Extraskeletal Osteosarcoma with Cystic Appearance in an Aged Sprague–Dawley Rat

Abstract

Extraskeletal osteosarcoma, arising spontaneously from the subcutis of the left abdomen and having a cystic appearance, was found in an untreated male Sprague–Dawley rat during a carcinogenicity study. At 76 weeks of age, the tumor mass had grown to 50 × 110 × 140 mm, and the animal exhibited severe anemia related to the complication of ulceration with hemorrhage. The tumor displayed irregular ossification at the walls of a cyst-like space filled with much yellowish fluid and necrotic cellular debris. Histopathologically, the tumor consisted of sheets of large, plump osteoblast-like cells, which produced broad irregular trabeculae of osteoid and calcified osseous tissue. Since extraskeletal osteosarcoma with a cystic appearance, has not been reported in animals, except for the telangiectatic type, our case shows an extremely rare type.

Case Report

Osteosarcoma is the most common primary malignant bone tumor in man with a poor prognosis, rapid growth, and frequent distant metastasis, especially to the lung usually through the blood stream (Yoshida et al., 1989; Enzinger and Weiss, 1995). The frequency of spontaneous development is generally low in laboratory animals, and extraskeletal osteogenic tumors occur more rarely in dogs, rats (Minato et al., 1988; Greaves and Barsoum, 1990; Litvinov and Soloviev, 1990; Mii et al., 1991; Woodard, 1997), and humans in whom only 1–2% of all soft tissue sarcomas and approximately 2–4% of all osteosarcomas occur (Rosenberg and Heim, 2002). Even in the NTP historical data of F344 control rats, the incidences of all osteosarcomas in the musculoskeletal system, including bone, were 24/3885 in males (0.6%) and 12/3933 (0.3%) in females (Haseman et al., 1990).

Our case was a male rat of the Jcl:SD(CD) strain, one of the untreated group in a carcinogenicity study for safety assessment conducted in the Toxicology Research Laboratories of Fujisawa Pharmaceutical Co., Ltd. (present name: Astellas Pharma, Inc). The animal, housed in a polycarbonate cage with woody bedding under barrier conditions of 23 ± 2°C room temperature, 55 ± 5% relative humidity, and a 12 hour light-dark cycle, had been given the standard radiation-sterilized laboratory diet (CA-1, CLEA Japan, Inc., Tokyo) and chlorinated tap water ad libitum. Adequate consideration was given to avoid unnecessary anxiety or pain to the animal. All animal care and procedures were performed in accordance with the ”Guideline for Animal Experimentation” (Japanese Association for Laboratory Science, 1987).

The animal was routinely observed for clinical signs twice a day, was weighed once a week during the study period, and grew normally until 1 year of age when its body weight was 791 g. The tumor had been detected at 58 weeks of age, measuring approximately 5 mm in diameter at the subcutis of the left abdomen. The animal wasted gradually to 683 g at 72 weeks of age with decrease of food consumption, exhibiting paralysis of the left hind limb, hypolocomotion, and dyspnea gasping. At 76 weeks of age, the tumor had attained a size of 50 × 110 × 140 mm, and the animal, displaying severe anemia related to the hemorrhaged ulceration complicated with this mass, was euthanized and necropsied carefully. Over 40 of the systemic organs including this tumor were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for routine histopathologic examinations. Roentgenographs of the mass were taken after fixation using the Rotanode type DRX80A (Toshiba, Japan). Some of the sliced tissues of the mass were treated with a 10% formic acid-formalin solution for decalcification.

Additional cross-sections of the tumor mass were stained with Periodic Acid-Schiff, reticulin silver impregnation, and phosphotungstic acid hematoxylin and immunostained using a labelled-streptavidin-biotin method (LSAB Universal Kit, Dako, Glostrup, Denmark), anti-swine vimentin (clone: V9, 1:40, Dako), and anti-keratin/cytokeratin (clone: AE1, AE3, prediluted, Nichirei, Tokyo, Japan) antibodies. For ultrastructural observations, some of the formalin-fixed tumor tissues were cut into small pieces, fixed secondarily in 2.5% glutaraldehyde and 2% osmium tetroxide, embedded in epoxy resin, sectioned, and stained on-grid with uranyl acetate and lead citrate.

