Spontaneous rhabdomyosarcomas in aged Mastomys ...

Spontaneous rhabdomyosarcomas in aged Mastomys (Praomys coucha) Hiroo Madarame, Yoshinori Kashimoto, Tsuyoshi Kawamoto, Toyonaga & Yoshihisa Hasegawa

Shinya

Department of Laboratory Animal Science, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan

Summary Two spontaneous tumours in the hind leg of 2 aged Mastomys were diagnosed as poorly differentiated rhabdomyosarcomas_ In both cases, the neoplastic cells had no evidence of cross-striation, but had a positive reaction for muscle-specific proteins. This is the second report of rhabdomyosarcomas in Mastomys, and the first described in detail. Keywords Praomys coucha (Mastomys); skeletal muscle; immunohistochemistry; rhabdomyosarcoma

Praomys coucha (Mastomys), formerly called Praomys (Mastomys) natalensis, are rodents of intermediate size between mice and rats. However, they differ in their disease pattern from other rodents. Mastomys are especially unique in respect to neoplasms, with a high percentage of lymphoepithelial thymomas, gastric carcinoids, parathyroid adenomas, and prostatic tumours, but an absence of tumours in the brain, lungs, and mammary glands ISolleveld et 01. 1982). Spontaneous rhabdomyosarcoma is one of the rarest neoplasms in mice, rats, hamsters, and Mastomys (Soga 1978, Faccini et a1. 1990, McDonald & Hamilton 1990, Carlton & Engelhardt 1991, Maekawa 1991). The only report of a rhabdomyosarcoma in Mastomys lacked a histopathological description (Oettle 1955). Recently we found 2 spontaneous rhabdomyosarcomas within the skeletal muscles of the leg. In this study, we describe the radiographic, histological, and immunohistochemical findings of the 2 naturally Correspondence to: Hiroo Madarame, Department of Laboratory Animal Science, School of Veterinary Medicine and Animal Sciences, Kitasato, University, Towada, Aomori 034, Japan Accepted 4 January 1995

tumour;

occurring rhabdomyosarcomas in aged Mastomys. To our knowledge, this is the first detailed report of rhabdomyosarcomas in Mastomys. Materials and methods Two untreated Mastomys were housed in a conventional animal room maintained at 22 ± 2°C and 60 ± 5% relative humidity with 12/12-h light/dark cycle. They had been fed a commercial pelleted diet (CL-2, CLEA, Japan) and tap water ad libitum. There was no known exposure to carcinogens. One male Mastomys weighing 84.82g died at 643 days ICase 1) while another male Mastomys weighing 90.91 g was sacrificed at 533 days (Case 2). In case I, only the right hind leg was X-rayed after postmortem examination, while in case 2, the whole body was radiographed before postmortem examination. For histological examinations, all tissue samples of primary tumours and metastases were fixed in 10% neutral buffered formalin lfor a maximum of 1 week). embedded routinely in paraffin, cut at 4 J.tm, and stained with haematoxylin and eosin (HE). Histochemically, selected sections of the Laboratory Animals (1995) 29, 464-469

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tumours and metastases were stained with periodic acid-Schiff(PASLMasson's trichrome, and phosphotungustic acid haematoxylin (PTAH). Serialsections used for histochemistry were also placed on silanized slides for immunohistochemical staining. Three different immunohistochemical stains were used on the paraffin embedded sections. Immunoreactivity to mouse monoclonal antibody against desmin, an intermediate filament in muscle tissue (mouse antihuman desmini DAKO D33) and mouse monoclonal antibody against myoglobin (mouse anti-human myoglobinj ZYMED ZOOl) were demonstrated using the labelled avidin-biotin method (LAB-SA)(Zymed's HISTOSTAIN-SP kit, Zymed Labs, CA, USA). Immunoreactivity to rabbit polyclonal antibody against myoglobin (rabbit antihuman myoglobinj DAKO)was demonstrated using the avidin-biotin-peroxidase complex (ABC)method (Histo Scan Kit, Biomeda, CA, USA). As a positive control, a desmin control slide (human leiomyosarcomaj DAKO) and a myoglobin control slide (human skeletal musclej DAKO)were stained simultaneously with the above specimens. Results

