Avian Pathology (December 2003) 32(6), 613 /616
Rhabdomyosarcoma in a racing pigeon (Columba livia) H. Ferna´ndez-Bellon, J. Martorell, R. Rabanal and A. Ramis* Departament de Medicina i Cirurgia Animals, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona, 08193-Bellaterra, Barcelona, Spain
An adult racing pigeon (Columba livia) was presented with a subcutaneous mass on the ventral aspect of the right wing. A fine-needle aspirate and radiographic study of the mass were suggestive of highly invasive sarcomatous neoplasm. Euthanasia was decided because of the poor prognosis. Necropsy confirmed the highly invasive nature of the neoplasm, which also occupied a large portion of the right breast. There also was extensive osteolysis of the sternum with neoplastic invasion of the left breast and the coelomic cavity. Histopathology revealed a highly cellular, poorly demarcated, unencapsulated invasive sarcoma. Immunohistochemistry was positive for muscle actin, and myoglobin, weakly positive for vimentin, and negative for desmin, neuron-specific enolase and S-100 protein, suggesting a diagnosis of undifferentiated rhabdomyosarcoma.
Introduction Benign or malignant neoplasms originating from striated or smooth muscle are observed occasionally in captive and free-ranging birds. Rhabdomyosarcomas have often been described as irregular, elevated, lobulated, relatively firm subcutaneous swellings of the wing or shoulder that limit the use of the wing (Latimer, 1994). Reported sites of origin also include the eye (Dukes & Pettit, 1983), tongue and eyelid (Latimer, 1994), dorsum (Raphael & Nguyen, 1980), and head (Duncan & Fitzgerald, 1997), but they are rarely observed in birds (Reece, 1992; Schmidt, 1997) and have not been described in pigeons. Microscopically, these neoplasms are composed of a pleomorphic population of fusiform to elongated cells. Anisokaryosis may be prominent with plump oval-to-elongated nuclei, and some elongated or ‘strap cells’ will retain cross striations typical of skeletal muscle cells (Latimer, 1994). Confirmation of the diagnosis requires the use of electron microscopy or immunohistochemistry. In the first case, the most useful diagnostic feature is the presence of myofilaments. In the latter case, antibodies that have proven useful include those against vimentin, desmin,
actin, myoglobin, myosin and titin in several species of mammals (Cooper & Valentine, 2002); and muscle actin, vimentin and desmin in several species of birds (Frazier et al., 1993; Duncan & Fitzgerald, 1997; Hafner et al., 1998; Ijzer et al., 2002). In the present paper we describe a primary rhabdomyosarcoma in the ventral musculature of the right wing. To our knowledge this is the first case of the description of this tumour in a pigeon with confirmation by immunohistochemical techniques.
Case History An adult racing pigeon (Columba livia) was presented to the veterinary practice with a firm, non-ulcerated, lobulated, subcutaneous mass on the ventral aspect of the right wing that encompassed the distal humerus and proximal ulna and radius. The size and localization of the mass precluded flight, although the general condition of the bird was good. A fine-needle aspirate of the mass was obtained and cytological examination
*To whom correspondence should be addressed. Tel: /34 93 5811597. Fax: / 34 93 5813142. E-mail:
[email protected] Received 1 April 2003. Provisionally accepted 1 May 2003. Accepted 10 June 2003 ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/03/60613-04 # 2003 Houghton Trust Ltd DOI: 10.1080/03079450310001610712
614 H. Ferna´ndez-Bellon et al.
Materials and Methods The tumoural mass and all major organs and tissues were fixed in 4% neutral buffered formalin, processed routinely, sectioned at 4 mm and sections were stained with haematoxylin and eosin, and phosphotungstic acid haematoxylin. Additional immunohistochemical stains were carried out on the tumoural tissue. The primary antibodies, antigen retrieval methods, and incubation conditions used are described in Table 1. Biotinylated goat anti-mouse or goat anti-rabbit antibodies (as appropriate) were used as secondary antibodies, followed by the avidin / biotin complex immunoperoxidase procedure (Naish et al ., 1989). Diaminobenzidine with 0.5% H2O2 in imidazol buffer (pH 7.2, 0.1 M) was used as chromogen. Replicated sections of tumoural tissue were immunostained using normal rabbit serum (S-100 protein, desmin, myoglobin) and normal mouse serum (NSE, vimentin, muscle actin) instead of the primary antiserum, as negative controls. Finally, sections of leg muscles of the same bird were used as positive controls.
