Demonstration of the Muscle Type of Intermediate Filament Protein, Desmin, as a Diagnostic Aid

Transcription

1 Alveolar Rhabdomyosarcoma Demonstration of the Muscle Type of Intermediate Filament Protein, Desmin, as a Diagnostic Aid M. MIETTINEN, MD, V.-P. LEHTO, MD, R. A. BADLEY, PhD, and I. VIRTANEN, MD From the Department of Pathology, University of Helsinki, Haartmaninkatu 3, SF Helsinki 29, Finland and Unilever Research, Colworth Laboratory, Shambrook, Bedford, United Kingdom Three cases of soft-tissue sarcomas with the characteristic histologic features of alveolar rhabdomyosarcoma, but lacking cytoplasmic cross-striations, were studied ultrastructurally and immunohistochemically to confirm the diagnosis and evaluate the histogenesis. The results showed that it was not possible to judge the skeletal muscle derivation of the cells at the ultrastruc- tural level. However, immunohistochemically, the results ofevery case were positive for desmin-the muscle type of the intermediate filament protein. The results suggest that demonstration of desmin may be a helpful adjunct tool in the diagnosis of poorly differentiated alveolar rhabdomyosarcomas. (Am J Pathol 1982, 108: ) ALVEOLAR RHABDOMYOSARCOMA was introduced by Riopelle and Thieriault' and by Enterline and Horn2 as a round cell sarcoma with peculiar alveolar-like arrangement of the tumor cells between the fibrous septa. Larger series have since shown that only a minority of neoplasms with such histologic features show cross-striations in the cells, and thus the ascertainment of the skeletal muscle nature of these tumors has remained difficult.3 In the majority of the cases,46 the same problem also is obvious at the ultrastructural level. Therefore, it may be asked whether all these neoplasms are truly of skeletal muscle cell origin. In this report we demonstrate the presence of the desmin type of cytoskeletal intermediate filaments in 3 cases of alveolar rhabdomyosarcoma. The presence of these filaments reflects the muscle cell derivation of these tumors and demonstrates the usefulness of anti-desmin antibodies as a tool in the diagnosis of poorly differentiated sarcomas. Materials and Methods Three cases of alveolar rhabdomyosarcoma were studied immunohistochemically and by means of light and electron microscopy. The following are short case reports. Case 1: An 11-year-old girl had a swelling at the base of the nose, and a subcutaneous tumor 2 cm in diameter was extirpated. Two local recurrences developed within a year. One of the recurrences was available for this study. Case 2: A 23-year-old woman had noticed a tumor 3 cm in diameter on her left foot. Simultaneously, she found an inguinal tumor, which was interpreted histologically as a metastasis. The tumor from the foot was extirpated and studied in detail. Case 3: A 17-year-old girl had noticed a tumor that had grown rapidly in the vulva. The tumor, which was 6 cm in diameter, was removed. The patient died 8 months later because of widespread metastases. Paraffin blocks were available for all of the tumors. Hematoxylin and eosin (H&E), phosphotungstic acid hematoxylin (PTAH), and periodic acid-schiff (PAS) staining with and without prior diastase treatment was used in all cases. Supported by grants from the Finnish Medical Research Council and the Association of Finnish Life Insurance Companies. Accepted for publication April 2, Address reprint requests to M. Miettinen, MD, University of Helsinki, Haartmaninkatu 3, SF Helsinki 29, Finland /82/ $01.10 i American Association of Pathologists 246