Gross appearance Case 1 The tumour presented subcutaneously as a large poorly demarcated smooth mass (3.5 x 3.8 x 2.3 em) in the subcutis and muscles of the right hind leg. The bones and joints were involved in the tumour mass without any macroscopical bone fractures. The cut surface of the tumour showed fragile tissue of various colours with several areas of necrosis. White and

firm nodules, the size of sesame seeds, were scattered throughout the lungs. Also a tumour mass (1.2x 1.3 x 1.0 em) was present between the rectum and the dorsal abdominal wall. Both iliac lymph nodes were unclear and seemed to be involved in this mass. Case 2 A firm raised mass (3.5x3.9x4.1 em), was located subcutaneously on the left hind leg

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between the thigh and the knee. It was easily dissected from the intact skin, but the border between the tumour mass and the normal muscle was obscure. On the cut surface, the centre of the tumour was haemorrhagic and necrotic. All lobes of the lungs contained the scattered grayish white nodules (2-3 mm). The left iliac lymph node was severely enlarged (0.075g) compared to the right iliac lymph node (0.008 g). Radiographic features In case 1, a partial radiolucent zone was observed in the distal femur. In case 2, the bones appeared normal and the metastatic foci in the lungs were not visible radiographically. Histopathology Case 1 In the primary lesion of the hind leg, the tumour exhibited no particular growth pattern and the stroma of the tumour was scanty. The margins were indistinct and neoplastic cells had infiltrated into the adjacent normal muscles and bone. In the central part of the tumour mass, necrosis and haemorrhage were fairly common. The tumour cells consisted mainly of round, oval or fusiform cells with a single darkly staining nucleus, strongly eosinophilic cytoplasm, and a distinct cell border. The nuclei had coarse chromatin with indistinct nucleoli and varied in size, shape and location. There was considerable mitotic activity. Uninucleated and multinucleated giant cells were scattered, and the nuclei in the latter were not arranged in a row (Fig. 1). The cytoplasm of each tumour cell type was abundant and deeply eosinophilic, but acceptable cross-striation was not observed even with the PTAH stain. In the metastatic foci from the lungs and the abdomen, the main type of tumour cell was mononuclear, similar to that of the primary lesion. In the lung, tumour emboli in the arterial branches were apparent. Case 2 In the left hind leg, the border between the tumour mass and normal muscles was not clear, and the neoplastic cells were intermixed

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round to oval to spindle shaped with considerable eosinophilic cytoplasm, and one or two nuclei (Type II). The third type was a multinucleated giant cell with bizarre nuclei and abundant eosinophilic cytoplasm without cross-striations. The nuclei of these cells were mostly distributed randomly around the cytoplasm and rarely arranged in tandem. These giant cells were scattered between the other 2 types of tumour cells, and sometimes aggregated small cell nests (Type ill) (Fig. 2). Histochemically, only a few tumour cells had PAS-positive granules in the cytoplasm, but none had crossstriations even when stained by PTAH stain. The left iliac lymph node was completely replaced by type I and II tumour cells, and some tumour cells had invadedthe surrounding adipose tissue. The right one had a normalappearing lymph node structure. In the lungs there were many tumour emboli in the arteries and disseminated metastatic foci in the alveoli. Some tumour

Fig 1 Rhabdomyosarcoma, hind leg, case 1. Uninucleated or multinucleated giant cells with

bizarre nuclei and abundant eosinophilic cytoplasm. HE. Line represents 30/Lm

with the normal muscle cells peripherally. Necrosis and haemorrhage were fairly common in the primary tumour. However, in the metastatic foci in the lung, and the left iliac lymph node, only some small necrotic areas were detectable microscopically. A considerable variation in cell types and cellularity was present in different fields of the primary tumour. Most of the tumour cells were distributed randomly without any specific cellular arrangements. However, in some parts the tumour cells showed a lacework pattern separated by anastomosing vascular channels. The tumour cells were divided into 3 principal types. The first type was a relatively small round mononucleated cell with a small rim of pale or faintly amphophilic cytoplasm, and indistinct cell borders. In this type of cell, the nucleus/ cytoplasm ratio and the mitotic ratio were high (Type I). The second type of tumour cell varied from