Figure 1. Fine-needle aspirate of the tumoural mass. Cluster of cells showing anysocytosis and anisokaryosis, with cytoplasmic vacuolation. A multinucleated cell is also present. Diff-Quick Stain. Bar/16 mm.
revealed numerous exfoliated cells arranged singly or in clusters. Cells were round to oval, and were variable in size with an irregularly basophilic cytoplasm that was finely vacuolated in some cells. Nuclei were often oval, but some were very large and irregularly shaped, with one or two prominent nucleoli. Multinucleated cells and mitoses were also observed. There was also a moderate amount of a pink material between the cells (Figure 1). The cytological diagnosis was a malignant sarcomatous neoplasm of stromal cells. Radiographic examination under general anaesthesia with isofluorane was performed to determine the extent of the neoplasm. There was a large soft tissue mass, increased opacity in the right humerus and ulna, and a lytic lesion involving the distal humerus near the joint. A lateral radiographic view revealed increased opacity in the sternum and pectoral muscle area. Because of the diagnosis of a sarcomatous malignant neoplasm, euthanasia was selected. At necropsy, the neoplastic growth was multilobular with multiple pinkish grey spherical nodules of different sizes (0.5 to 3 cm.). The growth was associated with osteolysis of the humerus and ulna, and invaded a large part of the right breast. The tumour invaded through the sternum at multiple sites with extension into the left breast and into the precardial thoracic cavity. The internal organs did not exhibit apparent lesions. Table 1.
Histologically, sections of the mass were highly cellular, unencapsulated, and invasive. The tumour was composed of anaplastic fusiform to elongated cells arranged in irregular sheets that lacked apparent stroma, and showed focal areas of necrosis. Neoplastic cells had abundant eosinophilic cytoplasm but lacked cross striations, as was demonstrated on replicated sections stained with phosphotungstic acid haematoxylin. Anisokaryosis was prominent, with plump oval-to-elongated nuclei, coarsely clumped to vacuolated chromatin and prominent nucleoli. Mitotic figures were common, some of them irregular in shape. Cells containing multiple nuclei were also common (Figure 2). Neoplastic cells were immunopositive for muscle actin and myoglobin, but negative for desmin. Immunostaining for myoglobin was homogeneous and intense, while immunostaining for actin was intense only in scattered neoplastic cells (Figure 3). Staining for vimentin, an intermediate filament that labels a variety of mesenchymal cells, was homogeneous, albeit much lighter than for myoglobin. Staining for the remaining cell markers (desmin, S-100, NSE) was absent in the tumour cells. In the sections of leg muscle, used as positive controls, striated muscle cells were vimentin negative, and desmin, muscle actin and myoglobin positive. As expected, peripheral nerves (Schwann cells) were S-100 and NSE positive; and nonmuscle mesenchymal cells and vascular endothelial cells were vimentin positive.
Primary antibodies, antigen retrieval methods, and incubation conditions used in the immunohistochemical tests performed
Antibody Desmin Muscle actin Myoglobin Neuron-specific enolase (NSE) S-100 protein Vimentin a
Results
Host species Rabbit Mouse Rabbit Mouse Rabbit Mouse
DakoCytomation Denmark A/S, Glostrup, Denmark.
Target species Chicken Human Human Human Cow Pig
Source a
DakoCytomation DakoCytomationa DakoCytomationa DakoCytomationa DakoCytomationa DakoCytomationa
Antigen retrieval
Dilution
/ Saponin Saponin / / /
1:50 1:100 1:350 1:800 1:400 1:50
Rhabdomyosarcoma in a pigeon 615
Figure 2. Haematoxylin and eosin section of the tumour. Markedly anaplastic cells arranged in irregular sheets between remnants of striated muscular fibres. Neoplastic cells contain eosinophilic cytoplasm without cross striations and exhibit marked anisokaryosis of plump oval-to-elongated nuclei; numerous mitotic figures and occasional multinucleated cells are present. Bar /56 mm.
Figure 3. Immunoreactivity to actin in a section of the tumoural mass. Immunostaining for actin was intense only in scattered neoplastic cells. Note positive staining of a multinucleated cell, and an atypical mitosis in the centre of the image. Avidin /biotin complex immunoperoxidase technique, diaminobenzidine chromogen, haematoxylin counterstain. Bar/27 mm.
Of the immunohistochemical markers used on this neoplasm, the pattern of positive staining most clearly matched the staining characteristics of rhabdomyomas and rhabdomyosarcomas, since neoplastic cells were muscle actin and myoglobin positive and S-100 and NSE negative (True, 1990; Cooper & Valentine, 2002). However, the negative staining for desmin and positive staining for vimentin presented a possible discrepancy between our results and the expected staining pattern of rhabdomyosarcomas. This apparent inconsistency can be resolved since antigens are expressed differently depending on the degree of differentiation of the rhabdomyocytes, probably reflecting the sequence of expression of antigens in the developing rhabdomyocytes. Vimentin is expressed early in the rhabdomyocytic development but is later lost, while desmin and actin are also expressed early but persist (Carter et al., 1990; Wijnaendts et al., 1994). In fact, in domestic mammals, vimentin is often expressed in interstitial cells and is usually absent from well-differentiated rhabdomyosarcomas, but may be expressed in poorly differentiated rhabdomyoblasts (Cooper & Valentine, 2002). Also, desmin has been undetectable in some pleomorphic rhabdomyosarcomas in humans (Furlong et al., 2001). Thus, the weak homogeneous positive staining for vimentin, and absence of staining for desmin in the neoplastic cells in this case can be interpreted as a sign of poor differentiation. The irregular staining for actin could be due to the heterogeneity of the neoplastic cells, with positive cells being those most well differentiated, whereas cells negative for actin would be more anaplastic (Cooper & Valentine, 2002). The intense homogeneous staining for myoglobin observed in this case is difficult to account for under this scenario, as this protein is expressed late during differentiation (Carter et al., 1990). We can conclude that the histologic features and the immunocytochemical profile of the tumour cells were consistent with a diagnosis of a poorly differentiated pleomorphic rhabdomyosarcoma.