2 Vol. 108 * No. 2 Specimens for electron-microscopic examination were taken from formalin-fixed tissue in 2 cases and from a dewaxed piece of a paraffin block in 1 case. Small tissue pieces were postfixed in 0.507o osmium tetroxide in 0.1 M phosphate buffer, at ph 7.2; dehydrated in acetone, and embedded in Epon 812. Five to 10 blocks were made in each case, and a cellular area was chosen for thin sections with the use of semithin sections of 11 1 stained with toluidine blue. Thin sections were poststained with lead citrate and uranyl acetate and examined in a Hitachi HS-7S electron microscope at an acceleration voltage of 50 kv. One block and 3-5 grids of each case were examined. Desmin was purified from chicken gizzards according to Small and Sobieszek.' Antiserum was raised in rabbits and purified on a desmin-sepharose CL4B column.8 The purified antiserum stained typically frozen sections of skeletal muscle and cultured rhabdomyosarcoma cells, but not fibroblasts or fibrosarcoma cells,10 fibrosarcoma, or adipose tissue sections ALVEOLAR RHABDOMYOSARCOMA 247 viewed by immunofluorescence microscopy. On the other hand, tumors consisting of vimentin-positive cells, like epithelioid sarcoma, were negative for desmin.9 Similarly, no cross-reactivity of the desmin antibodies with keratin, neurofilament proteins, or glial fibrillary acidic protein could be found.10 Immunohistochemical staining for desmin was performed as follows. The 3-5 ua-thick paraffin sections were deparaffinized and treated with 0.4% pepsin in 0.01 N HCl for 2 hours. Endogenous peroxidase was blocked by incubation of the slides for 30 minutes in methanol containing 0.20% H2O2. After being washed, the slides were incubated with rabbit antidesmin antibodies (25 ig/ml). The slides then were exposed to a biotinylated antirabbit immunoglobulin antiserum, (dilution 1: 500), avidin (dilution 1: 1000), and biotinylated horseradish peroxidase complex.11 The reagents were purchased from Vector Laboratories (Vectastain, Burlingame, Calif). We developed the peroxidase reaction by in- Figure 1-Alveolar rhabdomyosarcoma shows round, medium-sized, relatively uniform cells with formation of alveolar or glandlike structures between the connective tissue septa. No rhabdomyoblastic features are evident light-microscopically. (H&E, x 400) (With a photographic reduction of 2%)

3 248 MIETTINEN ET AL cubating the slides in 0.01% H202 and '3- diaminobenzidine tetrahydrochloride (Fluka AG, Buchs, Switzerland) for 10 minutes in the dark. The slides were counterstained for nuclei with Meyer's hematoxylin for 1 minute, rinsed, and mounted in Aquamount. The results of control stainings, made with preimmune serum or serum run through the desmin-sepharose 4B column, were negative. Light Microscopy Results The tumors were closely similar at the light microscopic level, showing rounded, medium-sized uniform cells with diffusely hyperchromatic nuclei, usually containing inconspicuous nucleoli and having sparse, but occasionally more copious, eosinophilic cytoplasm. The cell cohesion often was poor, and the clefts had an alveolar or glandlike appearance (Figure 1). At some areas, the tumors had a solid pattern (Figure 2). Thick fibrous septa containing blood vessels irregularly lobulated the tumor tissue. Mitoses were numerous; there were three to four in the highpower field in the cellular areas. In 2 cases, the tumor infiltrated and destroyed adjacent striated muscle tissue. Many cells showed cytoplasmic diastase-labile PAS-positivity (ie, glycogen). Cytoplasmic cross-striations after PTAH staining were not found in any of the cases. Electron Microscopy (EM) The tumor cells were uniform in appearance, having rounded nuclei and evenly distributed chromatin. The cytoplasm was sparse and contained only few organelles, such as short strands of rough endoplasmic reticulum, and few mitochondria. Clusters of polyribosomes were often numerous. In 1 case, some neoplastic cells facing the collagenous stroma were endowed with a well-defined basal lamina (external lamina) (Figure 3). No specific signs suggesting rhabdomyosarcoma, such as myofilament bundles or sarcomeres, were seen in the cells of any of the tumors. Immunohistochemical Findings AJP * August 1982 All 3 cases showed cytoplasmic desmin positivity, especially in the cells lining the alveolar clefts. Some neoplastic giant cells with abundant cytoplasmic desmin staining were seen in 1 case (Figure 4). In solid 1w -to wir "I I.4 0-0? 0 -t.4p, 4 0 Is "N. Figure 2-Alveolar rhabdomyosarcoma may show a solid pattern of growth at places. (H&E, x 400)

