. At the peripheral area of the tumor, the tumor cells proliferated solid and blood vessels were in a minority. There were increasing tendencies for the number and size of blood vessels toward the central area of the tumor. In the necrotic central part of the tumor, the cells survived only around the areas containing blood vessels (Fig. 1). The tumor consisted of small round, spindle and intermediate form cells. The small round cells, which made up a major portion of the tumor, showed trabecular, sheet and pericytoma-like patterns. They had scant eosinophilic cytoplasm and hyperchromatic round to oval nuclei. Some nuclei were small and irregularly shaped; occasionally, the nuclei showed a budding shape in a part of the sheet pattern area. In addition, numerous mitotic figures were found in the round cells. In the sheet pattern area, large oval cells with a washy eosinophilic cytoplasm were occasionally observed. The spindle cells were observed at the peripheral area of the tumor or surrounded an island-shaped pattern area. They showed a bundle pattern and had an eosinophilic cytoplasm and a cigar-shaped nucleus, and mitotic figures were less frequently observed. The intermediate form cells were polygon to spindle in shape, their nuclei were small and oval and they were observed between the small round cell and the spindle cell areas. The boundary of these cells was not clear, and amount of fibrous matrix varied in these areas; however, there was a large amount of fibrous matrix in the sheet pattern area. Blastic round cells, which had scant amphophilic cytoplasm and more chromatin-rich, uniformly shaped nuclei, were also observed in a small portion of the tissue. They showed an island-shaped pattern, and the cell boundaries were clear. The tumor contained several blood vessels, and pseudorosettes consisting of tumor cells around blood vessels were found in the sheet pattern area (Fig. 2).

Fig. 1.

Lower magnification of the mass. Close to skin (a). Far from the skin (b). At the peripheral area of the tumor, the tumor cells proliferated solid. There was an increasing tendency for number and size of blood vessels toward the central area of the tumor. The central part of the mass was necrotic. a, b: HE staining. Bars=1 mm (a, b).

Fig. 2.

Histological findings of the mass. The mass showed 5 different proliferative patterns. The trabecular pattern area consists of small round cells (a), and thick argentophilic fibers were observed between the trabecular (e). In the sheet pattern area, frequent mitotic figures (b) and small round cells that had budding shapes (arrowheads) and/or small nuclei were seen (b inset). Also thick or thin argentophilic fibers were prominent in the sheet pattern (f). Round cells also formed pericytoma-like pattern (c) and island-shaped proliferative areas (in the center of d) with a few argentophilic fibers (g, h), and the surrounding adjacent area showed a bundle pattern of spindle cells (d). a–d: HE staining. e–h: Watanabe’s silver impregnation. Bars=50 μm (a–h).

Histochemically, thick argentophilic fibers were observed between the cells in the trabecular and pericytoma-like pattern areas, and thin fibers were observed between the cells in the sheet and bundle pattern areas using Watanabe’s silver impregnation (Fig. 2). A generous amount of collagenous fibers was observed between the cells in the sheet and bundle pattern areas using Masson’s trichrome staining (Fig. 3). All tumor cells were Grimelius negative. Large oval cells that had PAS-positive granules in their rich cytoplasms were occasionally seen in the sheet pattern area.

Fig. 3.

Fig. 3. Histological findings (Masson’s trichrome staining). a) A generous amount of collagenous fibers was observed between the cells in the sheet pattern area. b) A small amount of collagenous fibers was observed between the cells in the island-shaped pattern area. Bars=50 μm.

Most of the tumor cells in the trabecular, sheet, bundle and pericytoma-like patterns and invaded area were positive for PCNA and vimentin. The small round cells in the trabecular area were more strongly positive for vimentin than in other areas. However, the blastic round cells in the island-shaped pattern area were negative for vimentin. S-100 protein produced a positive reaction for a few neoplastic cells in the trabecular pattern area. Few cytokeratin AE1/AE3-positive cells were noted in the sheet, pericytoma-like and bundle pattern area. In these cells, cytokeratin AE1/AE3 gave a characteristic dot-like positive result as a juxtanuclear globe, and coexpression with vimentin was comfirmed by double immunostaining. The positive reaction for cytokeratin MNF116 was similar to the result for cytokeratin AE1/AE3. No positive reaction was observed for the other immunostains, including those for CD31, CD99, chromogranin A, cytokeratin 20, LCA, melanoma PNL2, neurofilament, NSE, P63, synaptophysin, von Willebrand factor and α-SMA (Fig. 4, Table 1).

