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The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on proliferation and differentiation of the human embryonal rhabdomyosarcoma cell line RD was investigated. The proliferation of RD cells is drastically and reversibly inhibited by 100 nM TPA. The effect is evident after 24 h of treatment and is maximal after 50-70 h. The reduction of proliferation in treated cells is followed by increased expression of differentiative characters such as a large increase in muscle myosin expression and in the binding of 125I-alpha-bungarotoxin. Moreover TPA induces the appearance of myotube-like structures, which contain bundles of thick and thin myofilaments along with Z bodies. The described effects are not observed if the TPA-containing medium is replaced daily, thus suggesting that these effects might be related to substances secreted by treated cells. The phosphorylation of three proteins is significantly stimulated by TPA within minutes of its administration to RD cells. Although with a different pattern, the stimulation of protein phosphorylation is still clearly detectable after 6 days of incubation with TPA. These results on human rhabdomyosarcoma cells are, to our knowledge, the first evidence for a growth-inhibiting and a differentiative effect of TPA on a solid tumor of mesodermal origin.

The effects of mitomycin C (MTC) alone and in combination with radiation on R1H rhabdomyosarcomas in WAG/Rij rats were studied. Growth delay was determined from tumor volume growth curves and proportion of tumor and host cells from flow cytometry measurements. Only minor growth delays were obtained with MTC at doses near the toxic level while irradiation applied in three fractions of 10 Gy in three weeks yielded growth delays of 27, 44, and 51 days measured at volumes of 2, 5, and 10 cm3, respectively. The combination of 1 micrograms/kg b.w. MTC and 10 Gy irradiation given in three weekly fractions resulted in 21, 28, and 29 days growth delay for the above tumor volumes. The lower effect of combination as compared to irradiation alone is discussed with the flow cytometry data. These data show that early after start of treatment the proportion of tumor cells remains constant in contrast to radiation alone where the fraction of tumor cells decreases because of influx of granulocytes and macrophages into the tumor. In contrast to the reduced effect on tumors the combination treatment was considerably more toxic for the animals than the single treatments.

The diagnostic utility of several antibodies against desmin and their optimal staining conditions have not been systematically evaluated. Sections of paraffin-embedded tissues from 584 cases were stained with a monoclonal antibody against desmin (Clone DER 11 from DAKO), using an avidin-biotin-peroxidase technique. The results were tabulated and compared with those from previous reports. The following observations were made: (1) When pronase digestion was performed before staining, desmin was equally demonstrable in tissues fixed in formalin, Zenker's, Bouin's, or B5 fixative; however, desmin staining was lost or significantly diminished in tissues fixed in absolute ethyl alcohol. In contrast, when pronase was not used, a positive staining was demonstrated only in tissue fixed in absolute ethyl alcohol. (2) Positive staining was found in normal muscle (92 of 92 cases), leiomyoma (12 of 13), rhabdomyoma (6 of 6), rhabdomyosarcoma (31 of 31), leiomyosarcoma (18 of 26). (3) Desmin was never found in epithelia, normal mesenchymal tissue other than muscle, tumors stimulating rhabdomyosarcoma, and epithelial tumors. (4) A positive staining was documented in 1 tumor (a fibrous histiocytoma) of 42 benign predominantly spindle cell tumors and in 8 of 89 predominantly spindle cell sarcomas. (5) Desmin was never documented in myoepithelial cells but stained myofibroblasts in 2 of 12 examples of granulation tissue and in 29 of 67 samples containing tumor-associated desmoplasia. The authors' data on the diagnostic sensitivity and specificity of the evaluated antibody should improve the use of desmin in diagnostic pathology.

