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Gingival Rhabdomyosarcoma Accompanied by an Immature Myogenic Population Immunoreactive for α-Smooth Muscle Actin in a Dog
J. Comp. Path. 2013, Vol. 149, 48e52
Available online at www.sciencedirect.com
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NEOPLASTIC DISEASE
Gingival Rhabdomyosarcoma Accompanied by an Immature Myogenic Population Immunoreactive for a-Smooth Muscle Actin in a Dog M. Kimura*, K. Suzuki†, Y. Fujii*, R. Yamamoto*, M. Shibutani* and K. Mitsumori* *Laboratory of Veterinary Pathology and † Laboratory of Veterinary Toxicology, Tokyo University of Agriculture and Technology, Tokyo, Japan
Summary A 3-year-old female shih tzu was presented with a white to dark red mass arising from the gingiva. Because of the rapid and invasive growth of the mass, the dog was humanely destroyed. Microscopically, round to polygonal anaplastic cells with strongly eosinophilic cytoplasm grew in an alveolar pattern separated by fibrous stroma. Mitotic figures were numerous. Multinucleated cells and ‘strap cells’ were observed, but cross striation and glycogen accumulation were absent. Immunohistochemically, the tumour cells were positive for vimentin, desmin, muscle-specific actin and MyoD1, and a small number of tumour cells were positive for a-smooth muscle actin (a-SMA). Based on the morphological and immunohistochemical features, the gingival mass was diagnosed as alveolar rhabdomyosarcoma accompanied by a-SMA-positive immature myogenic cells. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; gingiva; immunohistochemistry; rhabdomyosarcoma
Rhabdomyosarcomas are rare, malignant, striated muscle tumours, which have been detected in the head, neck, urogenital tract, retroperitoneum, limbs, larynx and heart of dogs. Cases of striated muscle tumours arising in the oral cavity have been reported (Salisbury and Lantz, 1988), but details of whether these tumours were malignant were not provided. Because rhabdomyosarcomas consist of pleomorphic neoplastic cells and many cases are undifferentiated tumours lacking cross striation (Parham, 2001; Cooper and Valentine, 2002), immunohistochemistry (IHC) is required to identify the origin of these tumours and to distinguish them from other soft tissue sarcomas (Cooper and Valentine, 2002). Most rhabdomyosarcomas are negative for a-smooth muscle actin (a-SMA), but some rhabdomyosarcomas expressing a-SMA have been reported in people (Furlong et al., 2001; Moon et al., 2005) and a cow (Aoyagi et al., 2001). A canine case of a-SMA-positive spindle cell rhabdomyosarcoma lacking the polygoCorrespondence to: K. Suzuki (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.11.233
nal tumour cells typical of rhabdomyosarcomas has been documented in the frontal region of the skull (Roza et al., 2010). We report a case of canine gingival rhabdomyosarcoma accompanied by a-SMA-positive polygonal cells. A 3-year-old female shih tzu presented with a mass in the maxillary region and was referred to a veterinary hospital. The mass was 1.5 cm in diameter and was located in the gingiva of the left maxillary cuspid region and infiltrated the hard palate. After approximately 1 week, the dog was again referred because of enlargement of the mass (3 5 cm). There was focal ulceration and discolouration of the surface of the mass. Cryotherapy using liquid nitrogen was used to treat the mass. After 1 month, the mass had extended into the left orbit, resulting in proptosis. The dog was humanely destroyed and a sample of the mass was submitted for microscopical examination. The sample was fixed with 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE), periodic acideSchiff (PAS), Ó 2012 Elsevier Ltd. All rights reserved.
