Zbl. Vet. Med. A, 24, 542-551
(1977)
@ 1977 Verlag Paul Parey, Berlin und Hamburg
ISSN 0300-871 l/ASTM-Coden: ZVRAAX
From the Department of Veterinary Pathology State University of Ghent, Belgium Director: Prof. Dr. J . Hoorens
The Ultrastructure of Rhabdomyosarcoma in a Dog BY C. MEYVISCH, H. THOONEN and J. HOORENS With 10 figures and one table (Received for publication December 4 , 1976)
Introduction Rhabdomyosarcoma is a rather uncommon tumor in domestic animals. In human pathology rhabdomyosarcomas are rare in adults but occur more frequent as malignant tumors of soft tissue in children. In the dog 14 cases of rhabdomyosarcoma have been described and are summarized in Table I. Ultrastructural studies on human rhabdoniyosarcomas have mainly been restricted to the embryonal type of rhabdomysarcoma, while KORENYI-BOTH et al. (1968) and HORVAT et al. (1970) reported electron microscopic studies of a human adult pleomorphic rhabdomyosarcoma. In domestic animals the only ultrastructural report of a spontaneous rhabdomyosarcoma has to our knowledge been given by PETERand KLUGE(1970). These authors describe a tumor in the mouth of a dog, which they classify as an undifferentiated rhabdomyosarcoma, since they could not demonstrate any cross-striation. Because of the limited data on the ultrastructural features of naturally occurring rhabdomyosarcoma in animals, a correlated light and electron microscopic study of a pleomorphic rhabdomyosarcoma showing unquestionable cross-striation seemed worthwhile reporting. Materials and Methods For histological examination pieces of the neoplasm were fixed in 10 O/o formalin and Zenker's acetic fluid. Sections were stained with haematoxylineosin, Van Gieson and phosphotungstic acid haematoxylin. For electron microscopy multiple blocks of the original tumor of the limb and pieces of the lung metastasis were prefixed in 2.5 O/o glutaraldehyde in 0.1 M cacodylate buffer and postfixed in 2 O / o OsO, in the same buffer for two hours. They were dehydrated through ascending concentrations of acetone and embedded in SPURR (1969) medium. Sections cut at 2 ,u were stained with toluidine blue for light microscope correlation. Thin sections cut with glass
The Ultrastructure of Rhabdomyosarcoma in a Dog
543
knives were stained with uranylacetate and lead citrate and examined with a ZEISS EM 9 electron microscope. Results Case Report A 5 year old male Malinois Shepherd was presented at the small animal clinic for an acute swelling of the muscle of the left thigh. The animal showed no lamenness. The swelling was diffuse and by palpation hard and nearly painless. A biopsy was taken and a malignant sarcoma, probably a rhabdomyosarcoma, was diagnosed. The tumor was still growing and the left hind leg became oedematous. Metastases of the tumor were seen on radiographs of the thorax. Due to the poor prognosis, the owner requested euthanasia. Necropsy O n autopsy a tumor (15 X 10 X 12 cm) was located in the left thigh, caudo-lateral of the femur (Fig. 1). O n the cut surface the tumoral mass appeared greyish white, with small areas of necrosis and haemorrhages. The whole left hind leg was swollen by an extensive subcutaneous oedema. The left inguinal lymph node was enlarged (7 X 6 X 5 cm). In the lung numerous foci of metastasis were present.
Fig. 1. Macroscopic aspect of the tumor
Fig. 2. Light microscope picture of lymph node metastasis showing strap-like cells with peripheral alignment of nuclei HE X 600
544
MEYVISCH. THOONEN and HOORENS
Histopathology The primary tumor appeared not well circumscribed, as groups of tumor cells were seen infiltrating the surrounding muscle tissue. Small necrotic and haemorrhagic areas were randomly spread in the tumor. Considerable cellular pleomorphism was evident (Fig. 2). The neoplastic cells ranged from oval to spindle-shaped. Nuclei were mostly large and round with a very clear, sometimes vacuolated, karyoplasm. In some areas mitotic figures were abundant. Scattered throughout the tumor a limited number of round to oval giant cells with large or multiple nuclei were seen. Elongated strap-like cells with abundant acidophilic cytoplasm contained several nuclei with prominent nucleoli, arranged in a row of three or more. In most cases these strap-like cells contained longitudinal fibrils and, especially in the metastasis, their cytoplasm showed, in a few cases, a faint cross-striation after phosphotungstic acid haematoxylin staining. These cross-striations were more distinct in semi-thin sections ( 2 p) stained with toluidine blue (Fig. 3).
Fig. 3. Light microscops picture of a 2!( section, showing clear cross-striation. Toluidine blue X 750
Fig. 4. T w o undifferentiated myoblasts (Mb), closely applied t o the surface of a rhabdomyoblast containing organized rnyofilaments (Mf) a n d short segments of sarcoplasmic reticulum (arrow) X 5,600
The Ultrastructure of Rhabdomyosarcoma in a Dog
545
Electron microscopy Ultrastructural examination of the tumor revealed several cell types. The majority consisted of undifferentiated mesenchymal cells, some of which were associated with each other in rows of 2 to 4 cells. Their cytoplasm contained abundant free ribosomes and polysomes but only few elements of endoplasmic reticulum. The mitochondria, most of them swollen and vesiculated, were poorly preserved. The large euchromatic nuclei contained small peripheral condensations of chromatin. In a few cells the nuclear membrane showed deep invaginations. One or two centrally located prominent nucleoli were always present (Fig. 4). In addition to, and dispersed between these undifferentiated mesenchymal cells, rhabdomyoblasts with varying degrees of differentiation were found. They could be clearly identified by the presence of specific myofibrils (thick and thin filaments arranged in alternate fashion) in their cytoplasm. The number of myofibrils per cell varied greatly: when present only in small numbers the filaments were mostly situated in the immediate vicinity of the nucleus (Fig. 5).
