Soft Tissue Sarcomas - Europe PMC

American Journal of Pathology, Vol. 144, No. 4, April 1994 Copyright © American Society for Investigative Pathology

Myogenic Regulatory Protein Expression in Adult Soft Tissue Sarcomas A Sensitive and Specific Marker of Skeletal Muscle Differentiation

Giovanni Tallini,* David M. Parham,t § Peter Dias,t Carlos Cordon-Cardo,* Peter J. Houghton,t and Juan Rosai* From the Department of Pathology,* Memorial Sloan-Kettering Cancer Center, New York, New York; the Departments of Pathology,t and Biochemical & Clinical Pharmacology,* St. Jude Children's Research Hospital, Memphis, Tennessee, and the Department of Pathology,5 The University of Tennessee, Memphis, Tennessee

Myogenic regulatory protein (MyoDi) is a DNAbinding nuclear protein that initiates myogenesis in mesenchymal stem ceUs. Its expression has proved a very sensitive marker of myogenic differentiation in malignant tumors of childhood. In this study, the reliability ofMyoD1 expression as marker of skeletal muscle differentiation has been tested in 38 cases of adult-type sarcomas. Frozen sections were stained with the monoclonal antibody 5.8A (MAb 5.8A) developed against recombinant wild-type murine MyoD1. Routinely processedformalin-fxed sectionsfrom the same cases were reviewed and immunostained with a panel of antibodies commonly used to detect muscle differentiation. myoglobin, fast-myosin, desmin, muscle-specifrc actin, a-smooth muscle actin. Four cases positively stained with MAb 5.8A. three were high-grade spindle ceU sarcomas, the fourth case was an alveolar soft-part sarcoma. Only tumors in which skeletal muscle histogenesis was inferred on the basis of histology and/or immunohistochemistry with conventional muscle markers stained positively for MAb 5.8A. Al the other tumors tested, including leiomyosarcomas (11), liposarcomas, (10, including four dedifferentiated liposarcomas), flbrosarcomas (three), malignantftbrous histiocytomas (two), synovial sarcomas (two), and malignantperipheral nerve sheath tumors (two),

were negativefor MAb 5.8A. The high level of sensitivity and specijtcity of MyoD1 expression indicates the value of this marker in the diagnosis of soft tissue sarcomas. (AmiJPathol 1994, 144:

693-701)

Rhabdomyosarcomas represent a heterogeneous group of mesenchymal neoplasms in which the dominant line of differentiation is that of rhabdomyoblasts. Considerable differences in age of incidence, site of origin, cytogenetics, and clinical behavior exist among the different histological subtypes of rhabdomyosarcomas (ie, embryonal, botryoid, alveolar, and pleomorphic) popularized by Horn and Enterline more than 30 years ago1 and since then generally accepted.2 The first subtype to be recognized was the rhabdomyosarcoma of skeletal muscle, which corresponds to the pleomorphic (or adult type) of the current classification.3 The tumor (as the original name implied) has an obvious anatomic origin from myotome-derived striated muscle and is therefore usually located in an extremity, typically affects middle-aged adult patients, and has to be differentiated from other much more common pleomorphic sarcomas such as malignant fibrous histiocytoma or liposarcoma. It soon became apparent that this neoplasm has a "quite different if not opposite appearance" compared to the other varieties of rhabdomyosarcoma,4 which arise in children or adolescents. In contrast to pediatric rhabdomyosarcomas, the study of pleomorphic rhabdomyosarcomas of adult paSupported in part by National Cancer Institute (NCI) Cancer Center Support (CORE) grant P30 CA21765, NCI grant CA 23099, and by American Lebanese Syrian Associated Charities (ALSAC). Accepted for publication December 7, 1993. Address reprint requests to Dr. Juan Rosai, Chairman, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