Gingival Rhabdomyosarcoma in a Dog
phosphotungstic acid haematoxylin (PTAH) and Masson’s trichrome. Microscopically, the mass was located in the gingival submucosa and consisted of pleomorphic, round to polygonal anaplastic tumour cells containing variably-sized, atypical, hypochromatic nuclei with 1e3 prominent nucleoli and strongly eosinophilic cytoplasm. The neoplastic cells showed solid growth and formed alveoli separated by fibrous septa. Many of the central cells were discohesive. The cells of the fibrous septa were slightly atypical spindle cells that had distinct nuclei, prominent nucleoli and were arranged in a fascicular pattern (Fig. 1). Mitotic figures were frequently observed in the polygonal cells (mitotic index 5.6 per 400 field). Multinucleated cells and ‘strap cells’ were also observed. Cross striation and glycogen accumulation were lacking on PTAH or PAS staining, respectively. In the central area of the mass there was necrosis with infiltration of inflammatory cells, predominantly neutrophils. Infiltration of inflammatory cells (mainly lymphocytes and plasma cells) was observed in the superficial layer of the gingival mucosa. IHC was performed using the VECTASTAINÒ Elite ABC kit (Vector Laboratories Inc., Burlingame, California, USA) and the avidinebiotineperoxidase complex (ABC) method. Primary mouse monoclonal antibodies used were specific for vimentin (clone V9; 1 in 100 dilution; Dako, Glostrup, Denmark), cytokeratin (clone AE1/AE3; 1 in 50 dilution, Dako), desmin (clone D33; 1 in 100 dilution; Thermo Fisher Scientific Inc., Rockford, Illinois, USA), musclespecific actin (clone HHF35; 1 in 50 dilution;
Fig. 1. Neoplastic cells show solid growth and form alveoli separated by fibrous septa, with many of the central cells appearing discohesive. The cells of the fibrous septa are slightly atypical spindle cells with distinct nuclei and prominent nucleoli. They are arranged in a fascicular pattern. HE. 100.
49
Dako), MyoD1 (clone 5.8A; 1 in 50 dilution; Dako), a-SMA (clone 1A4; 1 in 100 dilution; Dako), calponin (clone hCP; 1 in 10,000 dilution; Chemicon International, Temecula, California, USA), smoothelin (clone R4A; MAB3242; 1 in 100 dilution; Chemicon), melan A (clone A103; 1 in 50 dilution; Dako) and proliferating cell nuclear antigen (PCNA; clone PC10; 1 in 200 dilution; Dako). Primary rabbit polyclonal antibodies used were specific for S100 protein (1 in 400 dilution; Dako) and Iba1 (1 in 100 dilution; Wako Pure Chemical Industries Ltd., Osaka, Japan). Antigen retrieval was performed by microwaving in 10 mM citrate buffer (pH 6.0) at 90 C for 10 min (desmin, a-SMA, cytokeratin, PCNA and S100) or autoclaving in 10 mM citrate buffer (pH 6.0) (vimentin, calponin, smoothelin and melan A) or in Tris/ethylenediaminetetraacetic acid buffer (pH 9.0) (Target Retrieval SolutionÔ, pH 9.0; Dako) (MyoD1) at 121 C for 10 min. Sections were treated with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidase activity. The sections were blocked with normal goat or horse serum at room temperature for 30 min. Each section was incubated with primary antibody at 4 C overnight, with the secondary antibodies against mouse or rabbit immunoglobulin (Ig) G applied at room temperature for 30 min. The ABC reagent was applied at room temperature for 30 min. Labelling was ‘visualized’ with 3,30 -diaminobenzidine (DAB) as chromogen and counterstaining was with haematoxylin. Double immunohistochemical labelling for MyoD1 and a-SMA was also performed. MyoD1 antibody was first applied using the ABC method as above. To block antibody cross-reactivity, the slide was heated by autoclaving in 10 mM citrate buffer (pH 6.0) before the second round of IHC. Subsequently, a-SMA was detected using the avidinebiotinealkaline phosphatase complex (ABCeAP) method using the VECTASTAINÒ ABCeAP kit (Vector Laboratories), and labelling was visualized using VectorÒ Blue Alkaline Phosphatase Substrate Kit I (Vector Laboratories). As positive control tissues, canine cases of melanoma (melan A) and cutaneous histiocytoma (Iba1; Ide et al., 2011), and sections of normal dog skeletal muscle (desmin, muscle-specific actin and MyoD1) and skin (other antibodies) were used. Sections incubated with 0.1 M phosphate buffered saline in place of the primary antibody were used as negative controls. Both polygonal tumour cells and stromal spindle cells expressed vimentin. The majority of polygonal cells and some spindle cells were strongly positive for desmin (Fig. 2). A strong positive reaction for muscle-specific actin was observed in the cytoplasm of the polygonal cells, while the spindle cells were
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Fig. 2. Polygonal tumour cells labelled strongly for desmin expression. IHC. 400.