Weakly (RMS8) and highly (RMS0) metastatic rat rhabdomyosarcoma cells were assayed for their interaction with hyaluronate. The cells in subconfluent cultures were incubated with 35S methionine, the cells were fractionated and the labelled proteins were separated by affinity chromatography on hyaluronate-Sepharose and by HPLC. The RMS8 cells expressed about twice the amount of labelled hyaluronate-binding proteins seen in the RMS0 cells. The molecular sizes of the main hyaluronate-binding proteins were similar in both cell types. Unlike the RMS0 cells, the RMS8 cells took up exogenous, radioactively labelled hyaluronate at 4 degrees C in a saturable and specific way with high affinity. Cells were also incubated with 3H glucosamine. The isolation of the glycosaminoglycans from these cultures by ion-exchange chromatography indicated that the RMS8 cells retained more endogenous 3H hyaluronate in their pericellular domain than did the RMS0 cells. The attachment of trypsinized cells could be inhibited with exogenous hyaluronate, indicating that the proteins with affinity for hyaluronate may act as hyaluronate-binding sites on these cells.

Transplantable rat rhabdomyosarcoma RA-2 selected for affinity to lung tissue was cloned in vivo, and DNA contents of interphase nuclei was cytophotometrically measured in progenies of 121 clonogenic cells implanted in the lungs. Clone populations have shown a high karyotypic heterogeneity, karyotypic structure being unchanged from the 16th to the 28th selection cycles investigated. Karyotypic heterogeneity is reproduced due to the high rate of genome mutations in the progenies of clonogenic cells (11.10-2-14.10-2 per cell per generation). Cells with increased numbers of chromosomes arose 3 fold more frequently than cells with decreased chromosome numbers, tumor cell mutability being non-equal in different clones.

Using the indirect immunoperoxidase method, alpha 1-fetoprotein (AFP) can be demonstrated in paraffin sections of tumours by light-microscopy. Among 160 tumour preparations it was demonstrated in five of six endodermal sinus tumors, 19 of 40 teratomas, the three hepatoblastomas and 9 of 16 primary liver-cell carcinomas. Among 15 rhabdomyosarcomas, 15 Wilms tumours, 15 neuroblastomas and 15 other malignant tumours only one rhabdomyosarcoma contained AFP. No AFP was demonstrated in 35 various benign tumours and various other human tissue samples. Distribution of AFP-producing cells in tumour tissue is generally focal samples. In malignant teratomas AFP was found only in areas which corresponded to an endodermal sinus tumour or an embryonic carcinoma. The practical significance of the results consists of (1) demonstration and localisation of AFP in tumour tissue; (2) aiding the differential diagnosis and classification of tumours and their metastases; and (3) recognition of pathogenetic connection between embryonic carcinoma and endodermal sinus tumour.

Immunoperoxidase staining for myoglobin was performed on formalin-fixed, paraffin-embedded tissues of normal human skeletal muscle and rhabdomyosarcomas in order to assess its potential diagnostic value. Myoglobin-positive cells were observed in normal adult and fetal muscles, and in 13 of 26 rhabdomyosarcomas. In normal skeletal muscle, the intensity of staining for myoglobin varied in individual cells. Of the 13 myoglobin-positive rhabdomyosarcomas tested, 6 showed cross striations in tumor cells, whereas the others did not. Myoglobin-positive tumor cells were not demonstrated in two rhabdomyosarcomas in which cross striations were observed. In other types of tumors examined that sometimes resembled rhabdomyosarcomas, no myoglobin was detected. Thus tests for myoglobin by the immunoperoxidase method seem helpful in identifying rhabdomyosarcomas and confirming their histological diagnosis.

Thirty two human tumours, mainly neurogenic, have been investigated for the presence of adenovirus-related RNA sequences. 3H-labelled tumour virus DNA probes derived from human adenoviruses types 2 and 12, bovine adenovirus type 3, and avian adenovirus CELO were hybridized in-situ on tumour kryostat sections under conditions that detect complementary RNA. Tumour virus-related RNA was detected in 62% of all tumours tested, but was not detectable in normal human brain tissues. Expression of tumour virus-related RNA was found in 2/4 astrocytomas, 2/4 metastatic brain carcinomas, 2/2 glioblastomas, 1/1 melanoma, 5/7 meningiomas, 4/4 neurinomas, 1/2 oligodendrogliomas, and 1/1 rhabdomyosarcoma. The presence of adenovirus-related RNA in the majority of human neurogenic tumours may reflect a viral involvement in the pathogenesis of these tumours.