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tients has been relatively neglected and the morphological and immunohistochemical criteria required for the diagnosis are less defined.5 During the past decade, extensive studies have revealed the existence of a family of genes, termed the "MyoD family", which encodes a series of DNAbinding proteins that act as nodal points for the initiation of muscle differentiation in mesenchymal cells.6 One of these genes, MyoDl, converts a number of different cell types into muscle cells.6 Because MyoDl expression in mesenchymal stem cells appears at the earliest stages of commitment to a striated muscle phenotype, a number of studies have investigated its activity in rhabdomyosarcoma. In these studies, MyoDl protein has proved to be the most sensitive marker for detection of myogenic differentiation in "small, round, blue-cell tumors of childhood", including desmin-negative undifferentiated neoplasms.7 Monoclonal antibodies (MAbs) to the MyoDl protein have been developed and characterized for diagnostic purposes.8 In the present study, we further evaluate the utility of MyoDl as a diagnostic marker of rhabdomyosarcoma in other soft tissue sarcomas of adults using MAb 5.8A.

Materials and Methods Frozen tissue from 38 cases diagnosed as adult mesenchymal neoplasms between 1985 and 1990 were obtained from the tumor bank of the Department of Pathology of Memorial Hospital, (see Tables 1 and 2). These tissues had been snap-frozen within 1 hour of surgery and stored at -70 C. Criteria for selection of cases were: availability of adequate frozen tissue samples, histological diagnosis of sarcoma, and patient's age at time of diagnosis of 18 or older. The age (range: 18 to 87 years; median: 52 years) and sex distribution (22 males; 16 females) of the patients, the site of the tumors (23 trunk/retroperitoneum, 13 extremities, two head/neck), and the histological diagnoses are representative of the adult soft tissue sarcomas seen at Memorial Hospital and are comparable to those of reported series from other institutions.2 Tissue from two pediatric embryonal rhabdomyosarcomas, one lung adenocarcinoma, one infantile myofibromatosis, and one adult fibromatosis were added to the study as additional controls. Cryostat sections from the cases selected for the study were stained with MAb 5.8A developed against recombinant wild-type murine MyoDl protein. The MAb 5.8A, developed and characterized by Dias et al, is an immunoglobulin G1 antibody that recognizes an epitope in the carboxyl terminus region of the MyoDl protein (amino acid residues 180 to 189).8 Immunoperoxidase stains with MAb 5.8A were performed in the laboratory of the Department of Pathol-

ogy at St. Jude Children's Research Hospital without prior knowledge of the histological diagnosis. All of the histology slides and the pathology reports in the files of Memorial Hospital from the cases selected for this study were reviewed in parallel, without knowledge of MyoDl expression results. In cases in which the original histological diagnosis was questionable, new hematoxylin and eosin (H&E) sections were cut and additional slides were stained with a panel of conventional MAb that included vimentin, desmin, muscle-specific actin (HHF35), a-smooth muscle actin, myoglobin, and skeletal fast myosin.

Immunohistochemistry Cryostat Sections/MYOD1-IPX Six-p cryostat sections were obtained from freshfrozen blocks of tumor stored at -70 C. The blocks were prepared by immersing fresh tissue embedded in OCT (Miles, Elkhart, IN) into liquid nitrogen. Once obtained, particular care was paid to prevent thawing of the sections by keeping them in styrofoam containers packed with dry ice. These were shipped via overnight mail to Memphis, TN, where MyoDl immunostaining was performed. After storage at -70 C, the sections were immersed in 1 % v/v paraformaldehyde in phosphate-buffered saline, pH 7.4, for 20 minutes. MyoDl immunostaining was then performed with MAb 5.8A as previously described.8 Staining was accomplished by using an Elite Vectastain kit (Vector Laboratories, Burlingame, CA). Light green SF (Eastman, Rochester, NY) was used as counterstain. A case of embryonal rhabdomyosarcoma was used as a positive MyoDl control. For negative controls, the immunoglobulin G1 antibody anti-CD45RB (anti-leucocyte common antigen, dilution 1:100, clone PD7/26, Dako Corp., Carpinteria, CA) was substituted for MAb 5.8A. Paraffin Sections Routinely processed, formalin-fixed sections were immunostained using a streptavidin biotinylated horseradish peroxidase technique9 with MAbs to: vimentin (Dako, 1:100 dilution), desmin (Dako, 1:200 dilution), HHF35 (Enzo, 1:20,000 dilution), a-smooth muscle actin (Sigma Chemical Co., St. Louis, Ml 1:20,000 dilution), myoglobin (Biogenics 1:3,000 dilution), skeletal fast myosin (Sigma 1:100,000).