Fig. 5. Rhabdomyoblast with commencing accumulation of filaments close to the nucleus. x 10.800
Fig.6. Rhabdomyoblast with myofibrils in sarcomere pattern, with formation of I- and A zones, Z lines and vesicles of sarcoplasmic reticulum (arrow) x 10,800 Zbl. Vet. Med., Keihe A, Bd. 24, H e f t 7
38
546
MEYVISCH, THOONEN a n d HOORENS
Fig. 7. Detail of portions of a n undifferentiated myoblasr (Mb) and a rhabdomyoblast containing organized myofilaments (Mf) a n d intermingled intermediate filaments (If) x 16,800
Fig. 8. Rhabdomyoblast with myofilaments in contraction: K: collagen fibers in intercellular space. Large a r r o w ; fat droplet. Inset: vesicle of sarcoplasmic reticulum between myofilaments. X 16.200
Occasional rhabdomyoblasts showed the characteristic banding of striated muscle. Typical A bands, I bands and 2 lines were clearly discernable (Fig. 6), but in general the highly differentiated rhabdornyoblasts showed a disorganized pattern of muscle banding and often the thick (myosin) and thin (actin) filaments were less closely packed as compared to normal muscle (Fig. 7). Tumor cells with myofibrils showing only A and 2 bands resembled muscle cells in contraction (Fig. 8). The sarcoplasmic reticulum was poorly developed, even in the more differentiated rhabdomyoblasts. Only a few intersections of it appeared as short tubules (Fig. 4) o r vesicles, next to o r within the myofilarnent bundles (Fig. 6 , inset Fig. 8).
The Ultrastructure of Rhabdomyosarcoma in a Dog
547
Fig. 9. Degenerating rhabdomyoblast containing regions with well organized fibrils and amorphous dark staining patches. x 9,100
Fig. 10. Detail of a rnultinucleate giant cell with a fine fibrillar matrix and bundles of thick filaments. X 11,850 ?S*
548
MEYVISCH, THOONEN and HOORENS
In a few areas cells were found filled with randomly oriented tightly packed filaments. Such tumor cells often contained cytoplasmic areas where the fibrillar pattern was lost and they appeared as amorphous dark staining patches, enclosing coalesced cell organelles (Fig. 9). The “multinucleated giant cells” seen with the light microscope appeared by electron microscopy as rounded to elongated structures, mostly containing several peripherally located nuclei. They were clearly separated from the surrounding cells by an empty space. The clear cytoplasm consisted of a fine filamentous matrix. Cell organelles, except for a few mitochondria and small clusters of ribosomes, wcre almost absent. Some of the multinucleated giant cells contained bundles of (thick) filaments (Fig. 10). A small number of the rhabdomyoblasts examined contained lipid droplets. Microtubules were only rarely encountered. Throughout the tumor a small number of fibroblasts with abundant rough endoplasmic reticulum and a small spindle-shaped nucleus were found. The interstitial stroma between the undifferentiated tumor cells or rhabdomyoblasts contained mature collagen fibers in many places.
Discussion Rhabdomyosarcomas originate from mesenchymatous tissue which has retained the capacity of differentiation into striped muscle. The maturation of undifferentiated rumor cells to striated muscle has often been reported to mimic the course of normal myogenesis (BOCKER and STEGNER, 1975; MORAL E et ~ al., 1972; HO~TZER, 1970). During this process, the development of the primitive rhabdomyoblasts into’ mature rhabdomyoblasts is accompanied by the appearance of specific myofilaments of actin (thin) and myosin (thick), which by their specific mutual arrangement form myofilament bundles, first dispersed in the cytoplasm and later in sarcomeres, which give the typical cross-striation of skeletal muscle. In this respect, the undifferentiated tumor cells - presumptive rhabdomyoblasts (BOCKERand STEGNER, 1975) -, the mononuclear cells with small bundles of filaments and the elongated polynucleated strap-cells with aligned sarcomeres - myotubes - can be compared to cells in different stages of m yogenesis. It must be kept in mind though that degenerative processes may occur in the tumor cells at any stage of their development from primitive mesenchymal cell to fully developed muscle cell (FRIEDMAN and BIRD, 1969; WEBB,1971). We think that the multinucleated giant cells of this rhabdomyosarcoma must be interpreted in that way: they probably arise as a consequence of a defect in myogenesis at the level of cell elongation. Indeed, in ultrastructural morphology these rounded multinucleated structures are identical to the myosacs described during myogenesis in cell cultures (HOLTZER,1970; ISHIKAWA et al., 1968). The cells illustrated in Fig. 10 constitute another example of tumor cell degeneration; in this case, highly differentiated rhabdomyoblasts are concerned, with degenerating sarcomeres and electrondense zones of “coagulative necrosis”. The location and the anatomical and histological characteristics of this canine tumor are consistent with those of pleomorphic rhabdomyosarcoma in man. At the ultrastructural level positive identification of specific myofilaments - thick and thin - alternatively arranged can establish the diagnosis of rhabdomyosarcoma (MORALES et al., 1972).