We performed immunoperoxidase studies in 29 cases of rhabdomyosarcoma from the Intergroup Rhabdomyosarcoma Study I using antisera against actin, myosin, myoglobin, alpha-actinin, and tropomyosin. Although each of these antisera reacted with some of the tumors, none reacted with all of the tumors, and some tumors showed no reactivity. Antimyosin reacted with more tumors than any of the others, while antiactin and antimyoglobin were about equally sensitive. Antitropomyosin and anti-alpha-actinin reacted with few of the tumors. The better-differentiated tumors were more likely to react compared with the poorly differentiated tumors.

A small number of differentiated tumor cells could be found besides a great number of undifferentiated ones. Abnormal dense granules were found in the nuclei of undifferentiated tumor cells, not in differentiated ones. Non-specific filaments were seen in the cytoplasm of undifferentiated tumor cells. Differentiated tumor cells demonstrated various stages of myofibrillar structures such as A, I, and Z bands, many glycogen granules, mitochondria and a basement membrane. Polyglucose particles synthesized from glycose-1-phosphate by phosphorylase activity were located in the cytoplasmic matrix of undifferentiated and differentiated tumor cells and in the karyolymph of undifferentiated tumor cells. Polyglucose partices increased in number according to the degree of differentiation of tumor cells.

Nine cases of malignant "triton" tumors, based on the coexistence of rhabdomyoblasts and Schwann cell elements, were analyzed clinicopathologically and immunocytochemically. All tumors were stained for myoglobin and S-100 protein by the immunoperoxidase technique. Six of the nine patients were in the third or fourth decade of life. Six cases were associated with von Recklinghausen's disease, and the tumors in two cases grew along nerve trunks. The malignant tumors showed a predilection for the thigh and buttock. Six of the seven deaths occurred within two years of the initial treatments. Strongly positive staining for S-100 protein was observed in three tumors, with transitional zones between the sarcomas and peripheral neurofibroma-like areas, as well as in two tumors composed predominantly of rhabdomyoblastic elements. In four other cases the tumors were only weakly positive for protein S-100. Intracytoplasmic myoglobin was present in all cases. Tumors composed predominantly of rhabdomyosarcomatous elements occurred in four patients, including two children with von Recklinghausen's disease. These results, considered with other findings, suggest that malignant "triton" tumors may not be as rare as previously believed.

The intermediate filament cytoskeleton of various types of human soft tissue tumors was analyzed by immunofluorescence microscopy with the use of specific antibodies against cytokeratins, vimentin, and desmin, as well as by one- and two-dimensional gel electrophoresis of high-salt buffer- and detergent-resistant cytoskeletal preparations. All leiomyomas as well as a leiomyosarcoma contained desmin. Leiomyomas of both gastrointestinal and uterine derivation and the retroperitoneal leiomyosarcoma showed strong reaction for desmin in the smooth muscle cells, but the latter two exhibited also vimentin staining. In embryonal rhabdomyosarcomas, desmin prevailed in the large, apparently well-differentiated rhabdomyoblasts; whereas the smaller, less differentiated tumor cells preferentially contained vimentin. Cells of malignant fibrous histiocytomas were characterized by their content of vimentin as the only intermediate filament protein present. In alveolar soft part sarcoma, a rare tumor of hitherto unknown histogenesis, vimentin and desmin co-existed within the same tumor cells, indicating, together with chemical determinations, the myogenic derivation of this neoplasm. The results show that immunologic and biochemical analysis of proteins associated with the intermediate filament cytoskeleton is a useful adjunct in the diagnosis of diverse neoplasms, particularly those with equivocal histologic features, and thus aids in the histogenetic classification of soft tissue tumors.