Fig 2 Rhabdomyosarcoma, hind leg, case 2. The multinucleated giant cells (Type III). HE. Line represents 30 /Lm

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cells were also detected on the pleura. These metastatic foci were composed of only type I and ITcells. Immunohistochemistry Mouse anti-human desmin monoclonal antibody In case I, desmin-positive reaction was observed in some tumour cells in both the primary tumour and metastatic foci. In the positive cells, there were many fine granules in the cytoplasm. . In case 2, the positive reaction was detected diffusely in the cytoplasm of type ITand ill cells, but type I cells did not have a positive reaction in the primary tumour and metastases (Fig. 3). Rabbit anti-human myoglobin polyclonal antibody In case I, there was a positive reaction in only a few cells of the primary tumour but no positive cells in the metastatic foci. In case 2, the positive reaction was detected uniformly in the cytoplasm of Fig 4 Rhabdomyosarcoma, hind leg, case 2. Myoglobin-positive substance in type II and III cells. Immunostaining of myoglobin. Mayer's haematoxylin counterstain. Line represents 20 ILm

some type ITand ill cells in the primary tumour and the metastases (Fig. 4). Mouse anti-human myoglobin monoclonal antibody No myoglobin-positive reaction was observed by monoclonal antibody against myoglobin in both case 1 and 2.

Discussion

Fig 3 Rhabdomyosarcoma, hind leg, case 2. Desmin-positive reaction in type" and '" cells. Immunostaining of desmin. Mayer's haematoxylin counterstain. Line represents 20 ILm

There are 2 convincing histopathological criteria for the diagnosis of rhabdomyosarcoma in rodents: the first one is the demonstration of the cross striation in the tumour cells by some special stains, and another is the identification of the muscle specific proteins by immunohistochemistry (Faccini et aI. 1990, McDonald &. Hamilton 1990, Maekawa 1991, Carlton et aI. 1992). Tumour cells with cross striations are sufficient evidence of rhabdomyosarcoma, but they are extremely rare and sometimes difficult to find (McDonald &. Hamilton

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1990, Carlton & Engelhardt 1991), as with our 2 cases. On the other hand, immunohistochemistry is also an important clue for the differential diagnosis of rhabdomyosarcomas in rodents: most rhabdomyosarcomas have a positive reaction against myoglobin, actin, and desmin (Carlton et al. 1992.). Above all, myoglobin is a specific marker for the rhabdomyosarcomas of mouse and rat origin (Takahashi et al. 1986, Takahashi et al. 1988, McDonald & Hamilton 1990), and desmin is also an excellent diagnostic marker for rat rhabdomyosarcomas induced by Ni3S2 (Altmannsberger et al. 1985). In this study, both tumours showed a positive reaction for the mouse anti-human desmin monoclonal antibody and for the rabbit anti-human myoglobin polyclonal antibody. However, they did not react positively to the mouse anti-human myoglobin monoclonal antibody. The rabbit polyclonal antibody against human myoglobin has been proved to be a diagnostic aid for identification of rodent rhabdomyosarcomas [Takahashi et al. 1986, Germann et al. 1994), although the antigenicity of rodent myoglobin is not the same as that of human myoglobin (Kagen & Gurevich 1967J. The mouse monoclonal antibody against human myoglobin may react more specifically with human muscles than rabbit anti-human myoglobin polyclonal antibody. Therefore, the tumour cells in these 2 cases may have contradictory results between the monoclonal and polyclonal antibody against human myglobin. In rats, Yamashiro et al. (1980) separated the experimentally induced rhabdomyosarcomas into three subtypes based on their histopathological and cytological characteristics: well differentiated, poorly differentiated, and embryonal. The former 2 subtypes are similar, but represent a different level of differentiation; the ratio of the strap-like multinucleated cells in the tumour components is an important factor for subtyping of rhabdomyosarcomas. The subclassifying term 'embryonal' is based on the resemblance to its human counterpart, e.g. myxoid or mucus-producing tumour areas mixed with the compact tumour areas [Yamashiro et ai. 1980). In the present 2 cases, the strap-like