Results The results of immunostaining are listed in Tables 1 and 2. Four cases of the adult soft tissue sarcomas in

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Table 1. MyoDi-Positive Cases

Original diagnosis

Case # 1

Age/sex 65M

Arm

Rhabdomyosarcoma

2

64M

Thigh

3

18M

Arm

4

31 M

Chest wall

Malignant fibrous histiocytoma (high grade) Leiomyosarcoma (high grade) Alveolar soft part sarcoma

VIM

vimentin; MSA

Location

=

muscle-specific actin (HHF35); DES

=

Table 2. MyoDl-Negative Cases

Diagnosis Leiomyosarcoma (including gastrointestinal stromal tumor) Liposarcoma Fibrosarcoma Malignant fibrous histiocytoma Malignant peripheral nerve sheath tumor Synovial sarcoma Ewing/undifferentiated small cell sarcoma Angiosarcoma Chondrosarcoma (extraskeletal)

Number of cases 11

10 3 2 2 2 2 1 1

this study stained positively with MAb 5.8A (Table 1; Figures ld, 2d, 3d, 4d) and are described below in detail.

Case 1 This was a high-grade sarcoma that developed in the posterior aspect of the right arm of a 65-year-old male. The tumor measured 8 x 6 x 5.5 cm and was described as a fairly well circumscribed mass with a lobulated, tan cut surface, and foci of glistening myxoid tissue. Microscopically, most of the tumor was very cellular and consisted of uniform undifferentiated cells with ovoid to spindled nuclei and a relatively small amount of cytoplasm. Up to 12 mitoses could be counted per high-power field (HPF, 400x). In some areas, the cells were more elongated, displaying a bright eosinophilic fibrillary cytoplasm and a fascicular arrangement (Figure la). Rare isolated cells possessed large bizarre nuclei and abundant eosinophilic cytoplasm. Paucicellular myxoid foci and "geographic" areas of necrosis were also present. The original diagnosis was "high-grade spindle and giant cell sarcoma, best classified as rhabdomyosarcoma". Ultrastructural examination of a sample of tissue from areas of the tumor with a predominance of undifferentiated cells was non-contributory. Strong reactivity for vimentin, HHF35, and desmin (Figure 1, b and c) demonstrated muscular differentiation of the tumor, but a differential diagnosis between rhabdo- or

Revised

diagnosis

Positive immunostains

Myosarcoma (leiomyosarcoma vs. rhabdomyosarcoma) Myosarcoma, consistent with rhabdomyosarcoma

VIM, MSA, DES

Rhabdomyosarcoma, spindle cell type Same

VIM, MSA, DES, myoglobin, myosin SMA (focal), DES

VIM, DES, SMA (focal)

(punctate) desmin; SMA

=

a-smooth muscle actin.

leiomyosarcoma was not possible (a-smooth muscle actin, fast myosin, and myoglobin were all negative). Therefore, our revised diagnosis was that of myosarcoma, not further specified. At the last follow-up, the patient was alive with lung metastasis, 14 months after the original diagnosis.

Case 2 This tumor was located in the anterior aspect of the left thigh of a 64-year-old male patient. The mass was 18 x 15 x 8 cm and well circumscribed, with a tan, fleshy cut surface and large areas of necrosis. Histologically, cells were predominantly spindled and arranged in poorly defined fascicles; numerous nuclei were hyperchromatic and pleomorphic (Figure 2a), with up to 11 mitoses per HPF. The cytoplasm of the larger cells was deeply eosinophilic, but cross striations were not identified. The first biopsy specimen was diagnosed as "high-grade leiomyosarcoma". This diagnosis was later changed to "malignant fibrous histiocytoma" after evaluation of the resection specimen, immunoperoxidase stains, and noncontributory electron microscopic results. After review of the original slides, additional blocks were cut and immunostained. The majority of the tumor cells showed an intense dotlike paranuclear positivity for desmin (Figure 2c). In addition, diffuse desmin reactivity was present in the larger pleomorphic cells (Figure 2c). Rare isolated neoplastic elements stained with a-smooth muscle actin (Figure 2b). The other muscle markers were negative. The tumor was reclassified by us as "myosarcoma, compatible with pleomorphic rhabdomyosarcoma". At the last follow up visit, the patient was alive with metastases to the lung, 12 months after the original diagnosis.