The Ultrastructure of Rhabdomyosarcoma in a Dog
549
In the present case of rhabdomyosarcoma all stages of myogenesis could be traced back in the tumoral muscle cells, even up to aggregation of myofilaments into large bundles with a sarcomere organisation. This typical banding of the myofilaments has only occasionally been described at the ultrastructural level in human embryonal rhabdomyosarcoma (FRIEDMAN et al., 1965; KROLL,1967; MORALESet al., 1972) and experimental tumors and BIRD,1969). Our findings are in contrast with (CLARKE, 1969; FRIEDMAN those of previous ultrastructural studies of pleomorphic rhabdomyosarcomas by KORENYI-BOTH et al. (13); HORVAT et al. (1970) and PETER and KLUGE (1970), who claim that cells of pleomorphic rhabdomyosarcomas are less differentiated than those of the embryonal forms. Both at the light microscope and electron microscope level definite crossstriations (see Table I) can rarely be found in the pleomorphic form of this tumor of muscular origin. Even in the embryonal type of rhabdomyosarcoma cross-striations have been demonstrated in only about 50 O/o of the published
Table 1 Sex M M F M
F
M F F F F F F M F
Breed Boxer English Setter Great Dane St. Bernard Bassett Germ. Sheperd Corgi
St. Bernard St. Bernard Miniature Poodle St. Bernard St. Bernard Doberman Pinscher Mixed breed
Localisation
Cross striation
Reference
1 high Thigh Urinary bladder II 11
Mouth Pharynx Urinary bladder II
a II
II II
Maxillary area
-
-
+ +
PETER LADDS BRODEV KELLY 1973 KELLY KELLY KELLY HALCIWELL 1974 SEIBOLD 197L
Berichtigung: In der 3. Spalte ,Breed" muR es in der 6. Zeile richtig heii3en: Germ. Sheherd
cases. In our opinion the problem of sampling tissue for electron microscopy plays here a major role and careful search for cross-striations on semi-thin sections is certainly necessary.
Summary During light microscopic examination of biopsy material from a canine tumor in the thigh, cells of muscle-type appearance were seen and the possibility of rhabdomyosarcoma was considered. A few indistinct cross-striations were seen in paraffin sections and in 2 ,LA sections of plastic-embedded material they could be demonstrated more clearly. A detailed electron microscope study revealed muscle cells in different stages of myogenesis and removed all doubts about the diagnosis of rhabdomyosarcoma, since distinct cross-striations with A, I and Z bands typical for striped muscle were seen. The fact that tumor cells with cross-striations could also be found in a lung- and lymph node metastasis excluded the possibility that they were elements of degenerating or regenerating muscle tissue.
550
MEYVISCH, THOONEN and HOORENS
Acknowledgements Appreciation is expresscd to Mr. J. P. LOGGHEfor technical assistance with ultramicrotomy and with the illustrations. We are indebted to the “Clinic of Small Animals” (Director: Prof. Dr. D. MATTHEEUWS) for supplying the clinical history of the case. Zusammenfassung Die Rhabdomyosarkom-Ultrastruktur des Hundes Bei der lichtmikroskopischen Untersuchung von Biopsiematerial eines Schenkeltumors eines Hundes wurden muskelzelltypische Elemente gefunden und daher das Vorliegen eines Rhabdomyosarkoms vermutet. In Paraffinschnitten konnten undeutliche Querstreifungen festgestellt werden, welche in Schnitten von in Plastik eingebettetem Material deutlicher zur Darstellung kamen. Elektronenoptische Untcrsuchungen lieflen quergestreifte Muskelzellen mit A-, J- und 2-Banden i n verschiedenen Phasen der Myogenese erkennen, womit das Vorliegen eines Rhabdomyosarkoms gesichert war. D a quergestreifte Tumorzellen auch in Lungen- und Lymphknotenmetastasen gefunden wurden, handelte es sich bei den fraglichen Tumorzellen nicht um degeneriertes oder regeneriertes Muskelzellgewebe. Resume Ultrastructure du rhabdomyosarcome du chien Une recherche au microscope optique avec un matkriel d’une biopsie d’une tumeur d’une cuisse chez un chien a montrk les ClCments cellulaires musculaires typiques permettant de soupsonner un rhabdomyosarcome. Les striations furent peu nettes avec les coupes faites dans la paraffine; elles furent meilleures avec le matkriel coulk dans du plastique. Les recherches au microscope klectronique ont permis de reconnattre les cellulet; musculaires strikes avec des bandes A, J et 2 dans diffkrentes phases de la rriyogknkse; cela a confirmk la prksence d’un rhabdomyosarcome. Etant donnk que des cellules tumorales strikes furent kgalement trouvkes dans des mktastases pulmonaires et ganglionnaires, il ne s’agissait pas de tissu musculaire dkgknkrC ou rkgCnCrk. Resumen La ultraestructura del rabdomiosarcoma en el perro Con ocasi6n del examen fotomicrosc6pico de material de biopsia de un tumor sito en el muslo de un perro, se hallaron elementos tipicos de cklula muscular, sospechindose la pres’encia de un rabdomiosarcoma. Se pudieron apreciar en cortes de parafina unas pocas estriaciones transversales indistintas, que se logra demostrar con una claridad mayor en secciones de material encamado en plistico. Los eximenes electr6nico6pticos permitieron reconocer cklulas musculares estriadas con bandas A, J y C en fases diversas de miogknesis, con lo cual se hallaba asegurada la presencia de un rabdomiosarcoma. Puesto que se encontraron tambikn cklulas tumorales estriadas en metistasis pulmonares y ganglionares, en las cklulas tumorales problemiticas no se trataba de tejido miocelular degenerado o regenerado.