A case of rhabdomyosarcoma of the embryonal alveolar type arising from the prostate in a 6-year-old boy was reported with a immunohistochemical survey. Tumor cells in the biopsied inguinal lymph node were weakly positive for myoglobin and CPK-mm and showed positive staining for neuron specific enolase (NSE). At autopsy, an immunohistochemical examination demonstrated that the tumor cells, especially differentiated elongated cells, were strongly positive for myoglobin, CPK-mm and NSE. An enzyme immunoassay for three distinct subunits of NSE revealed a 320 fold increase in the beta subunit of enolase which is specific for heart and skeletal muscle. The diagnostic significance of immunohistochemical methods for rhabdomyoblasts was discussed.

Three entirely different tumor types were investigated biochemically for the presence and characteristics of endogenous carbohydrate-binding proteins in an inbred Brown Norway rat, an outbred Sprague-Dawley rat, and an outbred Han:NMRI mouse. The patterns under investigation included specificities for alpha- and beta-galactosyl, alpha-mannosyl, and alpha-fucosyl moieties, respectively, and specificities for heparin, analyzed by affinity chromatography on resins with immobilized sugars or glycoproteins and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The patterns were divided into categories according to dependence of the binding activity on the presence of Ca2+ and dependence on extraction conditions. Rhabdomyosarcoma revealed only Ca2+-independent activities, i.e., activities with specificity for beta-galactosides at a molecular weight of 12,000, with specificity for alpha-galactosides at molecular weights of 29,000, 43,000, and 45,000, with specificity for heparin at molecular weights of 13,000 and 16,000, and with specificities for mannose and fucose at molecular weights ranging from 62,000 to 70,000. For the spontaneous mammary adenocarcinoma the pattern was entirely different and more diverse, including species with the Ca2+ requirement. Extracts with the use of 0.2 M NaCl (salt) and 2% Triton X-100 (detergent) from teratoma contained at least nine different carbohydrate-binding proteins. The only similarities between the pattern of endogenous carbohydrate-binding proteins from teratoma and from mammary adenocarcinoma were beta-galactoside-binding proteins, one with a Ca2+ requirement and one without a Ca2+ requirement, and the heparin-binding proteins. These heparin-binding proteins were the only types of carbohydrate-binding proteins common to all three tumor types. The analysis indicates that certain bands represented newly identified proteins capable of binding to galactose-, mannose- or fucose-containing glycoconjugates, respectively. When assayed with rabbit erythrocytes, the different fractions showed agglutination activity. They can thus be termed "endogenous lectins." The use of endogenous lectin patterns as potential diagnostic markers in addition to the corresponding changes in the glycoconjugate composition is proposed.

The diagnosis of orbital rhabdomyosarcoma (RMS) in childhood gives rise to several clinical and anatomo-pathological problems. Antibodies recognizing structural proteins and cytoskeletal components have been shown to increase the diagnostic accuracy of different neoplastic lesions. In this study we examined anatomo-clinically and, where possible, by means of immunohistochemistry and electron microscopy, a series of 14 cases of orbital RMS in childhood. In the 12 cases studied by immunohistochemistry, desmin was always present, although showing variable patterns, and alpha-sarcomeric actin was found in 10 cases. alpha-Smooth muscle actin was always absent. The other markers tested (myoglobin, polyclonal actin, vimentin and enolase) proved unreliable for several reasons. We conclude that antibodies against desmin and alpha-sarcomeric actin are useful for the diagnostic definition of RMS. In addition, immunohistochemical analysis supplies data regarding the degree of tumor differentiation and may be applied to monitor radio- and chemotherapy.