Case 3 This case, had been originally diagnosed as "fibrosarcoma, low grade". This diagnosis had been changed first to "synovial sarcoma" after multiple lo-

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Figure 1. Case 1: Histological appearance of the spindle cell sarcoma (a, H&E, 200x). Thve tumor is strongly positive for HHF35 (b, 200x ) and desmin (C, 200X). Staining with MAb 5.8A (d, 200X).

cal recurrences, and later to that of "leiomyosarcoma, high grade", on the basis of a strong, diffuse positivity for HHF35. The tumor was located on the right forearm of an 18-year-old male patient, was very firm, with a tan-white, irregularly whorled cut surface, and measured 16 cm in greatest diameter. Histologically, the lesion consisted of bundles and small fascicles of elongated cells with uniform ovoidal nuclei and relatively abundant, coarse, eosinophilic cytoplasm (Figure 3a). Nineteen mitoses were counted per 10 HPF No tissue was available for electron microscopy. After an initial dramatic response to chemotherapy, amputation of the right arm was performed because of uncontrollable local recurrences. At the last follow-up, the patient was alive with a large recurrent tumor in the chest wall and axillary lymph node metastasis, 53 months after the first diagnosis. All the original slides were reviewed and additional blocks were cut and immunostained. The tumor reacted positively with all the conventional markers used in this study (vimentin, desmin, muscle-specific actin, myoglobin, fast myosin), with the exception of a-smooth muscle actin (Figure 3, b and c). The diagnosis was therefore changed by us to "rhabdomyosarcoma, spindle cell type".10

Case 4 This tumor had been removed from the soft tissues of the chest wall of a 31 -year-old male patient. The lesion was 10.5 cm in greatest diameter, with a fleshy, tanpink cut surface. Microscopically and ultrastructurally, the typical nesting architecture and cytological appearance (Figure 4a), plus the characteristic membrane-bound dense and rhomboid granules seen ultrastructurally were typical of alveolar soft part sarcoma. The patient was alive with lung metastasis, 73 months after the original diagnosis. Staining of additional sections with our panel of MAb disclosed rare tumor cells positive for a-smooth muscle actin (Figure 4b). Desmin (Figure 4c) and vimentin showed dotlike paranuclear positivity in a minority of tumor cells. Other muscle markers were negative.

Other Tumors All other tumors were negative for MyoD1 (Table 2). Reactivity for vimentin, desmin, HHF35, and ae-smooth muscle actin, confirmed the smooth muscle

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Figure 2. Case 2: Histological appearance of the pleomorphic spindle cell sarcoma (a, H&E, 200X). actin (b, 400X) and diffuselyfor desmin (C, 400X). Staining with MAb 5.8A (d, 200X).

origin of the 11 cases of leiomyosarcomas. Focal positivity for a-smooth muscle actin and/or desmin, in addition to vimentin positivity, was detected in other MyoDl -negative high-grade sarcomas (four cases of dedifferentiated liposarcoma and one case of malignant peripheral nerve sheath tumor). The remaining sarcomas studied were vimentin-positive but negative for all the muscle markers. The two cases of pediatric rhabdomyosarcoma, included in the study as additional controls, expressed the MyoDl antigen. The first, a classic embryonal rhabdomyosarcoma, also expressed vimentin, desmin, and HHF35; the second, diagnosed as pleomorphic subtype of embryonal rhabdomyosarcoma, co-expressed desmin, HHF35, a-smooth muscle actin (focally), myoglobin, and fast myosin.

