Spontaneous Rhabdomyosarcoma in a Young Sprague-Dawley Rat

Abstract

An eight-week-old male Sprague-Dawley (SD) rat showed signs of emaciation, and masses were found in the subcutis around the cervical and thoracic regions. At necropsy, a multilobular mass, 2.2 x 1.8 x 2.0 cm in size, had grown from the left neck into the thoracic cavity. On a cutting surface, masses were firm and whitish to tan, with necrotic and hemorrhagic plaques. Microscopically, masses were composed of multiple nodules of tumor cells that were incompletely encapsulated with fibrous connective tissue. The tumor cells exhibited varied forms, from spindle to globoid shapes with minimal to abundant eosinophilic cytoplasm, and appeared as large, multinucleated cells; many of the tumor cells were vacuolated. Histochemistry results revealed that tumor cells exhibited some cross-striation in the cytoplasm using PTAH staining. There were some multinuclear tumor cells with vacuoles located around the nuclei, and these vacuoles showed reddish staining by the periodic acid Schiff (PAS) method. Immunohistochemical staining also expressed intense granular cytoplasmic staining for desmin and myoglobin, with highly positive staining for PCNA, whereas α-smooth muscle actin (α-SMA) was negative. Based on the pathology results, a spontaneous rhabdomyosarcoma with pleomorphic type was diagnosed in a young SD rat.

Introduction

Rhabdomyosarcomas as malignant tumors of striated muscle are generally classified as embryonal, botryoid, alveolar, and pleomorphic types. The accurate classification of these tumors in humans is prognostically important. The best outcomes are with the botryoid type, and the worst with the alveolar type (Cooper and Valentine 2002). Spontaneous rhabdomyomas and rhabdomyosarcomas are rare in humans and in laboratory rats. Histologically, they are composed of primitive muscle fibers, or myotubes (Conner 1994). Adult rhabdomyoma predominantly occurs in elderly persons (median age about sixty years) with a male predilection (3:1 male-to-female ratio) (Cronin et al. 2000). The head and neck area is by far the most common site. These neoplasms occur mainly in the subcutaneous tissue within the head and neck region, because they arise from the musculature of the third and fourth branchial arches (Sørensen et al. 2006).

Fetal rhabdomyoma is even rarer than adult rhabdomyoma in humans. These two types of tumors have been classified as cardiac and extracardiac rhabdomyomas. A case of a newborn with cardiac rhabodomyoma was recently reported by Karnak et al. (2007). The diagnosis of rhabdomyoma can be suggested by modern methods such as computerized tomography, magnetic resonance imaging, and echocardiography. In this case, histological examination revealed that the tumor was composed of large, vacuolated cells, some of which were showing a spindle cell appearance, which is consistent with rhabodomyoma (Karnak et al. 2007).

In laboratory rats, rhabdomyosarcoma occurrence seems to be related to strain and age. The higher incidence of rhabdomyosarcoma in older rats is in contrast to the distribution of rhabdomyosarcoma in other species (Conner 1994). In the present study, we examined a rare rhabdomyosarcoma that occurred in the neck and thoracic areas in a young Sprague-Dawley (SD) rat by using histology and histochemical and immunohistochemical staining methods.

Materials and Methods

Forty male SD rats, four weeks old, were purchased from the National Laboratory Animal Center, Taipei, Taiwan. They were kept in suspended stainless steel wire mesh cages (two per cage) and were supplied with a standard nonpurified diet (Lab Diet 5001 Rodent diet, PMI Nutrition International, LLC, St. Louis, MO, USA) and ion reverse water (Milipore, Billerica, MA, USA) ad libitum in an environmentally controlled room (temperature 22°C–25°C, relative humidity 50%–70%, and on a twelve-hour light–dark cycle). All animals received humane care according to the guidelines of Guidebook for the Care and Use of Laboratory Animals (Yu et al. 2004).