In order to simulate more closely conditions in which resistance to vincristine (VCR) is selected in human solid tumors, a human rhabdomyosarcoma grown as a xenograft in immune-deprived mice has been selected for resistance in situ. Karyotype analysis showed the resistant line, HxRh18/VCR-3, to have a diploid modal number, with no apparent translocations, whereas the predominant population in the parental, sensitive HxRh18 xenograft demonstrated a modal number near-tetraploid with many marker chromosomes. From the rapid rate at which resistance was selected and from karyotypic evidence, data strongly suggest that HxRh18/VCR-3 was a subpopulation within the parent tumor. When grown in the same host, HxRh18/VCR-3 tumors accumulated less drug, and the rate of [3H]VCR loss was 5-fold greater than in HxRh18 tumors. Thus, accumulation and retention of [3H]VCR in HxRh18/VCR-3 resistant tumors was identical to that of [3H]vinblastine (VLB) in HxRh18 xenografts. HxRh18 xenografts are intrinsically resistant to VLB. Analysis by high-performance liquid chromatography of [3H]VCR:protein complexes in HxRh18 cytosols indicated one binding species (Mr 95,000 to 116,000), probably the tubulin heterodimer. Of interest was the observation that beta-tubulin species, identified on Western blots by monoclonal antibody, differed in these tumors. In HxRh18/VCR-3, less acidic beta-tubulins of HxRh18 were decreased or absent, with three additional more acidic isoforms present in the resistant line. As vincristine may bind to the beta-subunit of tubulin, this may have importance to vincristine resistance in vivo.

Tumoral myosins were isolated from rat and rabbit rhabdomyosarcomas and compared with normal adult and fetal skeletal myosins. The synthetic filaments, the light-chain composition and the Ca2+ ATP-ase activity were studied. In the presence of Mg2+, normal myosins precipitated as bipolar filaments (0.5 micrometer), fetal and tumoral myosins, however, precipitated as long fusiform filaments (1 to 10 micron). SDS-PAGE revealed that tumoral myosins contain the same light-chains as fetal myosin (25000 and 18000 daltons, L25-L18). The third light-chain of the normal muscle myosin (16000 daltons, L16) was absent. In addition, Urea-PAGE revealed the absence of the phosphorylated form of the L18 in fetal and tumoral myosins. Ca2+ ATPase activity measurements performed in function of the Ca2+ concentration showed similarities between fetal and adult muscle myosins. The Ca2+-ATPase activity of tumoral myosins, however, was very low and slightly activated by increasing the Ca2+ concentration (0.01 to 10 mM). The investigation has shown that fetal and tumoral myosins are identical concerning the ultrastructure of their synthetic filaments and their light-chain composition. This was not so in regard to the Ca2+ ATPase activity. This is probably the result of the expression of a new myosin- or of one of its polypeptides-, which has a different Ca2+-ATPase activity.

Hybridoma cell lines were obtained from the fusion of NS-O myeloma cells with spleen cells of mice immunized with bovine fetal skeletal myosin. A stable hybridoma clone, BF-G6, produced immunoglobulin G1 k antibodies reacting specifically with embryonic-type myosin heavy chains present in fetal but not in neonatal or adult human skeletal muscle, as determined by enzyme immunoassay and immunoblot analysis. Fetal but not adult skeletal muscle fibers were stained by this monoclonal antibody in indirect immunofluorescence assays; smooth muscle cells and cardiac muscle cells, as well as non-muscle cells were also unreactive. Solid tumors of infants and children were tested for reactivity with BF-G6 by immunofluorescence and immunoperoxidase staining. Embryonic myosin heavy chain was expressed in rhabdomyosarcomas but not in other types of tumor, except for Wilms' tumor. Rhabdomyosarcoma cells isolated from a bone marrow metastasis and grown in vitro for several months were also labelled by BF-G6. Embryonic myosin heavy chain can thus be used as a specific differentiation marker of normal and neoplastic skeletal muscle tissue.