Discussion This study demonstrates the remarkable degree of specificity and sensitivity of MyoD1 as marker of skeletal muscle differentiation and its potential value in the proper classification of soft tissue sarcomas. Of the 38 cases analyzed, only three spindle cell sarcomas

The tumor reacts focally with a-smooth muscle

expressed MyoDl. In all of them, myogenic differentiation was confirmed after review of the cases. In one tumor diagnosed as "leiomyosarcoma" (case 3, see Table 1), staining of formalin-fixed sections with conventional markers convincingly demonstrated skeletal muscle differentiation and led to reclassification of this tumor as rhabdomyosarcoma. The remaining two sarcomas were high-grade tumors composed of a mixture of poorly or undifferentiated spindle cells with larger pleomorphic elements. They illustrate the difficulties sometimes encountered by the pathologist in the proper classification of these tumors. Features of myogenic differentiation may be present, but are so focal or so incompletely developed that the diagnosis often remains unsubstantiated. Limited samples as well as sampling errors greatly reduces the utility of electron microscopy. Similarly, the relative nonspecificity of desmin and actin positivity in the diagnosis of pleomorphic sarcomas reduces the value of these markers.11-13 In addition to spindle cell sarcomas, positivity for MAb 5.8A was detected in the only alveolar soft part sarcoma included in the study, thus confirming the relevance and specificity of previous

findings."4

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Figure 3. Case 3: Histological appearance of the spindle cell rhabdomyosarcoma (a, H&E, 200X). The tumor reacts positively for HHF35 (b, 200X) and desmin (C, 200X); myoglobin and fast myosin are also positive. Staining with MAb 5.8A (d, 200X ).

The diagnosis of rhabdomyosarcoma has always rested on the identification of neoplastic cells with features of striated muscle, ie, rhabdomyoblasts. In his original description of the cytological features of rhabdomyoblastic cells (rounded, strap-shaped, or racquet-shaped), Stout stated3 and later insisted15 that cytoplasmic cross- or longitudinal striations are a specific but not very sensitive morphological marker and therefore not strictly necessary for the diagnosis of rhabdomyosarcoma. Similarly, Masson concluded that pleomorphic rhabdomyosarcoma resulted from the "proliferation of degenerative elements incapable of reconstructing fibrils with double striations".4 Not surprisingly, the diagnosis of rhabdomyosarcoma became very popular in the 1950s and 1960s. The need for stricter criteria was expressed by Enterline1 and by Willis who advocated "great caution in making a diagnosis of rhabdomyosarcoma in an adult, save on impeccable grounds".16 The identification of cytoplasmic cross striations became a prerequisite for the classification of a pleomorphic neoplasm as rhabdomyosarcoma.17 The wide acceptance of malignant fibrous histiocytoma since the late 1970s and the introduction of immunohistochemistry as a routine complement to histological diagnosis "reduced the

pleomorphic rhabdomyosarcoma ranks to a few rare collector cases".18 Only in a small minority of tumors previously interpreted as rhabdomyosarcoma on the basis of conventional histology could the diagnosis be confirmed immunohistochemically,5 19,20 and the very existence of pleomorphic rhabdosarcoma was questioned. Desmin and HHF35 are sensitive markers of myogenic differentiation in rhabdomyosarcomas.21 Desmin is clearly detectable in human differentiating skeletal muscle cells as early as the eighth gestational week.22 However, both desmin and HHF35 are coexpressed in leiomyosarcomas and are of no help in distinguishing between striated and smooth muscle cells. Similarly, expression of actin isoforms is not entirely specific: a-smooth muscle actin is detectable in pleomorphic rhabdomyosarcomas with ultrastructural features of skeletal muscle differentiation23 and in embryonal rhabdomyosarcomas23 but can be undetectable in poorly differentiated leiomyosarcomas24; in addition, a-sarcomeric actin can be expressed in tumors classified as leiomyosarcoma on the basis of electron microscopy.24 In one of our MyoD 1-positive cases (case 1; see Table 1) that was positive for desmin and HHF35 but negative for the

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Figure 4. Case 4: Histological appearance of the alveolar soft part sarcoma (a, H&E, 400X). The tumorfocally reacts with a-smooth muscle actin (b, 400X) and desmin (C, 400X). Staining witb MAb 5.8A (d, 400X).