Before treatment, a male eight-week-old SD rat showed signs of emaciation, and large subcutaneous masses were palpable around the neck and thoracic areas. The subject animal was euthanized, and a complete necropsy was performed. The masses were fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E) stain. Histochemical examination was performed according to Masson Trichrome staining (MT), phosphotungstic acid-hematoxylin staining (PTAH), and periodic acid Schiff staining (PAS) methods. Immunohistochemistry was performed on deparaffinized samples using antibodies of desmin (1:4, Signet, Schaffhausen, Switzerland), myoglobin (rabbit polyclone, 1:100, abcom, Cambridge, UK), α-smooth muscle actin (SMA, Clone IA4, 1:100, Dako, Carpinteria, CA, USA), and proliferating cell nuclear antigen (PCNA, PC10: sc-56, 1:800, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for two hours at room temperature. Slides were then incubated in biotinylated secondary antibody (anti-mouse IgG antibody, 1:1000) for forty-five minutes followed by DAB substrate (Vectastain ABC kit, Dako, CA, USA) for thirty minutes, and then were counterstained with Mayer’s hematoxylin (Sigma, St. Louis, MO, USA) for three minutes.

Results

Necropsy showed that two masses had grown from the left neck into the thoracic cavity. They were well circumscribed with large blood vessels. At the neck, a multilobular mass, 2.2 x 1.8 x 2.0 cm in size, had grown from the left neck and invaded into the skeletal muscles and the thoracic cavity. In the thoracic cavity, one mass seemed encapsulated with thin fibrous tissue, and another smaller mass was compressed adjacent to the apex of the heart (Figure 1A). On the cutting surface, masses were firm and whitish to tan, with necrotic and hemorrhagic lesions (Figure 1B).

Microscopically, the masses were composed of multiple nodules of tumor cells that were incompletely encapsulated with fibrous connective tissue (Figure 2A). In the thoracic area, the borders of the tumor were indistinct, with tumor cells mixed with normal skeletal muscle fibers. The tumor cells consisted of large, polygonal cells forming sheets and divided by thin, fibrous septa with abundant microvasculation. The tumor cells exhibited varied forms, from spindle form to globoid, with minimal to abundant eosinophilic cytoplasm, and appeared as large, multinucleated cells (Figure 2B). Some of the multinucleated cells contained peripheral vesicles around the nuclei, with prominent nucleoli (Figure 2C). The tumor cell vacuoles might be a result of intracellular glycogen, which had been removed during tissue processing. Rare mitotic figures and necrotic foci were found in the tumor masses.

Fibrous connective tissue was stained positive with MT staining on the peripheral of the tumor mass, but negative in the central spindle tumor cells. The tumor cells exhibited some cross-striation in the cytoplasm using PTAH staining (Figure 2D). Cytoplasmic vesicles containing PAS-positive materials were also observed in some tumor cells. Immunohistochemically, the tumor cells also expressed intense granular cytoplasmic staining for desmin (Figure 2E) and myoglobin (Figure 2F), and were highly stained with proliferation marker PCNA. Meanwhile, α-SMA was positive only in the blood vessels, but negative staining occurred in the tumor cells.

The diagnosis of rhabdomyosarcoma was made according to histopathological findings; the tumor cells varied from spindle to globoid types with abundant eosinophilic cytoplasm and appeared as large, multinucleated cells. Striations were present in some tumor cells. Furthermore, from special staining, the tumor cells exhibited some cross-striation in the cytoplasm using PTAH staining. Some tumor cells with peripheral vesicles located around nuclei and the vacuoles showed reddish staining by the PAS method. The tumor cells also expressed intense granular cytoplasmic staining for desmin and myoglobin, whereas α-SMA was negative and was highly stained with proliferation marker PCNA.

For the differential diagnosis, the spindle-form tumor cells were negatively stained with MT staining, and the encapsulated fibrous connective tissue was strongly positively stained with MT on the periphery of the tumor mass, indicating that tumor cells did not originate from fibroblasts. The diagnosis of cross-striation within rhabdomyosarcoma is rarely recognized. However, it could be easily demonstrated by PTAH in this case. Multinucleate giant cells are commonly observed. Many of the tumor cells are vacuolated and might represent glycogen that was removed during tissue processing. In this case, the vacuoles had reddish staining by the PAS method. The observations in the present case and those in published reports suggest that rhabdomyosarcomas in young rats are distinct entities, characterized by very low incidences and a high degree of differentiation.

