An Undifferentiated Sarcoma in a Rat Resembling Extraskeletal Myxoid Chondrosarcoma in Man

Abstract

A soft tissue tumor occurring in the inguinal subcutaneous tissue was detected in a 109-week-old male F344 rat. Macroscopically, the tumor mass showed no skeletal relationship and a gelatinous multinodular appearance. Histologically, the tumor consisted of irregular lobules separated by scant fibrous septa. In each lobule, tumor cells were arranged in cords and strands in the plentiful myxoid stroma. The tumor cells showed marked pleomorphism, and had large, round to ovoid nuclei and abundant eosinophilic cytoplasm. Mitotic figures were often seen. Myxoid stroma of the tumor contained a large amount of acid mucopolysaccharides and collagen type II, which were demonstrated by histochemistry and immunohistochemistry respectively. The tumor cells showed positive immunoreactivities for vimentin and S100 protein. Ultrastructural examination revealed that the tumor cells had a large amount of mitochondria, Golgi complex and dilated rough endoplasmic reticulum containing amorphous material, and the myxoid stroma contained collagenous fibrils and proteoglycan particles. Based on these results, the present tumor in a rat resembled extraskeletal myxoid chondrosarcoma in man.

Introduction

Extraskeletal chondrosarcomas are rare malignant neoplasms arising from the soft tissue unrelated to synovium, cartilage or bone (Enzinger and Shiraki, 1972). In human beings, extraskeletal chondrosarcomas have been classified histologically into two subtypes, mesenchymal and myxoid types (Kempson et al., 2001). Most extraskeletal myxoid chondrosarcomas have been reported to show no chondroid tissue formation (Antonescu et al., 1998; Aigner et al., 2004). On the other hand, extraskeletal myxoid chondrosarcoma is uncommon in animals, and to our knowledge, only one case was reported in a cow (Yamamoto et al., 2005). The present report describes the gross, histological, histochemical, immunohistochemical, and ultrastructural characteristics of an undifferentiated sarcoma in an aged male F344 rat, which were consistent with those of human extraskeletal myxoid chondrosarcomas.

Case Report

Five-week-old F344 rats (70 males and 70 females) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). These rats were maintained to collect background data for 104-week carcinogenisity studies, therefore the animals did not receive any treatment while alive. They were housed 2 per cage in wire mesh cages, and maintained in a barrier system animal room with controlled temperature (20–25°C), humidity (30–70%), and light environment (light: 12 hr, dark: 12hr). They had free access to the standard diet for rats (MF, Oriental Yeast Inc., Tokyo, Japan) and tap water, and were cared for and treated humanely during the experiments in accordance with the Guidelines for Care and Use of Laboratory Animals at the Nippon Institute for Biological Science (1999). The present case was euthanatized under deep anesthesia with ether and subjected to a complete necropsy at the end of 104-week study.

Macroscopically, a reddish and softened mass (55 × 45 × 30 mm in size) was observed in a right inguinal subcutaneous portion. At the cut surface, the mass showed a gelatinous and irregularly multinodular appearance with several hemorrhagic foci. The mass did not involve the underlying bones or joints in the inguinal portion.

The mass and the major organs were fixed in 10% neutral buffered formalin. The specimens from the mass and other organs were embedded in paraffin, sectioned 4 μm thick, and stained with HE. The selected sections of the mass were stained with AB-PAS, AB with or without predigestion by hyaluronidase, and colloidal iron. Additional sections of the mass were stained immunohistochemically by the labeled polymer method (Histofine Simple Stain MAX-PO(G) and Rat MAX-PO(MULTI), Nichirei Biosciences, Inc., Tokyo, Japan) with the antibodies for cytokeratin, vimentin, myoglobin, α-SMA, neurofilament, MBP, S100 protein, GFAP, lysozyme, ED1, vWF and CD34, and the streptoavidin-biotin complex technique (Histofine SAB-PO(M) and PO(R) Kit, Nichirei Biosciences, Inc.) with the antibodies for collagen type I, II, III, VI and X. Characteristics (clone, dilution, and products) of the antibodies used and pretreatments for antigen retrieval were shown in Table 1. The sections were then counterstained with hematoxylin. To confirm the specificity of antibodies, normal tissue sections were used for positive controls, and the sections in which the primary antibodies were replaced by PBS (pH 7.2) were used for negative controls. For electron microscopy, small pieces from the formalin-fixed tumor tissue were rinsed in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide solution, and then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined by a Hitachi H-600L transmission electron microscope.

Histologically, the tumor formed irregularly lobulated structures which were separated by scant fibrous connective tissues and showed peripheral hypercellularity (Figure 1). The tumor cells were arranged in cords and strands, and surrounded by a large amount of myxoid stroma (Figure 2). No chondroid tissues were detected in the tumor. The tumor cells showed marked pleomorphism in size and a variety in shape, such as spindle, round, polygonal or stellate. The nuclei of the tumor cells were large, round to ovoid with obvious nucleoli. A large amount of eosinophilic cytoplasm included hyaline inclusions. Mitotic figures were frequently seen. Multinucleated atypical giant cells were occasionally observed (Figure 3).