Fig. 4.

Immunostaining for vimentin (a) and double immunostaining for pan-cytokeratin and vimentin (b, c). a) Most of the tumor cells were positive for vimentin, whereas the cells in the island-shaped pattern area were negative. b, c) In the tumor cells in the sheet, pericytoma-like and bundle pattern areas, cytokeratin AE1/AE3 (red) gave a characteristic positive result as a juxtanuclear globe in the cytoplasm and coexpressed vimentin (brown). Bars=50 μm (a), 20 μm (b), 10 μm (c).

Table 1.

Immunohistochemical Results

An electron microscopic examination was performed to examine the cytoplasmic components. Few organelleswere observed in most of the tumor cells in the trabecular, sheet, pericytoma-like and bundle pattern areas. The mitochondria were occasionally surrounded by dilated rough endoplasmic reticula. Thin filaments and free ribosomes were also noted. Few cells in the sheet pattern area had globular filamentous bodies (Fig. 5b). The nuclei of the round cells in the sheet pattern area were round to oval or irregularly shaped, and prominent nuclei and micronuclei were sometimes observed. Thin fibers were observed between the cells. No cell junction was observed in any portion of this tumor. The Island-shaped pattern area was not found by electron microscopic examination (Fig. 5).

Fig. 5.

Ultrastructural findings. Most of the tumor cells had few organelles. a) Protruded nuclei (arrowhead) and micronuclei were sometimes observed in the sheet pattern area. b) Globular filamentous bodies (arrowhead) were observed in the cytoplasm with low frequency. Bars = 4 μm (a), 2 μm (b).

In this case, a solitary large subcutaneous tumor was found spontaneously in a male GALAS rat. The tumor consisted of a multihued pattern of proliferating tumor cells. Most of the tumor cells were small, round or spindle shaped and positive for vimentin by immunostaining. Thin argentophilic fibers were wrapped around the individual cells, and a generous amount of collagenous fibers was observed between the cells in the sheet and bundle pattern areas. Therefore, this tumor would be of mesenchymal origin rather than an epithelial tumor. The neoplastic cells forming the island-shaped pattern area were considered to be poorly differentiated on the basis of their blastic blastic neuclear morphology and the negative staining for vimentin, cytokeratin AE1/AE3, S-100 and LCA. Cytokeratin AE1/AE3-positive juxtanuclear globes were observed as globular filamentous bodies by electron microscopic examination. Globular filamentous bodies consisting of cytokeratin have been reported in diverse neoplastic cells, including those in the neuroendocrine tumors like Merkel cell carcinoma. However, so far, there have been no reports on the coexpression of vimentin and cytokeratin in a juxtanuclear globe. Coexpression of both mesenchymal and epithelial intermediate filamentous protein markers was deciphered mesenchymal-epithelial transition because vimentin-positive cells made up the major portion, meanwhile cytokeratin positive cells were few and no cytokeratin single positive cells were observed.

Concerning the differential diagnosis, the negative result for Grimelius, chromogranin A, synaptophysin and NSE ruled out a neuroendocrine tumor. Amelanotic melanoma was excluded, although some cells were S-100 positive because no positive reaction for anti-melanoma PNL2 and no melanosome were observed. The histological finding that the tumor cells were arranged in a pericytoma-like pattern might be indicative of a hemangiopericytoma. However, as a pericytoma-like pattern can be found in various tumors, this pattern was not a major histologic pattern in the present case and tumor cells lack a basal lamina, the tumor could not be diagnosed as a hemangiopericytoma. Fibrosarcoma was also excluded from the tumor diagnosis, although a generous amount of collagenous fibers was observed between the cells in the sheet and bundle pattern areas; this is because the tumor showed a multihued pattern of proliferating tumor cells. Budding shaped nuclei and micronuclei were reported to indicate chromosomal instability, although this was not considered important enough for tumor diagnosis. In conclusion, the present case was diagnosed as a poorly differentiated mesenchymal tumor and classified as a sarcoma, NOS (not otherwise specified), due to lack of distinguishing characteristics to specify the origin of the tumor.