Discussion

Spontaneous fetal rhabdomyomas and rhabdomyosarcomas are rare in humans and in laboratory rats. As in this case, these neoplasms occur mainly in the subcutaneous tissue in the head and neck region. Histologically, they are composed of primitive muscle fibers, or myotubes (Cooper and Valentine 2002). These two types of tumors have been classified as cardiac and extracardiac rhabdomyomas (Davies et al. 2007). In laboratory rats, these tumors seem related to strain and age. There have been reports of rhabdomyosarcomas in young SD rats (four and eight weeks old) (Glaister 1981; Minato et al. 1983; Tageldin and Elamin 1981), and one reported spontaneous rhabdomyosarcoma in a young (nine-week-old) SD rat by Conner in 1994. Our case is similar, with a rhabdomyosarcoma in a young (eight-week-old) SD rat. The higher incidence of rhabdomyosarcoma in older rats is in contrast to that in younger rats. However, only six rhabdomyosarcomas were 7,818 Fisher 344 rats (0.08%) from control groups in 79 carcinogenicity studies reported by the National Toxicology Program (Hanseman et al. 1990). A historical control study of non-neoplastic and neoplastic lesions in 696 rats showed the morbidity of rhabdomyosarcoma and was 0.1% in F344/DuCrj rats (Iwata et al. 1991). Data from ten carcinogenicity bioassays of spontaneous neoplasms in control Wistar rats showed a rate of rhabdomyosarcoma occurrence of 0.15% in males and 0.29% in females (Walsh and Poteracki 1994).

In humans, rhabdomyomas account for only 2% of all skeletal muscle tumors (Weiss and Goldblum 2001). Most extracardiac rhabdomyomas are located in the head and neck region (Hansen and Katenkamp 2005), but a genital type of rhabdomyoma has been reported in the paratestis of a young adult (Davies et al. 2007). In mice, the pathogenesis of rhabdomyomas was studied by Meikle et al. (2005). They proposed that in the mouse compared with humans, loss of Tsc1 in the ventricular myocytes during the gestation period and the presence of progesterone might accelerate the growth of rhabdomyomas. Furthermore, Nanni et al. (2003)also have shown that activation of the HER-2/neu oncogene coupled with inactivation of the oncosuppressor gene p53 cause rhabdomyosarcoma in mice, and they suggested the interaction between HER family genes and the p53 pathway might be involved in the origin of human rhabdomyosarcoma. In adult rhabdomyomas, these tumors favor the head and neck regions, because they arise from the musculature of the third and fourth branchial arches. They often present as benign tumors. Cross-striation was rarely observed in the H&E staining section, but it was demonstrated by PTAH staining.

Adult rhabdomyosarcomas display SMA and desmin positivity in the tumor cells (Cooper and Valentine 2002). In the present case, however, the cells were negative with α-SMA, positive with desmin and myoglobin, and were highly stained with proliferation marker PCNA; thus, the tumor was recognized as a rhabdomyosarcoma. A previous report stated that rhabdomyomas in humans were positively stained with α-muscle–specific actin, desmin, and myoglobin, whereas dystrophin was expressed in the membranes (Sørensen et al. 2006). Immunohistochemistry confirms that the tumors are almost totally mature neoplasms of clonal myogenic origin. However, rhabdomyoma was negative for S-100 compared with the differential diagnosis of granular cell tumor (Sørensen et al. 2006). In this case, tumor masses were immunohistochemically stained with S-100 and showed negative reaction (data not shown). Finally, the differential diagnosis of rhabdomyosarcoma should be included with neck and thoracic masses in young animals.

Classification of sarcomas in rats was rarely compared in the past. According to WHO classification of soft tissue tumors of humans, the tumors could initially be divided into two categories: rhabdomyosarcomas and other sarcomas. The sarcomas in which more than 5% of cells showed nuclear staining for myoD1, or more than 10% showed cytoplasmic staining for myoglobin, were classified as rhabdomyosarcomas (Fletcher et al. 2002). In addition, Taguchi et al. (2006)have tried to classify their DMBA-induced sarcomas of rats with morphological, electron microscopic, and immunohistochemical methods and reported that all of the DMBA-induced sarcomas did not show an immunohistochemically various or colliding pattern in a tumor as for expression of myoD1, myoglobin, desmin, α-SMA, and S-100. Therefore, classification of the sarcomas is feasible, but some problems remain in the application of a classification between human rhabdomyomas and rat rhabdomyosarcomas.