The Ultrastructure of Rhabdomyosarcoma in a Dog
551
References BOCKER,W., and H. E. STEGNER, 1975: Mixed Miillerian tumors of the uterus. Ultrastructural studies on the differentiation of Rhabdomyoblasts. Virchows Archiv Abt. A Path. Anat., 363, 337-351. BRODEY,R. S., 1971 : Hypertrophic osteoarthropathy in the dog. A clinicopathologic survey of 60 cases. Journal of the American Veterinary Medical Association, 159, 1242-1256. CLARKE, M. A., 1969: The fine structure of methylcholanthrene-induced tumors in mice. Cancer (Phil.), 24, 147-157. COLLET,MM. P., and H. TISSEUR, 1949: Sarcome primitif du droit andrieur de la cuisse chez un chien. Bulletin de la SociCtC des Sciences VCtCrinaires de Lyon, 5 2 , 1-7. FRIEDMAN, I., D. F. M. HARRISON, M. M. TUCKER and E. S. BIRD, 1965: Electron microscopy of a rhabdomyosarcoma of the ear. Journal of Clinical Pathology, 18, 63-68. FRIEDMAN, I., and E. S. BIRD, 1969: Electron microscope investigation of experimental rhabdomyosarcoma. Journal of Pathology, 97,375-382. HALLIWELL, W. H., and N. ACKERMAN, 1974: Botryoid rhabdomyosarcoma of the urinary bladder and hypertrophic osteoarthropathy in a young dog. Journal of the American Veterinary Medical Association, 165, 911-913. HOLTZER, H., 1970: Myogenesis. In Cell Differentiation (Schjeide, 0. H., and J. de Vellis, Eds.). Van Nostrand Reinhold Company, New York. HORVAT, B. L., M. CAINES and E. R. FISHER,1970: The ultrastructure of rhabdomyosarcoma. American Journal of Clinical Pathology, 53, 555-564. ISHIKAWA, H., R . BISCHOFF and H. HOLTZER,1968: Mitosis and intermediate sized filaments in developing skeletal muscle. The Journal of Cell Biology, 38, 538-556. KELLY,D. F., 1973: Rhabdomyosarcoma of the urinary bladder in dogs. Veterinary Pathology, 10, 375-384. KORENYI-BOTH, A., K. LAPISund M. GALLAI,1968: Uber die Feinstruktur des undifferenzierten, adulten, pleomorphen rhabdomyosarkoms. Beitrage zur Pathologischen Anatomie, 138, 96-108. KROLL,A. A., 1967: Fine structural classification of orbital rhabdomyosarcoma. Investigative Ophthalmology, 6, 531-543. LADDS,P. W., and D. R. WEBSTER, 1971 : Pharyngeal rhabdomyosarcoma in a dog. Veterinary Pathology, 8, 256-259. MORALES, A. R., G. FINE and R. C. HORN,1972: Rhabdomyosarcoma: an ultrastructural appraisal. Pathology Annual 7, London Butler worths. OSBORNE, C. A., D. G. LOW, V. PERMAN and D. M. BARNES,1968: Neoplasms of the canine and feline urinary bladder: Incidence, etiologic factors, occurrence and pathologic features. American Journal of Veterinary Research, 29, 2041-2053. PETER,C. P., and J. P. KLUGE,1970: An ultrastructural study of a canine rhabdomyosarcoma. Cancer, 26, 1280-1288. SEIBOLD, H. R., 1974: Juvenile alveolar rhabdomyosarcoma in a dog. Veterinary Pathology, 11, 558-560. SPURR,A. R., 1969: A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research, 26, 31-43. STAMPS,P., and D. L. H A R R I S , 1968: Botryoid rhabdomyosarcoma of the urinary bladder of a dog. Journal of the American Veterinary Medical Association, 1 5 3 , 1064-1068. TEUNISSEN, G . H. B., and W. MISDORP, 1968: Rhabdomyosarcoma of the urinary bladder and fibromatosis of the extremities in a young dog. Zbl. Vet. Med., Reihe A, 15, 81-88. WEBB,J. N., 1971 : The development of human skeletal muscle with particular reference to muscle cell death. Journal of Pathology, 106,221-228. WORLEY,G., and J. R. GORHAM,1954: The comparative pathology of rhabdomyosarcoma with a report of a case in a dog. American Journal of Pathology, 30, 837-849. Address of authors: Drs. C. MEYVISCH, H. THOONEN, Prof. Dr. J. HOORENS, Dept. of Veterinary Pathology, State University of Ghent, Casinoplein 24, B-9000 Ghent/Belgium.