only slightly immunoreactive. The cytoplasm of the spindle cells and of a small population of the polygonal cells was positive for a-SMA (Fig. 3). The nuclei of the polygonal cells and some spindle cells were positive for MyoD. Polygonal cells that were positive for myogenic markers (desmin, muscle-specific actin and MyoD1) were negative for a-SMA. In double IHC, MyoD1-positive polygonal cells and spindle cells did not co-express a-SMA, and a-SMA-positive spindle cells did not co-express MyoD1 (Fig. 4). Both polygonal and spindle cell populations were positive for PCNA, but the distribution of PCNA-positive cells in the polygonal cell population (66.9%) was higher than in the spindle cell population (37.2%). Polygonal and spindle cells were negative for calponin, smoothelin, cytokeratin, S100 protein, melan A and Iba1.
Fig. 3. The cytoplasm of the spindle cells and some polygonal cells (arrows) are positive for a-SMA. IHC. 400.
Fig. 4. Nuclei of most of the polygonal cells are positive for MyoD1 (brown) and the cytoplasm of the spindle cells is positive for a-SMA (blue, arrowheads). There is a minor population of spindle cells lacking a-SMA, but expressing MyoD1 (arrows). Double IHC. 400.
Oral rhabdomyosarcoma is rare in the dog (Salisbury and Lantz, 1988) and these tumours must be differentiated from oral melanocytic tumours and other mesenchymal tumours of the oral cavity. In the present case, the differential diagnoses for the gingival mass included rhabdomyosarcoma, leiomyosarcoma, malignant fibrous histiocytoma (MFH) and amelanotic melanoma. The negative immunoreactivity for Iba1, S100 protein and melan A ruled out MFH and amelanotic melanoma (Ide et al., 2011; Smedley et al., 2011). Negative labelling for calponin and smoothelin, proteins expressed in smooth muscle, indicated that the tumour was not a leiomyosarcoma (Uranishi et al., 2001). In contrast, immunoreactivity for a-SMA, a marker for smooth muscle (Hayden et al., 1999), was observed in spindle cells and a small number of the polygonal cells. However, the a-SMApositive spindle cell population was negative for markers for rhabdomyosarcoma (MyoD1), in contrast to the typical MyoD1 immunoreactivity observed in the polygonal cells. With regard to cellular proliferation, the spindle cells of the stromal bundles showed 37.2% of PCNA-positive cells, similar to that of the spindle cell populations located beneath the mucosa and consisting of non-neoplastic myofibroblasts (42.2%), and lower than that in the polygonal cell population (66.9%). Based on the immunoreactivities and the histological features, the a-SMA-positive spindle cells growing within stromal bundles were considered to be non-neoplastic myofibroblasts, and the polygonal cells irrespective of a-SMA-immunoreactivity were interpreted as being rhabdomyosarcoma. The minor population of MyoD1-positive spindle cells lacking a-SMA-
51
Gingival Rhabdomyosarcoma in a Dog
expression might have been neoplastic cells infiltrating the fibrous septa. Mature striated muscles do not express a-SMA, but a-SMA-expression is observed in myoblasts during the early stage of muscle development (Springer et al., 2002) and tumour cells of human rhabdomyosarcoma expressing SMA mRNA were considered to mimic the early stage of myogenesis (Caplan et al., 1983; Schurch et al., 1994). In the present study, the absence of cross striation and glycogen accumulation and the immunoreactivity for MyoD1, which is expressed in the early stage of striated muscle development (Kobayashi et al., 2004), suggested that the gingival