4 Vol. 108 * No. 2 areas, the tumor cells stained positively for desmin only occasionally. The entrapped striated muscle cells were also positive for desmin (Figure 5). The results in fibrous septa were completely negative, and the background staining was minimal. Discussion The results and previous reports6 show that muscle derivation of poorly differentiated alveolar rhabdomyosarcomas is difficult to demonstrate by light and even by electron microscopy. However, the EM sampling was limited and no large cells (Figure 4) were found in the EM sections. In this study we show that desmin, the major subunit protein of the muscle type of intermediate filaments can be demonstrated immunohistochemically in paraffin-embedded tissues and used as a marker of muscle differentiation. However, desmin is not specific for skeletal muscle; it is also seen in some smooth muscle cells,"2 although not in all.'3'14 None of the histologic features of our cases, however, suggested leiomyosarcoma. ALVEOLAR RHABDOMYOSARCOMA 249 In this study, all 3 cases of alveolar rhabdomyosarcoma showed positive results for desmin. Because only a portion of the cells showed positive staining, it is understandable that ultrastructural methods showed no specific features. It seems likely that immunohistologic techniques are superior to ultrastructural methods in recognizing poorly differentiated cells of skeletal muscle derivation that may lack typical ultrastructural organization. Moreover, immunohistologic techniques more easily enable a wide and rapid sampling of specimens. Recently, Gabbiani et all' have shown desmin positivity in rhabdomyosarcoma by the immunofluorescence technique, and Virtanen et al'0 showed desmin positivity in cultured rhabdomyosarcoma cells. A precaution must be taken into account, however, when one is using immunohistochemical methods. One must ascertain that the cells evaluated for the diagnosis truly are neoplastic cells, because, for example, entrapped muscle cells also stain positively for desmin. The counterstain with hematoxylin is a valuable aid in the localization of positive staining in the tumor tissue. Recently, antibodies against different types of cy- Figure 3-At the ultrastructural level, the tumor cells are poorly differentiated with sparse organelles. No structures specific for a skeletal muscle cell can be discerned. This cell, facing a connective tissue septum, is endowed by a well-defined basal lamina (arrow). C = collagen. (x 23,000) (With a photographic reduction of 2%)

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6 Vol. 108 * No. 2 ALVEOLAR RHABDOMYOSARCOMA 251 toskeletal intermediate filament (IMF) proteins, have been used successfully in differential diagnosis of epithelial and mesenchymal liver tumors" and in the detection of glial cells in amniotic fluid in cases of anencephaly.17'18 In a recent survey, Gabbiani et all5 even proposed IMF antibodies as a general aid in distinguishing poorly differentiated tumors, because it appeared that tumor cells retained their specific type of IMF proteins.10 Myoglobin also has been shown to be a suitable marker for skeletal muscle differentiation19'20 with the use of immunoenzymatic technique. Corson and Pinkus20 showed myoglobin-positive neoplastic cells in 5 of 7 cases of alveolar rhabdomyosarcoma. Unlike desmin, myoglobin is specific for skeletal muscle and does not occur in smooth muscle cells. 18 It has been suggested recently that methods of immunofluorescence would be superior to methods based on immunoenzymatic techniques. 