(1977)
@ 1977 Verlag Paul Parey, Berlin und Hamburg
ISSN 0300-871 l/ASTM-Coden: ZVRAAX
From the Department of Veterinary Pathology State University of Ghent, Belgium Director: Prof. Dr. J . Hoorens
The Ultrastructure of Rhabdomyosarcoma in a Dog BY C. MEYVISCH, H. THOONEN and J. HOORENS With 10 figures and one table (Received for publication December 4 , 1976)
Introduction Rhabdomyosarcoma is a rather uncommon tumor in domestic animals. In human pathology rhabdomyosarcomas are rare in adults but occur more frequent as malignant tumors of soft tissue in children. In the dog 14 cases of rhabdomyosarcoma have been described and are summarized in Table I. Ultrastructural studies on human rhabdoniyosarcomas have mainly been restricted to the embryonal type of rhabdomysarcoma, while KORENYI-BOTH et al. (1968) and HORVAT et al. (1970) reported electron microscopic studies of a human adult pleomorphic rhabdomyosarcoma. In domestic animals the only ultrastructural report of a spontaneous rhabdomyosarcoma has to our knowledge been given by PETERand KLUGE(1970). These authors describe a tumor in the mouth of a dog, which they classify as an undifferentiated rhabdomyosarcoma, since they could not demonstrate any cross-striation. Because of the limited data on the ultrastructural features of naturally occurring rhabdomyosarcoma in animals, a correlated light and electron microscopic study of a pleomorphic rhabdomyosarcoma showing unquestionable cross-striation seemed worthwhile reporting. Materials and Methods For histological examination pieces of the neoplasm were fixed in 10 O/o formalin and Zenker's acetic fluid. Sections were stained with haematoxylineosin, Van Gieson and phosphotungstic acid haematoxylin. For electron microscopy multiple blocks of the original tumor of the limb and pieces of the lung metastasis were prefixed in 2.5 O/o glutaraldehyde in 0.1 M cacodylate buffer and postfixed in 2 O / o OsO, in the same buffer for two hours. They were dehydrated through ascending concentrations of acetone and embedded in SPURR (1969) medium. Sections cut at 2 ,u were stained with toluidine blue for light microscope correlation. Thin sections cut with glass
The Ultrastructure of Rhabdomyosarcoma in a Dog
543
knives were stained with uranylacetate and lead citrate and examined with a ZEISS EM 9 electron microscope. Results Case Report A 5 year old male Malinois Shepherd was presented at the small animal clinic for an acute swelling of the muscle of the left thigh. The animal showed no lamenness. The swelling was diffuse and by palpation hard and nearly painless. A biopsy was taken and a malignant sarcoma, probably a rhabdomyosarcoma, was diagnosed. The tumor was still growing and the left hind leg became oedematous. Metastases of the tumor were seen on radiographs of the thorax. Due to the poor prognosis, the owner requested euthanasia. Necropsy O n autopsy a tumor (15 X 10 X 12 cm) was located in the left thigh, caudo-lateral of the femur (Fig. 1). O n the cut surface the tumoral mass appeared greyish white, with small areas of necrosis and haemorrhages. The whole left hind leg was swollen by an extensive subcutaneous oedema. The left inguinal lymph node was enlarged (7 X 6 X 5 cm). In the lung numerous foci of metastasis were present.
Fig. 1. Macroscopic aspect of the tumor
Fig. 2. Light microscope picture of lymph node metastasis showing strap-like cells with peripheral alignment of nuclei HE X 600
544
MEYVISCH. THOONEN and HOORENS
Histopathology The primary tumor appeared not well circumscribed, as groups of tumor cells were seen infiltrating the surrounding muscle tissue. Small necrotic and haemorrhagic areas were randomly spread in the tumor. Considerable cellular pleomorphism was evident (Fig. 2). The neoplastic cells ranged from oval to spindle-shaped. Nuclei were mostly large and round with a very clear, sometimes vacuolated, karyoplasm. In some areas mitotic figures were abundant. Scattered throughout the tumor a limited number of round to oval giant cells with large or multiple nuclei were seen. Elongated strap-like cells with abundant acidophilic cytoplasm contained several nuclei with prominent nucleoli, arranged in a row of three or more. In most cases these strap-like cells contained longitudinal fibrils and, especially in the metastasis, their cytoplasm showed, in a few cases, a faint cross-striation after phosphotungstic acid haematoxylin staining. These cross-striations were more distinct in semi-thin sections ( 2 p) stained with toluidine blue (Fig. 3).
Fig. 3. Light microscops picture of a 2!( section, showing clear cross-striation. Toluidine blue X 750
Fig. 4. T w o undifferentiated myoblasts (Mb), closely applied t o the surface of a rhabdomyoblast containing organized rnyofilaments (Mf) a n d short segments of sarcoplasmic reticulum (arrow) X 5,600
The Ultrastructure of Rhabdomyosarcoma in a Dog
545
Electron microscopy Ultrastructural examination of the tumor revealed several cell types. The majority consisted of undifferentiated mesenchymal cells, some of which were associated with each other in rows of 2 to 4 cells. Their cytoplasm contained abundant free ribosomes and polysomes but only few elements of endoplasmic reticulum. The mitochondria, most of them swollen and vesiculated, were poorly preserved. The large euchromatic nuclei contained small peripheral condensations of chromatin. In a few cells the nuclear membrane showed deep invaginations. One or two centrally located prominent nucleoli were always present (Fig. 4). In addition to, and dispersed between these undifferentiated mesenchymal cells, rhabdomyoblasts with varying degrees of differentiation were found. They could be clearly identified by the presence of specific myofibrils (thick and thin filaments arranged in alternate fashion) in their cytoplasm. The number of myofibrils per cell varied greatly: when present only in small numbers the filaments were mostly situated in the immediate vicinity of the nucleus (Fig. 5).