mass was an immature type of rhabdomyosarcoma. In addition, the a-SMA-positive polygonal cell component was consistent with an immature cell population at an early stage of myogenesis. In man, rhabdomyosarcomas are classified into four types, embryonal, botryoid, alveolar and pleomorphic, and their classification is dependent on gross morphological features or histological patterns including that of a grape-like polypoid mass, myxoid matrix production and the formation of alveoli supported by a framework of dense fibrous stroma (Cooper and Valentine, 2002). Although any rhabdomyosarcoma may be pleomorphic, the pleomorphic type is limited to the variant that lacks areas showing an embryonal and alveolar pattern (Cooper and Valentine, 2002). Until recently, undifferentiated rhabdomyosarcomas consisting of aSMA-positive pleomorphic cells were identified as either pleomorphic or alveolar rhabdomyosarcomas (Schurch et al., 1994; Aoyagi et al., 2001; Furlong et al., 2001; Moon et al., 2005). A canine case of aSMA-positive rhabdomyosarcoma was documented as spindle cell rhabdomyosarcoma lacking the polygonal cell component (Roza et al., 2010). In contrast, weak or doubtful immunoreactivity for a-SMA was reported in one case of canine alveolar rhabdomyosarcoma; however, the type of immunoreactive cells was not presented (Cooper and Valentine, 2002). The histological feature of the gingival mass investigated in this study, namely the presence of neoplastic polygonal cells surrounded by fibrous stroma, was most similar to a human case of alveolar rhabdomyosarcoma (Moon et al., 2005). Therefore, the present study is the second documented case of canine alveolar rhabdomyosarcoma accompanied by a-SMA-positive polygonal tumour cells. The prognosis for human alveolar rhabdomyosarcoma is poor (Friling et al., 1994; Moon et al., 2005; Pirillo et al., 2011; Sharma et al., 2011) and considering the aggressiveness of the tumour in this study, it may be surmised that canine alveolar rhabdomyosarcoma may have a similar outcome.
References Aoyagi T, Saruta K, Asahi I, Hojo H, Shibahara T et al. (2001) Pleomorphic rhabdomyosarcoma in a cow. Journal of Veterinary Medical Science, 63, 107e110. Caplan AI, Fiszman MY, Eppenberger HM (1983) Molecular and cell isoforms during development. Science, 221, 921e927. Cooper BJ, Valentine BA (2002) Tumors of striated muscle. In: Tumors in Domestic Animals, 4th Edit., DJ Meuten, Ed., Iowa State Press, Ames, pp. 341e363. Friling R, Marcus M, Monos T, Moses M, Yassur Y (1994) Rhabdomyosarcoma: invading the orbit in an adult. Ophthalmic Plastic and Reconstructive Surgery, 10, 283e286. Furlong MA, Mentzel T, Fanburg-Smith JC (2001) Pleomorphic rhabdomyosarcoma in adults: a clinicopathologic study of 38 cases with emphasis on morphologic variants and recent skeletal muscle specific markers. Modern Pathology, 14, 595e603. Hayden DW, Klausner JS, Waters DJ (1999) Prostatic leiomyosarcoma in a dog. Journal of Veterinary Diagnostic Investigation, 11, 283e286. Ide T, Uchida K, Kagawa Y, Suzuki K, Nakayama H (2011) Pathological and immunohistochemical features of subdural histiocytic sarcomas in 15 dogs. Journal of Veterinary Diagnostic Investigation, 23, 127e132. Kobayashi M, Sakai H, Hirata A, Yonemaru K, Yanai T et al. (2004) Expression of myogenic regulating factors myogenin and myoD in two canine botryoid rhabdomyosarcomas. Veterinary Pathology, 41, 275e277. Moon HS, Kwon SW, Lee JH (2005) A case of alveolar rhabdomyosarcoma of the ethmoid sinus invading the orbit in an adult. Korean Journal of