Clinicopathological analyses revealed mainly severe anemia (red blood cell: 587 × 106/μL, hemoglobin: 10.8 g/dl, hematocrit: 34.8%), neutrophilia (white blood cell: 32.72 × 10³/μL, segmented neutrophil: 51.5%), and thrombocythemia (1490 × 10³/μL), as well as mild hypocalcemia (9.5 mg/dL). Macroscopical examination showed the well-circumscribed mass occupying the region from the left abdomen to the leg (Figure 1a) but not connected with any skeletal tissues including bone. In tumor sections, the characteristic cyst-like space, divided irregularly by ossified septa, was filled with yellowish fibrinoid fluid. Complicated separation of the tumor by irregularly ossified septa was confirmed by radiography (Figure 1b). Light microscopy revealed that the tumor was located in the subcutis of the left abdomen and leg (Figure 2 and 3a), separated from the surrounding muscle tissue by a zone of loose, myxoid fibrous tissue, and invading neither femoral muscles nor bone. The tumorous cells were characterized by round and spindle-type cells, both of which contained clear nuclei with scanty chromatin and one or two large nucleoli. The walls and separations of the tumor consisted of sheets of plump osteoblast-like cells, which produced broad irregular trabeculae of osteoid and calcifed osseous tissue at the margin of the tumorous mass (Figure 3b). The large pleomorphic cells with cytoplasmic eosinophilic granules were interspersed among a population of smaller spindle cells (Figure 3c).

These tumorous cells were individually surrounded by reticulin fibers but stained by neither PAS nor PTAH. Immunohistochemically, most tumorous cells displayed a positive reaction for anti-vimentin antibody, but not anti-cytokeratin antibody. The central cystic space of this tumor contained necrotic tissue. Mitotic figures and hemorrhagic changes were frequently disseminated. Electron microscopic evaluation revealed that the tumorous cells were bounded by irregular plasma membranes and contained scattered mitochondria, well-developed dilated rough endoplasmic reticulum, Golgi apparati, cytoplasmic vacuoles, and clusters of free ribosomes (Figure 4a). Prominent dilated rough endoplasmic reticulum was often filled with fine granular proteinaceous materials (Figure 4b). Tumorous cells produced abundant fibrillar matrices with collagenous bundles, within which were deposited highly electron-dense bodies like hydroxyapatite (Figure 4c). Occasionally, neoplastic cells exhibited intercellular tight junctions or zonulae occludens. In the other organs and tissues, careful pathological observations disclosed brochiolar/alveolar adenoma, systemic lymphoid atrophy of the thymus and mesenteric and mandibular lymph nodes, and increased hematopiesis in the femoral and sternal bone marrow. No distant metastases of the tumor were observed in any of the organs examined.

The characteristic production of bone matrix by this tumor corresponds well to that described in osteosarcoma (Yoshida et al., 1989; Greaves and Barsoum, 1990; Nojima et al., 1992; Witzel et al., 1992; Enzinger and Weiss, 1995). Moreover, our case showed a multicystic appearance with a great deal of necrotic cellular debris. In general, osteosarcoma exhibits several morphologic patterns in rats: osteoplastic, fibroblastic, osteoblastic, telangiectatic, and compound (Leininger and Riley, 1990). Among these types, telangiectatic osteosarcoma develops cystic structures in humans (Huvos et al., 1982; Spina et al., 1998; Park and Joo, 2001; Matsuno et al., 2002; Murphey et al., 2003; Sirikulchayanonta and Jaovisidha, 2005) and animals (Leininger and Riley, 1990; Pool, 1990; Liu and Thacher, 1991; Woodard, 1997; Brellou et al., 2004). This type of tumor in humans is characterized by multicystic spaces containing blood, lined with membranous septa and giant cells without any endothelial lining; the permeation of the tumor occurs between preexisting bony trabeculae lined by giant cells (Park and Joo, 2001; Matsuo et al., 2002; Murphey et al., 2003; Sirikulchayanonta and Jaovisidha, 2005). The telangiectatic type occurs commonly in vertebrae in rats (Leininger and Riley, 1990). In our case, cyst-like spaces were filled with many necrotic cellular debris and not lined by giant tumorous cells. Our case, therefore, most likely should not be diagnosed as the telangiectatic type.

Based on the anatomical location of the tumor and its lack of connection to the leg bone, our case likely originated from extraskeletal mesenchymal cells. This neoplasm might, however, differ from undifferentiated sarcoma with cystic appearances and malignant mesenchyoma with its osteoid formation and other mesenchymal components (Greaves and Barsoum, 1990; Litvinov and Soloviev, 1990; Mii et al., 1991). In the human, myositis ossificans, a benign osseous cystic tumor, is reported to exhibit malignant transformation (Enzinger and Weiss, 1995). Myositis ossificans is characterized by the presence of immature cellular areas in the center and more mature, ossifying areas at the periphery. Osteosarcoma, however, displays a more disorderly or haphazard growth pattern, often consisting of a complex mixture of differentiated and undifferentiated cellular areas with no subsidence of growth and infiltration into neighboring structures in a destructive manner (Enzinger and Weiss, 1995). Our case can be distinguished from myositis ossificans by a complex cellular mixture and a noninfiltrative growth pattern. Although osteosarcoma presents less commonly as a multicystic mass in laboratory animals (Ruben and Rohrbacher, 1986; Minato et al., 1988; Wadsworth, 1989; Patnaik, 1990), the present tumor might be related to multicentric necrosis due to blood-flow disturbance followed by development of multicystic figures.