Immunohistochemically, most of the tumor cells showed positive reactivities for vimentin and S100 protein (Figure 4) but no reativities for cytokeratin, myoglobin, α-SMA, neurofilament, MBP, GFAP, lysozyme, ED1, vWF and CD34.

The myxoid stroma of the tumor was strongly stained with AB and colloidal iron but not with PAS. The staining reaction for AB was reduced after hyaluronidase digestion. In the immunohistochemical examinations, collagenous fibers contained in the myxoid stroma showed focal positive reactivity for collagen type II (Figure 5) and diffuse positive reactivities for collagen type I, type III and type VI. No immunoreactivity for collagen type X was observed in the stroma.

Ultrastructurally, the tumor cells attached occasionally together with immature cell junctions. The nuclei of the tumor cells were round to ovoid with smooth nuclear membrane (Figure 6) and an obvious nucleolus. The cytoplasm contained large numbers of oval mitochondria, dilated rough endoplasmic reticulum (RER), and well developed Golgi complex (Figures 6 and 7). Dilated RER contained amorphous materials (Figure 7). Free surface of the tumor cells showed a various number of short cytoplasmic projections (Figures 6 and 7). Collagenous fibrils (Figure 8) and dense granular materials were seen in the myxoid stroma.

Histological characteristics, such as irregularly lobulated structures separated by scant fibrous septa, cord and strand arrangements, abundant eosinophilic cytoplasm of the tumor cells in the lobules, and a large amount of myxoid stroma commonly seen in the present tumor were described in extraskeletal myxoid chondrosarcomas in human beings (Enzinger and Shiraki, 1972; Fletcher et al., 1986; Antonescu et al., 1998; Harris et al., 2000; Kempson et al., 2001). The absence of chondroid tissue formation in the present tumor has also been described as one of characteristics of extraskeletal myxoid chondrosarcaoma in human beings (Enzinger and Shiraki, 1972; Antonescu et al., 1998; Kempson et al., 2001; Aigner et al., 2004). Marked pleomorphism and high mitotic activity of the tumor cells in the present case imply malignant potency of the tumor. These malignant features have been reported in human high-grade extraskeletal myxoid chondrosarcomas (Lucas et al., 1999).

Ultrastructures of the tumor cells such as large numbers of mitochondria, dilated RER containing amorphous material and well-developed Golgi complex in their cytoplasm, and short cytoplasmic projections and immature cell junctions at the cell surface, as well as positive immunoreactivities for vimentin and S100 protein in the present case are suggestive of chondroblastic properties of the tumor cells, coinciding with human extraskeletal myxoid chondrosarcomas (Antonescu et al., 1998; Kempson et al., 2001).

Histochemical examinations revealed that the myxoid stroma of the present tumor contained a large amount of acid mucopolysaccharides, as consistent with human extraskeletal myxoid chondrosarcomas (Enzinger and Shiraki, 1972; Fletcher et al., 1986; Harris et al., 2000; Kempson et al., 2001; Aigner et al., 2004). The acid mucopolysaccharides of the myxoid stroma were digested by hyaluronidase, suggesting the content of hyaluronic acid. According to the previous reports of human cases, there is a wide spectrum in sensitivities of the myxoid stroma to hyaluronidase digestion (Kempson et al., 2001). Collagenous fibrils and dense granular materials, i.e., proteoglycan particles, in the myxoid stroma are known as ultrastructural characteristics of the cartilage matrix. In addition, collagenous materials in the myxoid stroma showed positive immunoreacivity for collagen type II, but negative for collagen type X. It is well known that cartilage is unique in containing collagen type II. These results suggest that the present tumor would show an immature differentiation toward cartilage as described in human extraskeletal myxoid chondrosarcomas (Harris et al., 2000; Aigner et al., 2004). The presence of collagen type I, III and VI in the myxoid stroma of the present case indicates that the tumor possesses a primitive mesenchymal phenotype profile as shown in human cases (Aigner et al., 2004).

Myxoid variants of chondrosarcoma essentially show a skeletal relationship and a content of collagen type X (Kempson et al., 2001) missing in the present tumor. Myxoid liposarcoma represents a prominent plexiform, i.e., chicken wire, vascular pattern and lipoblasts/lipocytes in the tumor tissue (Kempson et al., 2001). However, the present tumor missed these histological features. The facts that the tumor cells showed a positive immunoreactivity for S100 protein, and negative for ED1 and lysozyme can differentiate the present tumor from the myxoid type of malignant fibrous histiocytoma. No immunoreactivity for myoglobin or no myofilaments in the cytoplasm of the tumor cells revealed that the present tumor would not be derived from muscular tissues. The present tumor showed a negative immunoreactivity for GFAP and detected ultrastructurally no external basal lamina around the tumor cells, being differentiated from malignant Schwannoma.

The present tumor detected in an aged male F344 rat has characterized by histological consistence with human extraskeletal myxoid chondrosarcoma, simultaneous positive immunoreactivities for collagen type II, S100 protein and vimentin, and ultrastructures such as microvillus-like cytoplasmic protrusions. Based on these features, we concluded that the present undifferentiated sarcoma in a rat corresponded to extraskeletal myxoid chondrosarcoma in man.