Ophthalmology, 20, 70e75. Parham DM (2001) Pathologic classification of rhabdomyosarcoma and correlations with molecular studies. Modern Pathology, 14, 506e514. Pirillo V, Cipriano Cecchi P, Tripodi M, Maier K, Rizzo P et al. (2011) Primary cerebral alveolar rhabdomyosarcoma in adult. Rare Tumors, 3, e26. Roza MR, Amorim RFB, Carneiro FP, Benatto N, Barriviera M et al. (2010) Aggressive spindle cell rhabdomyosarcoma in an 11-month-old boxer dog. Journal of Veterinary Medical Science, 72, 1363e1366. Salisbury SK, Lantz GC (1988) Long-term results of partial mandibulectomy for treatment of oral tumors in 30 dogs. Journal of the American Animal Hospital Association, 24, 285e294. Schurch W, Bochaton-Piallat ML, Geinoz A, Amore E, Laurini RN et al. (1994) All histological types of primary human rhabdomyosarcoma express a-cardiac and not a-skeletal actin messenger RNA. American Journal of Pathology, 144, 836e846. Sharma A, Bhutoria B, Guha D, Bhattacharya S, Wasim NA (2011) Fine needle aspiration cytology of metastatic alveolar rhabdomyosarcoma. Journal of Cytology, 28, 121e123. Smedley RC, Lamoureux J, Sledge DG, Kiupel M (2011) Immunohistochemical diagnosis of canine oral
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amelanotic melanocytic neoplasms. Veterinary Pathology, 48, 32e40. Springer ML, Ozawa CR, Blau HM (2002) Transient production of a-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation. Cell Motility and the Cytoskeleton, 51, 177e186.
Uranishi R, Baev NI, Kim JH, Awad IA (2001) Vascular smooth muscle cell differentiation in human cerebral vascular malformations. Neurosurgery, 49, 671e679. July 13th, 2012 ½ Received, Accepted, November 12th, 2012
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Gingival Rhabdomyosarcoma Accompanied by an Immature Myogenic Population Immunoreactive for a-Smooth Muscle Actin in a Dog M. Kimura*, K. Suzuki†, Y. Fujii*, R. Yamamoto*, M. Shibutani* and K. Mitsumori* *Laboratory of Veterinary Pathology and † Laboratory of Veterinary Toxicology, Tokyo University of Agriculture and Technology, Tokyo, Japan
Summary A 3-year-old female shih tzu was presented with a white to dark red mass arising from the gingiva. Because of the rapid and invasive growth of the mass, the dog was humanely destroyed. Microscopically, round to polygonal anaplastic cells with strongly eosinophilic cytoplasm grew in an alveolar pattern separated by fibrous stroma. Mitotic figures were numerous. Multinucleated cells and ‘strap cells’ were observed, but cross striation and glycogen accumulation were absent. Immunohistochemically, the tumour cells were positive for vimentin, desmin, muscle-specific actin and MyoD1, and a small number of tumour cells were positive for a-smooth muscle actin (a-SMA). Based on the morphological and immunohistochemical features, the gingival mass was diagnosed as alveolar rhabdomyosarcoma accompanied by a-SMA-positive immature myogenic cells. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; gingiva; immunohistochemistry; rhabdomyosarcoma