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multinucleated cell was not a major component of both tumours, and there were no myxoid or mucous-secreting tumour areas. Therefore, on the basis of this classification, the present 2 cases were diagnosed as poorly differentiated rhabdomyosarcomas. Spontaneous rhabdomyosascomas are very rare in laboratory rodents (Faccini et al. 1990, McDonald & Hamilton 1990, Carlton & Engelhardt 1991, Maekawa 1991). Although the incidence of rhabdomyosarcomas has varied with the study and strain of rodents, it is less than 0.5% (McDonald & Hamilton 1990, Carlton & Engelhardt 1991). A systematic survey of a total of 14991 males and females from several strains of mice and rats revealed 24 tumours of various organ systems as rhabdomyosarcomas, including primary, primary site not determined, and metastatic tumours [Hoar et al. 1985), while data from 92 male and 91 female Syrian hamsters showed no primary or secondary tumours of the skeletal muscle (Schmidt et al. . 1983J. Only a few case reports are available in any rodent species (Carlton & Engelhardt 1991, Maekawa 1991). Mastomys has frequently been used as an animal model for several tumours (Soga 1978). Soga reported 3 relatively rare tumours in the leg (chondromyxoid tumour, fibromatous tumour, and ganglioneuroma); however, no rhabdomyosarcomas were described [Soga 1978). Solleveld et al. (1982) also reported pleomorphic sarcomas in the musculature of the legs in aged Mastomys [2 in 57 males, and 2 in 84 females), although none of these were recognized as rhabdomyosarcomas (Solleveld et al. 19821. In Mastomys, the only reported rhabdomyosarcoma case occurred in the ear region, but there were no histopathological descriptions (Oettle 1955). Our report is the first to describe rhabdomyosarcoma in Mastomys in detail.

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MacKenzie WF, eds). San Diego, USA: Academic Press, pp 193-207 Oettle AG (1955) Spontaneous carcinoma of the glandular stomach in a laboratory stock of Rattus (Mastomys) natalensis. The South African Journal of Medical Sciences 20, 36 Schmidt RE, Eason RL, Hubbard GB, Young TT, Eisenbrandt DL (1983) Musculo-skeletal system. In: Pathology of Aging Syrian Hamsters. Boca Raton, VSA: CRC Press, pp 175-82 Soga J (1978) Relatively rare tumors in other organs and tissues. In: Praomys (Mastomys) natalensis: The Significance of Their Tumors and Diseases for Cancer Research (Soga J, Sato H, eds). Niigata, Japan: The Daiichi Printing, pp 127-36 Solleveld HA, van Zwieten MJ, Zurcher C, Hollander CF (1982) A histopathological survey of aged Praomys (Mastomys) natalensis. Journal of Gerontology 37, 656-65 Takahashi K, Maiata K, Shirasu Y (1986) Combined ultrastructural and immunohistochemical studies on a mouse rhabdomyosarcoma. The Japanese Journal of Veterinary Science 48, 473-79 Takahashi K, Maita K, Shirasu Y, Taniguchi H, Yoshikawa Y (1988) Anti-rat myoglobin antisera in the immunocytochemical diagnosis of rhabdomyosarcomas of rats. Veterinary Pathology 25, 337-42 Yamashiro S, Gilman JPW, Hulland TJ, Abandowitz HM (1980) Nickel sulphide-induced rhabdomyosarcomata in rats. Acta Pathologica Japonica 30, 9-22