other muscle markers, the distinction between leioand rhabdomyosarcoma was not possible, nor could it be inferred from the histological features of the conventional H&E sections. The utility of desmin and actin in the diagnosis of pleomorphic rhabdomyosarcomas is also greatly limited by their expression in a substantial number of pleomorphic liposarcomas and malignant fibrous histiocytomas.1 1-13 The importance of a more careful search for evidence of specific lines of differentiation in pleomorphic sarcomas has recently been advocated,20 but in the case of skeletal muscle differentiation such evidence can be difficult to substantiate with the investigative techniques currently available. Of this difficulty Stout was aware, well before the introduction of immunohistochemistry or electron microscopy. In the original description of pleomorphic rhabdomyosarcoma, he discussed the often focal nature of muscle differentiation, "the vagaries found in tumor myoblasts", the variability in the expression of myofibrils in cultured neoplastic or embryonic myoblasts in which cytoplasmic striations "appear, disappear, and reappear within a cell, sometimes rapidly and sometimes after long intervals", and he advocated the use of the term "undifferentiated myosarcoma" for those tumors with incompletely developed muscular differ-

entiation.3 The primitive nature of neoplastic myoblasts limit the usefulness of markers specific for skeletal muscle differentiation (eg, myoglobin, heavychain myosin, tropomyosin). These markers are not sensitive enough and are usually negative in poorly differentiated rhabdomyosarcomas in children,21 25d28 in the positive cases, staining is usually detectable only in a minority of larger and better differentiated rhabdomyoblasts.2125 27 Yet, positivity for at least one of these skeletal muscle specific immunohistochemical markers (myoglobin, myosin etc.) is a generally accepted necessary requirement for the classification of a pleomorphic sarcoma as rhabdomyosarcoma. Commitment and maintenance of skeletal muscle differentiation is regulated by a family of closely related genes (eg, myogenin, myf-5, mrf-4-herculin), of which MyoDl is one of the better characterized members.6 These genes encode for myogenic regulatory proteins that include a basic domain capable of binding DNA and that share a conserved sequence motif (the "helix-loop-helix" region) responsible for homoor heterodimer formation with other regulatory proteins. Myogenic regulatory genes are capable of auto- and cross-activation, resulting in the production of large amounts of active regulatory proteins able to

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determine or maintain skeletal muscle differentiation,6 possibly through direct interaction with the retinoblastoma protein.29 The MyoDl protein is a 45-kd nuclear phosphoprotein that seems to activate myogenesis by directly binding to the enhancer sequence of muscle-specific genes.30 It is expressed in normal skeletal muscle cells and in their precursors (ie, myoblasts)31 and in alveolar and embryonal rhabdomyosarcomas.8,32 Expression of MyoDl was detected in the only three spindle cell sarcomas in this study which were independently reclassified as myosarcomas after review of the diagnostic material. In two of the cases (1 and 2), tumors lacked cross striations or expression of myoglobin and myosin, but in support of muscle differentiation was the general morphological appearance and positive staining for desmin and actin. Positivity for MAb 5.8A confirmed skeletal muscle differentiation. In fact, with the exception of the case of alveolar soft part sarcoma, expression of MyoDl paralleled histological evidence of skeletal muscle differentiation in our study. The other sarcomas with distinctive lines of differentiation (leiomyosarcomas, malignant peripheral nerve sheath tumor, etc.) were negative, and so were tumors of less well established histogenesis (eg, synovial sarcoma). In conclusion, the assumption that expression of a regulatory nuclear protein like MyoDl defines rhabdomyosarcomas at the molecular level, regardless of the extent of cytoplasmic differentiation in tumor cells, has been tested in a representative sample of adult soft tissue sarcomas. Only tumors that were reclassified as rhabdomyosarcomas by other criteria or in which a skeletal muscle histogenesis was inferred on the basis of histology or immunohistochemistry with conventional muscle markers stained positively for MAb 5.8A. Although the number of cases tested is relatively small, the results suggest that MyoDl expression is a useful diagnostic marker of rhabdomyosarcomas in adult as well as pediatric neoplasms.

Acknowledgments The authors would like to acknowledge Mary Ann Gangi, Hallie Holt, and Elisha Jenkins for the immunohistochemical stains.