15 However, our results clearly show the usefulness of the peroxidase method in combination with conventional histologic staining, which enables the use of all histologic criteria, in addition to the immunohistochemical findings. We also found that the peroxidase technique used with untreated sections gave rather poor results, which could be markedly improved by protease treatment of the deparaffinized sections, as has been suggested previously with trypsin2'l22 and with pepsin.23 This method also appears to make it possible for one to carry out retrospective surveys using routine paraffin-embedded material. References 1. Riopelle JL, Theriault JP: Sur une forme Meconnue de sarcome des parties molles: Le rhabdomyosarcome alveolaire. Ann Anat Path 1966, 1: Enterline HT, Horn RC: Alveolar rhabdomyosarcoma: A distinctive tumor type. Am J Clin Pathol 1958, 29: Enzinger FM, Shiraki M: Alveolar rhabdomyosarcoma. An analysis of 110 cases. Cancer 1969, 24: Churg A, Ringus J: Ultrastructural observations on the histogenesis of alveolar rhabdomyosarcoma. Cancer 1978, 41: Mierau GW, Favara BE: Rhabdomyosarcoma in children: Ultrastructural study of 31 cases. Cancer 1980, 46: van Haelst UJGM: General considerations on electron microscopy of tumors of soft tissues, Progress in Surgical Pathology. Vol. 2. Edited by CM Fenoglio, M Wolff. New York, Masson Publishing USA, 1980, pp Small, JV, Sobieszek A: Studies on the function and composition of the 10-nm (IOOA) filaments of vertebrate smooth muscle. J Cell Sci 1977, 23: Badley RA, Woods A, Carruthers L, Rees DA: Cytoskeleton changes in fibroblast adhesion and detachment. J Cell Sci 1978, 43: Miettinen M, Lehto VP, Vartio T, Virtanen I: Epithelioid sarcoma: Ultrastructural and immunohistochemical features suggesting a synovial origin. Arch Pathol Lab Med (In press) 10. Virtanen I, Lehto VP, Lehtonen E, Vartio T, Stenman S, Kurki P, Wager 0, Small VJ, Dahl D, Badley RA: Expression of intermediate filaments in cultured cells. J Cell Sci 1981 :50, Guesdon JL, Ternync T, Avrameas S: The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1978, 27: Lazarides E: Intermediate filaments as mechanical integrators of cellular space. Nature 1980, 283: Frank ED, Warren L: Aortic smooth muscle cells contain vimentin instead of desmin. Proc Natl Acad Sci USA 1981:78, Gabbiani G, Schmid E, Winter S, Chaponnier C, de Chastonay C, Vandekerckhove J, Weber K, Franke WW: Vascular smooth muscle cells differ from other smooth muscle cells: Predominance of vimentin filaments and a specific-type actin. Proc Natl Acad Sci USA 1981:78, Gabbiani G, Kapanci Y, Barazzone P, Franke WW: Immunohistochemical identification of intermediatesized filaments in human neoplastic cells: A diagnostic aid for the surgical pathologist. Am J Pathol 1981, 104: Bannasch P, Zerban H, Schmid E, Franke WW: Characterization of cytoskeletal components in epithelial and mesenchymal liver tumors by electron and immunofluorescence microscopy. Virchows Arch 1981:36, Aula P, von Koskull H, Teramo K, Karjalainen 0, Virtanen I, Lehto VP, Dahl D: Glial origin of rapidly adhering amniotic fluid cells. Br Med J 1980:281, von Koskull H, Virtanen I, Lehto VP, Vartio T, Dahl D, Aula P: Glial and neuronal cells in amniotic fluid of anencephalic pregnancies. Prenatal Diagnosis 1981:1, Mukai K, Rosai J, Hallaway BE: Localization of myoglobin in normal and neoplastic human skeletal muscle cells using an immunoperoxidase method. Am J Surg Pathol 1979, 3: Corson JM, Pinkus GS: Intracellular myoglobin-a specific marker for skeletal muscle differentiation in soft tissue sarcomas: An immunoperoxidase study. Am J Pathol 1981, 103: Huang SN: Immunohistochemical demonstration of hepatitis B core and surface antigens in paraffin sections. Lab Invest 1975, 33: Denk H, Radazkiewitxz T, Witting CH: Immunofluorescence studies on pathologic routine material: Application to malignant lymphomas. Beitr Pathol 1976, 159: Brozman M: Immunohistochemical analysis of formaldehyde- and trypsin- or pepsin-treated material. Acta Histochem 1978, 63: Acknowledgments The skillful technical assistance of Ms. Raili Taavela and Ms. Pipsa Kaipainen is kindly acknowledged. Figure 4-Immunohistochemical staining for desmin shows cytoplasmic positivity in many cells. A multinucleated neoplastic giant cell with ample cytoplasm shows strong positivity (arrow). (Anti-desmin, counterstain with hematoxylin, x 400) Figure 5-Immunohistochemical staining for desmin. Entrapped striated muscle cells (left) show positive staining. The solid tumor area (right) shows some positively staining cells. (Anti-desmin, counterstained with hematoxylin, x 400)