Clinical and histopathological features of two cases of malignant rhabdoid tumor of the kidney are presented. One of these cases was also studied by electron microscopy. Histologically, both tumors consisted of an admixture of undifferentiated polygonal or elongated cells and cells with abundant eosinophilic cytoplasm frequently containing hyaline globular structures. Ultrastructurally, these cytoplasmic inclusions were composed of large masses of actin-size and intermediate-size filaments. The poor prognosis of this type of tumor is emphasized and histological criteria for differential diagnosis from other malignant renal tumors of childhood and adolescence are discussed.

Histological localization of myosins of the smooth and skeletal muscles was investigated in comparison with that of myoglobin by immunoperoxidase technique using the antibody against each of them in surgical specimens from spindle cell tumors and tumor-like lesions of the soft tissue. Skeletal muscle myosin was demonstrated in all of the cases of rhabdomyosarcoma, whereas myoglobin was found in 75% of the examined cases. Smooth muscle myosin was widely distributed not only in the tumor cells of smooth muscle origin such as leiomyosarcoma and angioleiomyoma, but also in the tumor cells showing myofibroblastic differentiation such as malignant fibrous histiocytoma and in the epithelial components of synovial sarcoma. The results showed that skeletal muscle myosin can be regarded as an excellent marker in the diagnosis of rhabdomyosarcoma and that smooth muscle myosin is a useful marker of leiomyosarcoma and leiomyomas, and of tumors with myofibroblastic differentiation.

Cathepsin D is an acidic endopeptidase that has been found in the cytoplasm of neurons and other cells. To test the hypothesis that the cellular content or distribution of cathepsin D may serve as a marker of neuronal differentiation in childhood neoplasia, we performed an immunocytochemical study of the cathepsin D content of tumors that occurred in 49 pediatric patients. We found diffuse, moderate to heavy staining primarily in differentiating neurogenous tumors and minimal staining in one Wilms' tumor, one Ewing's sarcoma, and one rhabdomyosarcoma. Benign reactive histiocytes stained intensely in most tumors. We concluded that although the presence of cathepsin D in malignant cells is not diagnostic of neural tumors, it may serve as a marker for cellular maturation in neuroblastomas and as a marker of macrophage infiltration in human tumors.

In view of the personal observation that malignant peripheral neuroectodermal tumours (MPNT) can present different histological growth patterns, 41 cases of MPNT were histologically and immunohistochemically studied. The median age of the 41 patients was 15 years (range: 9 months - 23 years). There were 27 males and 14 females. Most tumours (23/41) were located in the thoracopulmonary region. In 31/41 cases there was bone as well as soft tissue involvement. The following histopathological patterns were found: Ewing's sarcoma-like (n = 7), atypical Ewing's sarcoma-like (n = 4), neuroblastoma-like (n = 8), rhabdomyosarcoma-like (n = 8), and hemangiopericytoma-like (n = 1). In 2 cases combined patterns were noted, one tumour being characterized by neuroblastoma-like and Burkitt's lymphoma-like features. Most cases of MPNT differed from the cytological features of typical Ewing's sarcoma in that they contained hyperchromatic nuclei with distinct nucleoli. Some reticulin fibrils were found in between the cells of some cases. Immunohistochemically, 19/23 cases reacted positively to vimentin, 29/32 to neuron specific enolase (NSE), 16/28 to protein S-100, and 1/9 to glial fibrillary acidic protein. 12/24 cases reacted positively to NSE and protein S-100. Neurofilaments and desmin were not found in the formalin fixed material of the present study. The results show that most cases of MPNT can be distinguished from typical Ewing's sarcoma by cytological and histological findings. Differential diagnosis from atypical Ewing's sarcoma, neuroblastoma, and rhabdomyosarcoma is possible by immunohistochemistry.