Fig. 5. Rhabdomyoblast with commencing accumulation of filaments close to the nucleus. x 10.800
Fig.6. Rhabdomyoblast with myofibrils in sarcomere pattern, with formation of I- and A zones, Z lines and vesicles of sarcoplasmic reticulum (arrow) x 10,800 Zbl. Vet. Med., Keihe A, Bd. 24, H e f t 7
38
546
MEYVISCH, THOONEN a n d HOORENS
Fig. 7. Detail of portions of a n undifferentiated myoblasr (Mb) and a rhabdomyoblast containing organized myofilaments (Mf) a n d intermingled intermediate filaments (If) x 16,800
Fig. 8. Rhabdomyoblast with myofilaments in contraction: K: collagen fibers in intercellular space. Large a r r o w ; fat droplet. Inset: vesicle of sarcoplasmic reticulum between myofilaments. X 16.200
Occasional rhabdomyoblasts showed the characteristic banding of striated muscle. Typical A bands, I bands and 2 lines were clearly discernable (Fig. 6), but in general the highly differentiated rhabdornyoblasts showed a disorganized pattern of muscle banding and often the thick (myosin) and thin (actin) filaments were less closely packed as compared to normal muscle (Fig. 7). Tumor cells with myofibrils showing only A and 2 bands resembled muscle cells in contraction (Fig. 8). The sarcoplasmic reticulum was poorly developed, even in the more differentiated rhabdomyoblasts. Only a few intersections of it appeared as short tubules (Fig. 4) o r vesicles, next to o r within the myofilarnent bundles (Fig. 6 , inset Fig. 8).
The Ultrastructure of Rhabdomyosarcoma in a Dog
547
Fig. 9. Degenerating rhabdomyoblast containing regions with well organized fibrils and amorphous dark staining patches. x 9,100
Fig. 10. Detail of a rnultinucleate giant cell with a fine fibrillar matrix and bundles of thick filaments. X 11,850 ?S*
548
MEYVISCH, THOONEN and HOORENS
In a few areas cells were found filled with randomly oriented tightly packed filaments. Such tumor cells often contained cytoplasmic areas where the fibrillar pattern was lost and they appeared as amorphous dark staining patches, enclosing coalesced cell organelles (Fig. 9). The “multinucleated giant cells” seen with the light microscope appeared by electron microscopy as rounded to elongated structures, mostly containing several peripherally located nuclei. They were clearly separated from the surrounding cells by an empty space. The clear cytoplasm consisted of a fine filamentous matrix. Cell organelles, except for a few mitochondria and small clusters of ribosomes, wcre almost absent. Some of the multinucleated giant cells contained bundles of (thick) filaments (Fig. 10). A small number of the rhabdomyoblasts examined contained lipid droplets. Microtubules were only rarely encountered. Throughout the tumor a small number of fibroblasts with abundant rough endoplasmic reticulum and a small spindle-shaped nucleus were found. The interstitial stroma between the undifferentiated tumor cells or rhabdomyoblasts contained mature collagen fibers in many places.
Discussion Rhabdomyosarcomas originate from mesenchymatous tissue which has retained the capacity of differentiation into striped muscle. The maturation of undifferentiated rumor cells to striated muscle has often been reported to mimic the course of normal myogenesis (BOCKER and STEGNER, 1975; MORAL E et ~ al., 1972; HO~TZER, 1970). During this process, the development of the primitive rhabdomyoblasts into’ mature rhabdomyoblasts is accompanied by the appearance of specific myofilaments of actin (thin) and myosin (thick), which by their specific mutual arrangement form myofilament bundles, first dispersed in the cytoplasm and later in sarcomeres, which give the typical cross-striation of skeletal muscle. In this respect, the undifferentiated tumor cells - presumptive rhabdomyoblasts (BOCKERand STEGNER, 1975) -, the mononuclear cells with small bundles of filaments and the elongated polynucleated strap-cells with aligned sarcomeres - myotubes - can be compared to cells in different stages of m yogenesis. It must be kept in mind though that degenerative processes may occur in the tumor cells at any stage of their development from primitive mesenchymal cell to fully developed muscle cell (FRIEDMAN and BIRD, 1969; WEBB,1971). We think that the multinucleated giant cells of this rhabdomyosarcoma must be interpreted in that way: they probably arise as a consequence of a defect in myogenesis at the level of cell elongation. Indeed, in ultrastructural morphology these rounded multinucleated structures are identical to the myosacs described during myogenesis in cell cultures (HOLTZER,1970; ISHIKAWA et al., 1968). The cells illustrated in Fig. 10 constitute another example of tumor cell degeneration; in this case, highly differentiated rhabdomyoblasts are concerned, with degenerating sarcomeres and electrondense zones of “coagulative necrosis”. The location and the anatomical and histological characteristics of this canine tumor are consistent with those of pleomorphic rhabdomyosarcoma in man. At the ultrastructural level positive identification of specific myofilaments - thick and thin - alternatively arranged can establish the diagnosis of rhabdomyosarcoma (MORALES et al., 1972).