Discussion Immunohistochemistry is now the preferred diagnostic technique for confirmation of the diagnosis of rhabdomyosarcoma and has been utilized in a great number of recent studies (Cooper & Valentine, 2002). Histologically, the undifferentiated sarcomatous characteristics of this tumour could also have been consistent with fibrosarcoma or schwannoma. In fact fibrosarcoma and schwannoma are usually also positive for vimentin and negative for desmin, as were the cells of this tumour. Nonetheless, positive staining for myoglobin and muscle actin, and lack of immunostaining for S-100 suggest the diagnosis of rhabdomyosarcoma (True, 1990).
Acknowledgements The authors thank Dr Isidre Ferrer (Servei de Patologia, Hospital Princeps d’Espanya, Facultat de Medicina, Universitat de Barcelona) for the performance of the actin and myoglobin immunostainings. They also thank J. Merchan for the submission of the case.
References Carter, R.L., Jameson, C.F., Philp, E.R. & Pikerton, C.R. (1990). Comparative phenotypes in rhabdomyosarcomas and developing skeletal muscle. Histopathology, 17 , 301 /309.
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´ SUME´ RE Rhabdomyosarcome chez un pigeon voyageur (Columbia livia) Un pigeon voyageur adulte (Columbia livia ) soumis a` consultation pre´sentait une masse sous-cutane´e a` l’aile droite en position ventrale. Une e´tude radiographique et une aspiration a` l’aide d’une aiguille fine de cette masse ont sugge´re´ qu’il s’agissait d’un ne´oplasme sarcomateux
tre`s fortement invasif. L’euthanasie de l’animal a e´te´ de´cide´e du fait du pronostic pessimiste. L’autopsie a confirme´ la nature tre`s invasive de ce ne´oplasme qui occupait une zone importante de la partie droite du bre´chet. Il y avait e´galement une importante oste´olyse du sternum avec une invasion ne´oplastique du coˆte´ gauche du bre´chet et de la cavite´ cœlomique. L’examen histopathologique a re´ve´le´ un sarcome invasif, non encapsule´, peu de´limite´ et fortement cellulaire. L’immunohistochimie a e´te´ positive pour l’actine du muscle et la myoglobine, faiblement positive pour la vimentine et ne´gative pour la desmine, l’enolase spe´cifique du neurone et la prote´ine S-100, sugge´rant un diagnostic de rhabdomyosarcome indiffe´rencie´.
ZUSAMMENFASSUNG Rhabdomyosarkom bei einer Reisetaube (Columbia livia) Eine erwachsene Reisetaube (Columba livia) wurde mit einer subkutanen Umfangsvermehrung auf der ventralen Seite des rechten Flu¨gels vorgestellt. Aspiration mit einer feinen Nadel und die ro¨ntgenologische Untersuchung der Umfangsvermehrung liessen ein hochgradig invasives sarkomato¨ses Neoplasma vermuten. Wegen der schlechten Prognose wurde die Euthanasie der Taube beschlossen. Die pathologisch-anatomische Untersuchung besta¨tigte die hochgradig invasive Natur der Geschwulst, die sich auch auf einen großen Teil der rechten Brust ausdehnte. Außerdem war eine extensive Osteolyse des Sternums mit neoplastischer Invasion der linken Brust und der Leibesho¨hle feststellbar. Die histopathologische Untersuchung ließ ein hochgradig zellula¨res, wenig abgesetztes, nicht eingekapseltes, invasives Sarkom erkennen. Die Immunhistochemie war positiv fu¨r Muskelaktin und Myoglobin, schwach positiv fu¨r Vimentin und negativ fu¨r Desmin, Neuron-spezifische Enolase und S-100-Protein, was die Diagnose undifferenziertes Rhabdosarkom nahelegt.
RESUMEN Rabdomiosarcoma en una paloma (Columba livia ) Se presento´ una paloma (Columba livia ) adulta con una masa subcuta´nea en la parte ventral del ala derecha. Una aspiracio´n con aguja fina y un estudio radiogra´fico de la masa sugirieron una neoplasia sarcomatosa altamente invasiva. Se decidio´ eutanasiar al animal debido al grave prono´stico. La necropsia confirmo´ la naturaleza invasiva de la neoplasia, que ocupaba una proporcio´n amplia del pectoral derecho. Tambie´n se observo´ una osteolisis extensa del esterno´n con invasio´n neopla´sica del pectoral izquierdo y de la cavidad celo´mica. La histopatologı´a revelo´ un sarcoma invasivo no encapsulado, densamente celular y mal demarcado. La inmunohistoquı´mica fue positiva para la actina y mioglobina muscular, de´bilmente positiva para la vimentina y negativa para la desmina, enolasa especı´fica de neuronas y la proteı´na S-100, lo que sugiere un diagno´stico de rabdomiosarcoma no diferenciado.