Rhabdomyosarcomas are rare, malignant, striated muscle tumours, which have been detected in the head, neck, urogenital tract, retroperitoneum, limbs, larynx and heart of dogs. Cases of striated muscle tumours arising in the oral cavity have been reported (Salisbury and Lantz, 1988), but details of whether these tumours were malignant were not provided. Because rhabdomyosarcomas consist of pleomorphic neoplastic cells and many cases are undifferentiated tumours lacking cross striation (Parham, 2001; Cooper and Valentine, 2002), immunohistochemistry (IHC) is required to identify the origin of these tumours and to distinguish them from other soft tissue sarcomas (Cooper and Valentine, 2002). Most rhabdomyosarcomas are negative for a-smooth muscle actin (a-SMA), but some rhabdomyosarcomas expressing a-SMA have been reported in people (Furlong et al., 2001; Moon et al., 2005) and a cow (Aoyagi et al., 2001). A canine case of a-SMA-positive spindle cell rhabdomyosarcoma lacking the polygoCorrespondence to: K. Suzuki (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.11.233
nal tumour cells typical of rhabdomyosarcomas has been documented in the frontal region of the skull (Roza et al., 2010). We report a case of canine gingival rhabdomyosarcoma accompanied by a-SMA-positive polygonal cells. A 3-year-old female shih tzu presented with a mass in the maxillary region and was referred to a veterinary hospital. The mass was 1.5 cm in diameter and was located in the gingiva of the left maxillary cuspid region and infiltrated the hard palate. After approximately 1 week, the dog was again referred because of enlargement of the mass (3 5 cm). There was focal ulceration and discolouration of the surface of the mass. Cryotherapy using liquid nitrogen was used to treat the mass. After 1 month, the mass had extended into the left orbit, resulting in proptosis. The dog was humanely destroyed and a sample of the mass was submitted for microscopical examination. The sample was fixed with 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE), periodic acideSchiff (PAS), Ó 2012 Elsevier Ltd. All rights reserved.
Gingival Rhabdomyosarcoma in a Dog
phosphotungstic acid haematoxylin (PTAH) and Masson’s trichrome. Microscopically, the mass was located in the gingival submucosa and consisted of pleomorphic, round to polygonal anaplastic tumour cells containing variably-sized, atypical, hypochromatic nuclei with 1e3 prominent nucleoli and strongly eosinophilic cytoplasm. The neoplastic cells showed solid growth and formed alveoli separated by fibrous septa. Many of the central cells were discohesive. The cells of the fibrous septa were slightly atypical spindle cells that had distinct nuclei, prominent nucleoli and were arranged in a fascicular pattern (Fig. 1). Mitotic figures were frequently observed in the polygonal cells (mitotic index 5.6 per 400 field). Multinucleated cells and ‘strap cells’ were also observed. Cross striation and glycogen accumulation were lacking on PTAH or PAS staining, respectively. In the central area of the mass there was necrosis with infiltration of inflammatory cells, predominantly neutrophils. Infiltration of inflammatory cells (mainly lymphocytes and plasma cells) was observed in the superficial layer of the gingival mucosa. IHC was performed using the VECTASTAINÒ Elite ABC kit (Vector Laboratories Inc., Burlingame, California, USA) and the avidinebiotineperoxidase complex (ABC) method. Primary mouse monoclonal antibodies used were specific for vimentin (clone V9; 1 in 100 dilution; Dako, Glostrup, Denmark), cytokeratin (clone AE1/AE3; 1 in 50 dilution, Dako), desmin (clone D33; 1 in 100 dilution; Thermo Fisher Scientific Inc., Rockford, Illinois, USA), musclespecific actin (clone HHF35; 1 in 50 dilution;
Fig. 1. Neoplastic cells show solid growth and form alveoli separated by fibrous septa, with many of the central cells appearing discohesive. The cells of the fibrous septa are slightly atypical spindle cells with distinct nuclei and prominent nucleoli. They are arranged in a fascicular pattern. HE. 100.