The Ultrastructure of Rhabdomyosarcoma in a Dog
549
In the present case of rhabdomyosarcoma all stages of myogenesis could be traced back in the tumoral muscle cells, even up to aggregation of myofilaments into large bundles with a sarcomere organisation. This typical banding of the myofilaments has only occasionally been described at the ultrastructural level in human embryonal rhabdomyosarcoma (FRIEDMAN et al., 1965; KROLL,1967; MORALESet al., 1972) and experimental tumors and BIRD,1969). Our findings are in contrast with (CLARKE, 1969; FRIEDMAN those of previous ultrastructural studies of pleomorphic rhabdomyosarcomas by KORENYI-BOTH et al. (13); HORVAT et al. (1970) and PETER and KLUGE (1970), who claim that cells of pleomorphic rhabdomyosarcomas are less differentiated than those of the embryonal forms. Both at the light microscope and electron microscope level definite crossstriations (see Table I) can rarely be found in the pleomorphic form of this tumor of muscular origin. Even in the embryonal type of rhabdomyosarcoma cross-striations have been demonstrated in only about 50 O/o of the published
Table 1 Sex M M F M
F
M F F F F F F M F
Breed Boxer English Setter Great Dane St. Bernard Bassett Germ. Sheperd Corgi
St. Bernard St. Bernard Miniature Poodle St. Bernard St. Bernard Doberman Pinscher Mixed breed
Localisation
Cross striation
Reference
1 high Thigh Urinary bladder II 11
Mouth Pharynx Urinary bladder II
a II
II II
Maxillary area
-
-
+ +
PETER LADDS BRODEV KELLY 1973 KELLY KELLY KELLY HALCIWELL 1974 SEIBOLD 197L
Berichtigung: In der 3. Spalte ,Breed" muR es in der 6. Zeile richtig heii3en: Germ. Sheherd
cases. In our opinion the problem of sampling tissue for electron microscopy plays here a major role and careful search for cross-striations on semi-thin sections is certainly necessary.
Summary During light microscopic examination of biopsy material from a canine tumor in the thigh, cells of muscle-type appearance were seen and the possibility of rhabdomyosarcoma was considered. A few indistinct cross-striations were seen in paraffin sections and in 2 ,LA sections of plastic-embedded material they could be demonstrated more clearly. A detailed electron microscope study revealed muscle cells in different stages of myogenesis and removed all doubts about the diagnosis of rhabdomyosarcoma, since distinct cross-striations with A, I and Z bands typical for striped muscle were seen. The fact that tumor cells with cross-striations could also be found in a lung- and lymph node metastasis excluded the possibility that they were elements of degenerating or regenerating muscle tissue.
550
MEYVISCH, THOONEN and HOORENS
Acknowledgements Appreciation is expresscd to Mr. J. P. LOGGHEfor technical assistance with ultramicrotomy and with the illustrations. We are indebted to the “Clinic of Small Animals” (Director: Prof. Dr. D. MATTHEEUWS) for supplying the clinical history of the case. Zusammenfassung Die Rhabdomyosarkom-Ultrastruktur des Hundes Bei der lichtmikroskopischen Untersuchung von Biopsiematerial eines Schenkeltumors eines Hundes wurden muskelzelltypische Elemente gefunden und daher das Vorliegen eines Rhabdomyosarkoms vermutet. In Paraffinschnitten konnten undeutliche Querstreifungen festgestellt werden, welche in Schnitten von in Plastik eingebettetem Material deutlicher zur Darstellung kamen. Elektronenoptische Untcrsuchungen lieflen quergestreifte Muskelzellen mit A-, J- und 2-Banden i n verschiedenen Phasen der Myogenese erkennen, womit das Vorliegen eines Rhabdomyosarkoms gesichert war. D a quergestreifte Tumorzellen auch in Lungen- und Lymphknotenmetastasen gefunden wurden, handelte es sich bei den fraglichen Tumorzellen nicht um degeneriertes oder regeneriertes Muskelzellgewebe. Resume Ultrastructure du rhabdomyosarcome du chien Une recherche au microscope optique avec un matkriel d’une biopsie d’une tumeur d’une cuisse chez un chien a montrk les ClCments cellulaires musculaires typiques permettant de soupsonner un rhabdomyosarcome. Les striations furent peu nettes avec les coupes faites dans la paraffine; elles furent meilleures avec le matkriel coulk dans du plastique. Les recherches au microscope klectronique ont permis de reconnattre les cellulet; musculaires strikes avec des bandes A, J et 2 dans diffkrentes phases de la rriyogknkse; cela a confirmk la prksence d’un rhabdomyosarcome. Etant donnk que des cellules tumorales strikes furent kgalement trouvkes dans des mktastases pulmonaires et ganglionnaires, il ne s’agissait pas de tissu musculaire dkgknkrC ou rkgCnCrk. Resumen La ultraestructura del rabdomiosarcoma en el perro Con ocasi6n del examen fotomicrosc6pico de material de biopsia de un tumor sito en el muslo de un perro, se hallaron elementos tipicos de cklula muscular, sospechindose la pres’encia de un rabdomiosarcoma. Se pudieron apreciar en cortes de parafina unas pocas estriaciones transversales indistintas, que se logra demostrar con una claridad mayor en secciones de material encamado en plistico. Los eximenes electr6nico6pticos permitieron reconocer cklulas musculares estriadas con bandas A, J y C en fases diversas de miogknesis, con lo cual se hallaba asegurada la presencia de un rabdomiosarcoma. Puesto que se encontraron tambikn cklulas tumorales estriadas en metistasis pulmonares y ganglionares, en las cklulas tumorales problemiticas no se trataba de tejido miocelular degenerado o regenerado.