Two cases of alveolar soft part sarcoma have been studied immunocytochemically using antisera against epithelial membrane antigen, lysozyme, keratins, S-100 protein, desmin, vimentin, fetal myosin, slow myosin, alpha-skeletal muscle actin, alpha-smooth muscle actin and myoglobin. The neoplastic cells were negative with all antisera employed with the exception of the alpha-skeletal muscle actin antiserum which stained the cytoplasm of numerous neoplastic elements, including the crystalloid rods, typical cytoplasmic inclusions of these tumours. It is suggested that the presence of this protein indicates rhabdomyoblastic differentiation of these tumours.

A correlated cytologic and histologic study of seven cases of embryonal rhabdomyosarcoma is presented. The diagnosis of rhabdomyosarcoma was established by light and electron microscopy and immunohistochemistry of the operative specimens. The cytologic appearance of the smears corresponded well with the histopathologic findings. Cytologically, two main cell types were distinguished: a predominant primitive, small round cell with scant cytoplasm and a large cell with an abundant cytoplasm, sometimes tadpole- or ribbon-shaped. The tumor cells were often enclosed in a background of mucosubstances. The lack of cytologic features proving rhabdomyoblastic differentiation, such as cross-striation, necessitates the use of additional methods in the cytologic diagnosis of embryonal rhabdomyosarcoma. The value of the embedding technique for ultrastructural analysis and immunohistochemistry in the demonstration of desmin in aspirates is emphasized in the diagnosis of embryonal rhabdomyosarcoma.

We report the case of an 18-year-old woman who had a small-cell malignant neoplasm of the left paranasal sinuses and who, 18 months later, developed malignant ascites. Immunoperoxidase stains of the ascitic fluid cells and, subsequently, the original tumor, identified it as embryonal rhabdomyosarcoma. Flow cytometric DNA analysis of the ascitic fluid demonstrated an aneuploid cell population that was also present in the paraffin-embedded tissue on which the original diagnosis had been made.

Anti-rat myoglobin (Mb) was prepared and used in the avidin-biotin-peroxidase complex (ABC) method on paraffin-embedded sections of nine soft tissue tumors (including two rhabdomyosarcomas) of rats. Distribution and nature of the reactive substance to Mb antiserum were compared to those of desmin antiserum. Rat Mb was isolated from the skeletal muscle; monospecificity of the rat antiserum was confirmed by the immunoblotting procedures. The Mb antiserum reacted specifically to normal and neoplastic striated muscle cells. Mb-staining reactions were present diffusely in the cytoplasm, while desmin-staining substances were localized at Z-bands or were diffuse in the cytoplasm as separated aggregates. Reaction to the Mb serum was also detected in cells of thick portions of Henle's loop and distal convoluted tubules.

Cell lysates of two established human tumor lines, a medulloblastoma (TE671), and a rhabdomyosarcoma (RD), contain mitogenic activity which elutes from heparin-Sepharose under conditions typical of class 1 heparin-binding growth factors, such as acidic brain fibroblast growth factor. The presence of this class of mitogen in both cell lines was confirmed by their chromatographic behavior on reversed-phase C3 columns, and by the ability of heparin to enhance their mitogenic activity. Using a specific synthetic DNA probe, RNA's were isolated from both cell lines by hybridization-selection, translated in vitro, and translated proteins affinity fractionated on heparin-Sepharose. The results demonstrate that TE671 and RD cell lysates contain mRNA's for mitogens related to acidic brain fibroblast growth factor, and also suggest that high molecular weight proteins exist that are closely related to, or are precursor forms of, the class 1 mitogens.