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2. Enzinger FM, Weiss SW: Soft Tissue Tumors. The C.V. Mosby Company, St. Louis, MO, 1988, pp 4-5,448-449 3. Stout AP: Rhabdomyosarcoma of the skeletal muscles. Ann Surg 1946, 123:447-472 4. Masson P: Human Tumors: Histology, Diagnosis and Technique. Wayne State University Press, Detroit, Ml, 1970, pp 275-286 5. DeJong ASH, Van Kessel-Van Vark M, Albus-Lutter CE: Pleomorphic rhabdomyosarcoma in adults: immunohistochemistry as a tool for its diagnosis. Hum Pathol 1987, 18:298-303 6. Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Blackwell TK, Turner D, Rupp R, Hollenberg S, Zhuang Y, Lassar A: The myoD gene family: nodal point during specification of the muscle cell lineage. Science 1991, 251:761-766 7. Dias P, Parham DM, Shapiro DN, Webber BL, Houghton PJ: Myogenic regulatory protein (MyoD1) expression in childhood solid tumors: diagnostic utility in rhabdomyosarcoma. Am J Pathol 1990, 137:1283-1291 8. Dias P, Parham DM, Shapiro DN, Tapscott SJ, Houghton PJ: Monoclonal antibodies to the myogenic regulatory protein MyoDl: epitope mapping and diagnostic utility. Cancer Res 1992, 52:6431-6439 9. Cartun RW, Pedersen CA: An immunocytochemical technique offering increased sensitivity and lowered cost with a streptavidin-horseradish peroxidase conjugate. J Histotechnol 1989, 12:273-275 10. Cavazzana AO, Schmidt D, Ninfo V, Harms D, Tollot M, Carli M, Treuner J, Betto R, Salviati G: Spindle cell rhabdomyosarcoma: a prognostically favorable variant of rhabdomyosarcoma. Am J Surg Pathol 1992, 16:229-235 11. Lawson CW, Fisher C, Gatter KC: An immunohistochemical study of differentiation in malignant fibrous histiocytoma. Histopathology 1987, 11:375-383 12. Miettinen M: Antibody specific to muscle actins in the diagnosis and classification of soft tissue tumors. Am J Pathol 1988, 130:205-215 13. Hirose T, Kudo E, Hasegawa T, Abe J-l, Hizawa K: Expression of intermediate filaments in malignant fibrous histiocytomas. Hum Pathol 1989, 20:871-877 14. Rosai J, Dias P, Parham DM, Shapiro DN, Houghton P: MyoDl protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature. Am J Surg Pathol 1991, 15:974-981 15. Stout AP: Tumors of the soft tissues. Atlas of Tumor Pathology, fascicle 5. Armed Forces Institute of Pathology, 1953, pp 89-98 16. Willis RA: Pathology of Tumours, 3rd ed. London, Butterworths, 1960, pp 744-747 17. Enterline HT: Histopathology of sarcomas. Sem Oncol 1981, 8:133-155 18. Ross J, Hendrickson MR, Kempson RL: The problem of poorly differentiated sarcoma. Sem Oncol 1982, 9:467-483 19. Molenaar WM, Oosterhuis AM, Ramaekers FCS: The

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1986, 110:776-778 26. Schupham R, Gilbert EF, Wilde J, Wiedrich TA: lmmunohistochemical studies of rhabdomyosarcoma. Arch Pathol Lab Med 1986, 110:818-821 27. Schmidt D, Reimann 0, Treuner J, Harms D: Cellular differentiation and prognosis in embryonal rhabdomyosarcoma. Virchows Arch 1986, 409:183-194 28. Eusebi V, Ceccarelli C, Gorza L, Schiaffino S, Bussolati G: Immunocytochemistry of rhabdomyosarcoma: the use of four different markers. Am J Surg Pathol 1986, 10:293-299 29. Gu W, Schneider JW, Condorelli G, Kaushal S, Mahdavi V, Nadal-Ginard B: Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell 1993, 72:309-324 30. Lassar AB, Buskin JN, Lockshon D, Davis RL, Apone S, Hauschka SD, Weintraub H: MyoD is a sequencespecific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell 1989, 58:823-831 31. Davis RL, Weintraub H, Lassar AB: Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987, 51:987-1000 32. Scrable H, Witte D, Shimada H, Seemayer T, Wang-Wuu S, Soukup S, Koufos A, Houghton P, Lampkin B, Cavenee W: Molecular differential pathology of rhabdomyosarcoma. Genes Chromosom Cancer 1989, 1:23-25