49
Dako), MyoD1 (clone 5.8A; 1 in 50 dilution; Dako), a-SMA (clone 1A4; 1 in 100 dilution; Dako), calponin (clone hCP; 1 in 10,000 dilution; Chemicon International, Temecula, California, USA), smoothelin (clone R4A; MAB3242; 1 in 100 dilution; Chemicon), melan A (clone A103; 1 in 50 dilution; Dako) and proliferating cell nuclear antigen (PCNA; clone PC10; 1 in 200 dilution; Dako). Primary rabbit polyclonal antibodies used were specific for S100 protein (1 in 400 dilution; Dako) and Iba1 (1 in 100 dilution; Wako Pure Chemical Industries Ltd., Osaka, Japan). Antigen retrieval was performed by microwaving in 10 mM citrate buffer (pH 6.0) at 90 C for 10 min (desmin, a-SMA, cytokeratin, PCNA and S100) or autoclaving in 10 mM citrate buffer (pH 6.0) (vimentin, calponin, smoothelin and melan A) or in Tris/ethylenediaminetetraacetic acid buffer (pH 9.0) (Target Retrieval SolutionÔ, pH 9.0; Dako) (MyoD1) at 121 C for 10 min. Sections were treated with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidase activity. The sections were blocked with normal goat or horse serum at room temperature for 30 min. Each section was incubated with primary antibody at 4 C overnight, with the secondary antibodies against mouse or rabbit immunoglobulin (Ig) G applied at room temperature for 30 min. The ABC reagent was applied at room temperature for 30 min. Labelling was ‘visualized’ with 3,30 -diaminobenzidine (DAB) as chromogen and counterstaining was with haematoxylin. Double immunohistochemical labelling for MyoD1 and a-SMA was also performed. MyoD1 antibody was first applied using the ABC method as above. To block antibody cross-reactivity, the slide was heated by autoclaving in 10 mM citrate buffer (pH 6.0) before the second round of IHC. Subsequently, a-SMA was detected using the avidinebiotinealkaline phosphatase complex (ABCeAP) method using the VECTASTAINÒ ABCeAP kit (Vector Laboratories), and labelling was visualized using VectorÒ Blue Alkaline Phosphatase Substrate Kit I (Vector Laboratories). As positive control tissues, canine cases of melanoma (melan A) and cutaneous histiocytoma (Iba1; Ide et al., 2011), and sections of normal dog skeletal muscle (desmin, muscle-specific actin and MyoD1) and skin (other antibodies) were used. Sections incubated with 0.1 M phosphate buffered saline in place of the primary antibody were used as negative controls. Both polygonal tumour cells and stromal spindle cells expressed vimentin. The majority of polygonal cells and some spindle cells were strongly positive for desmin (Fig. 2). A strong positive reaction for muscle-specific actin was observed in the cytoplasm of the polygonal cells, while the spindle cells were
50
M. Kimura et al.
Fig. 2. Polygonal tumour cells labelled strongly for desmin expression. IHC. 400.
only slightly immunoreactive. The cytoplasm of the spindle cells and of a small population of the polygonal cells was positive for a-SMA (Fig. 3). The nuclei of the polygonal cells and some spindle cells were positive for MyoD. Polygonal cells that were positive for myogenic markers (desmin, muscle-specific actin and MyoD1) were negative for a-SMA. In double IHC, MyoD1-positive polygonal cells and spindle cells did not co-express a-SMA, and a-SMA-positive spindle cells did not co-express MyoD1 (Fig. 4). Both polygonal and spindle cell populations were positive for PCNA, but the distribution of PCNA-positive cells in the polygonal cell population (66.9%) was higher than in the spindle cell population (37.2%). Polygonal and spindle cells were negative for calponin, smoothelin, cytokeratin, S100 protein, melan A and Iba1.
Fig. 3. The cytoplasm of the spindle cells and some polygonal cells (arrows) are positive for a-SMA. IHC. 400.
Fig. 4. Nuclei of most of the polygonal cells are positive for MyoD1 (brown) and the cytoplasm of the spindle cells is positive for a-SMA (blue, arrowheads). There is a minor population of spindle cells lacking a-SMA, but expressing MyoD1 (arrows). Double IHC. 400.