The Ultrastructure of Rhabdomyosarcoma in a Dog
551
References BOCKER,W., and H. E. STEGNER, 1975: Mixed Miillerian tumors of the uterus. Ultrastructural studies on the differentiation of Rhabdomyoblasts. Virchows Archiv Abt. A Path. Anat., 363, 337-351. BRODEY,R. S., 1971 : Hypertrophic osteoarthropathy in the dog. A clinicopathologic survey of 60 cases. Journal of the American Veterinary Medical Association, 159, 1242-1256. CLARKE, M. A., 1969: The fine structure of methylcholanthrene-induced tumors in mice. Cancer (Phil.), 24, 147-157. COLLET,MM. P., and H. TISSEUR, 1949: Sarcome primitif du droit andrieur de la cuisse chez un chien. Bulletin de la SociCtC des Sciences VCtCrinaires de Lyon, 5 2 , 1-7. FRIEDMAN, I., D. F. M. HARRISON, M. M. TUCKER and E. S. BIRD, 1965: Electron microscopy of a rhabdomyosarcoma of the ear. Journal of Clinical Pathology, 18, 63-68. FRIEDMAN, I., and E. S. BIRD, 1969: Electron microscope investigation of experimental rhabdomyosarcoma. Journal of Pathology, 97,375-382. HALLIWELL, W. H., and N. ACKERMAN, 1974: Botryoid rhabdomyosarcoma of the urinary bladder and hypertrophic osteoarthropathy in a young dog. Journal of the American Veterinary Medical Association, 165, 911-913. HOLTZER, H., 1970: Myogenesis. In Cell Differentiation (Schjeide, 0. H., and J. de Vellis, Eds.). Van Nostrand Reinhold Company, New York. HORVAT, B. L., M. CAINES and E. R. FISHER,1970: The ultrastructure of rhabdomyosarcoma. American Journal of Clinical Pathology, 53, 555-564. ISHIKAWA, H., R . BISCHOFF and H. HOLTZER,1968: Mitosis and intermediate sized filaments in developing skeletal muscle. The Journal of Cell Biology, 38, 538-556. KELLY,D. F., 1973: Rhabdomyosarcoma of the urinary bladder in dogs. Veterinary Pathology, 10, 375-384. KORENYI-BOTH, A., K. LAPISund M. GALLAI,1968: Uber die Feinstruktur des undifferenzierten, adulten, pleomorphen rhabdomyosarkoms. Beitrage zur Pathologischen Anatomie, 138, 96-108. KROLL,A. A., 1967: Fine structural classification of orbital rhabdomyosarcoma. Investigative Ophthalmology, 6, 531-543. LADDS,P. W., and D. R. WEBSTER, 1971 : Pharyngeal rhabdomyosarcoma in a dog. Veterinary Pathology, 8, 256-259. MORALES, A. R., G. FINE and R. C. HORN,1972: Rhabdomyosarcoma: an ultrastructural appraisal. Pathology Annual 7, London Butler worths. OSBORNE, C. A., D. G. LOW, V. PERMAN and D. M. BARNES,1968: Neoplasms of the canine and feline urinary bladder: Incidence, etiologic factors, occurrence and pathologic features. American Journal of Veterinary Research, 29, 2041-2053. PETER,C. P., and J. P. KLUGE,1970: An ultrastructural study of a canine rhabdomyosarcoma. Cancer, 26, 1280-1288. SEIBOLD, H. R., 1974: Juvenile alveolar rhabdomyosarcoma in a dog. Veterinary Pathology, 11, 558-560. SPURR,A. R., 1969: A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research, 26, 31-43. STAMPS,P., and D. L. H A R R I S , 1968: Botryoid rhabdomyosarcoma of the urinary bladder of a dog. Journal of the American Veterinary Medical Association, 1 5 3 , 1064-1068. TEUNISSEN, G . H. B., and W. MISDORP, 1968: Rhabdomyosarcoma of the urinary bladder and fibromatosis of the extremities in a young dog. Zbl. Vet. Med., Reihe A, 15, 81-88. WEBB,J. N., 1971 : The development of human skeletal muscle with particular reference to muscle cell death. Journal of Pathology, 106,221-228. WORLEY,G., and J. R. GORHAM,1954: The comparative pathology of rhabdomyosarcoma with a report of a case in a dog. American Journal of Pathology, 30, 837-849. Address of authors: Drs. C. MEYVISCH, H. THOONEN, Prof. Dr. J. HOORENS, Dept. of Veterinary Pathology, State University of Ghent, Casinoplein 24, B-9000 Ghent/Belgium.