Oral rhabdomyosarcoma is rare in the dog (Salisbury and Lantz, 1988) and these tumours must be differentiated from oral melanocytic tumours and other mesenchymal tumours of the oral cavity. In the present case, the differential diagnoses for the gingival mass included rhabdomyosarcoma, leiomyosarcoma, malignant fibrous histiocytoma (MFH) and amelanotic melanoma. The negative immunoreactivity for Iba1, S100 protein and melan A ruled out MFH and amelanotic melanoma (Ide et al., 2011; Smedley et al., 2011). Negative labelling for calponin and smoothelin, proteins expressed in smooth muscle, indicated that the tumour was not a leiomyosarcoma (Uranishi et al., 2001). In contrast, immunoreactivity for a-SMA, a marker for smooth muscle (Hayden et al., 1999), was observed in spindle cells and a small number of the polygonal cells. However, the a-SMApositive spindle cell population was negative for markers for rhabdomyosarcoma (MyoD1), in contrast to the typical MyoD1 immunoreactivity observed in the polygonal cells. With regard to cellular proliferation, the spindle cells of the stromal bundles showed 37.2% of PCNA-positive cells, similar to that of the spindle cell populations located beneath the mucosa and consisting of non-neoplastic myofibroblasts (42.2%), and lower than that in the polygonal cell population (66.9%). Based on the immunoreactivities and the histological features, the a-SMA-positive spindle cells growing within stromal bundles were considered to be non-neoplastic myofibroblasts, and the polygonal cells irrespective of a-SMA-immunoreactivity were interpreted as being rhabdomyosarcoma. The minor population of MyoD1-positive spindle cells lacking a-SMA-
51
Gingival Rhabdomyosarcoma in a Dog
expression might have been neoplastic cells infiltrating the fibrous septa. Mature striated muscles do not express a-SMA, but a-SMA-expression is observed in myoblasts during the early stage of muscle development (Springer et al., 2002) and tumour cells of human rhabdomyosarcoma expressing SMA mRNA were considered to mimic the early stage of myogenesis (Caplan et al., 1983; Schurch et al., 1994). In the present study, the absence of cross striation and glycogen accumulation and the immunoreactivity for MyoD1, which is expressed in the early stage of striated muscle development (Kobayashi et al., 2004), suggested that the gingival mass was an immature type of rhabdomyosarcoma. In addition, the a-SMA-positive polygonal cell component was consistent with an immature cell population at an early stage of myogenesis. In man, rhabdomyosarcomas are classified into four types, embryonal, botryoid, alveolar and pleomorphic, and their classification is dependent on gross morphological features or histological patterns including that of a grape-like polypoid mass, myxoid matrix production and the formation of alveoli supported by a framework of dense fibrous stroma (Cooper and Valentine, 2002). Although any rhabdomyosarcoma may be pleomorphic, the pleomorphic type is limited to the variant that lacks areas showing an embryonal and alveolar pattern (Cooper and Valentine, 2002). Until recently, undifferentiated rhabdomyosarcomas consisting of aSMA-positive pleomorphic cells were identified as either pleomorphic or alveolar rhabdomyosarcomas (Schurch et al., 1994; Aoyagi et al., 2001; Furlong et al., 2001; Moon et al., 2005). A canine case of aSMA-positive rhabdomyosarcoma was documented as spindle cell rhabdomyosarcoma lacking the polygonal cell component (Roza et al., 2010). In contrast, weak or doubtful immunoreactivity for a-SMA was reported in one case of canine alveolar rhabdomyosarcoma; however, the type of immunoreactive cells was not presented (Cooper and Valentine, 2002). The histological feature of the gingival mass investigated in this study, namely the presence of neoplastic polygonal cells surrounded by fibrous stroma, was most similar to a human case of alveolar rhabdomyosarcoma (Moon et al., 2005). Therefore, the present study is the second documented case of canine alveolar rhabdomyosarcoma accompanied by a-SMA-positive polygonal tumour cells. The prognosis for human alveolar rhabdomyosarcoma is poor (Friling et al., 1994; Moon et al., 2005; Pirillo et al., 2011; Sharma et al., 2011) and considering the aggressiveness of the tumour in this study, it may be surmised that canine alveolar rhabdomyosarcoma may have a similar outcome.
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Uranishi R, Baev NI, Kim JH, Awad IA (2001) Vascular smooth muscle cell differentiation in human cerebral vascular malformations. Neurosurgery, 49, 671e679. July 13th, 2012 ½ Received, Accepted, November 12th, 2012