Twenty-five rhabdomyosarcomas (RMSs), including 12 alveolar and 13 embryonal types, were immunohistochemically studied for the presence of different classes of intermediate filament proteins and muscle actins (MAs). For the most part, formaldehyde-fixed and paraffin-embedded tissue was used in immunostaining. All RMSs showed desmin and MAs, usually in a major portion of tumor cells. The number of MA-positive cells was sometimes higher than that of desmin-positive cells. Vimentin was present in all tumors studied in frozen sections. Eight of 12 alveolar RMSs showed small number of cytokeratin-positive neoplastic cells. Cytokeratin-positive cells were present less commonly in embryonal RMS (3/13 cases). The 68-kD neurofilament protein was found in frozen sections of two embryonal RMSs. The cytokeratin and neurofilament immunostaining could be reproduced by immunofluorescence technique. In addition, we studied three childhood sarcomas, which showed abundant desmin and MA immunostaining but did not conform to the ultrastructural criteria of RMS. Scattered cytokeratin-positive cells were found in two of these tumors, and neurofilaments were found in the two cases for which frozen sections were available. The results show that typical RMS may demonstrate immunohistological pleomorphism with cytokeratin and neurofilament immunoreactivity suggesting the presence of multidirectional differentiation. In addition, there are tumors that by morphology look like RMS and have muscle cell markers but cannot be verified as RMS by electron microscopy; also, these tumors seem to show immunohistological pleomorphism. The presence of nonmyoid markers in RMS should be considered when making immunohistological diagnosis of soft tissue sarcomas.

Monoclonal antibody HHF35 has previously been characterized biochemically as recognizing isotypes of actin (alpha and gamma) which are specific to muscle cells. In this study, the authors have investigated the normal and pathologic tissue distribution of HHF35-positive cells using the avidin-biotin immunoperoxidase method on methacarn-fixed, paraffin-embedded sections of human tissue. In addition to muscle tissues (smooth, skeletal, and cardiac) the antibody localizes to myoepithelium, as well as most of the capsular cells of several parenchymal organs, including liver, kidney, and spleen, with extension of the latter cells into the splenic trabeculaes. In pathologic tissues, the antibody localizes to cells, identified by some investigators as "myofibroblasts," in the stroma of certain tumors, within hyperplastic fibrous tissue responses ("fibromatoses") such as Dupuytren's contracture, and within fibrotic lung tissue. HHF35 also localizes to cells that proliferate within the intima in lesions of atherosclerosis and to a unique population of reactive mesothelial and submesothelial cells. Among tumors, it is positive only on leiomyomas, leiomyosarcomas, and rhabdomyosarcomas, and negative on all nonmuscle sarcomas. This antibody thus shows great potential utility as a diagnostic reagent in various pathologic conditions, most especially in the diagnosis of tumors of muscle origin.

Twenty seven cases of rhabdomyosarcoma from pathologic files of the Hokkaido University Hospital from 1975 to 1985 were reviewed and the presence of myoglobin and S-100 protein was investigated immunohistochemically. Their average age at diagnosis was 30.7 years (ranging from two months to 77 years) and there was a predilection for males in a ratio of 1.7:1. The common locations were the head and neck. Nine cases were positively stained for myoglobin, mainly in the cytoplasm of rhabdomyoblasts and 19 cases were positive for S-100 protein and alpha-subunit of S-100 protein in the cytoplasm of immature tumor cells as well as of rhabdomyoblasts. In seven cases, both of myoglobin and S-100 protein were demonstrated. Although S-100 protein is not strictly specific, however, these results suggest that the immunohistochemical demonstration of S-100 protein can be a useful diagnostic tool of rhabdomyosarcoma.

Tumour markers now have an established role in tumour diagnosis and patient management. However, antibodies used to detect these tumour markers have in some instances proved unreliable, with a low rate of sensitivity and specificity. In this study we wished to evaluate the role of a commercial antibody to myoglobin as a marker of rhabdomyosarcomas. The purpose of this investigation was to assess the sensitivity and specificity of myoglobin antiserum as a marker of rhabdomyosarcomas. This was performed by reacting a large number of tumours (sarcomas, carcinomas and melanomas) with a polyclonal anti human myoglobin antiserum. Staining was demonstrated in 60% of rhabdomyosarcomas. Only two tumours from a total of 226 non-skeletal muscle tumours showed a positive reaction (0.88%). One was a leiomyosarcoma and the other had been classified as an undifferentiated sarcoma but a rhabdomyosarcoma was included in its differential diagnosis. It is of interest that both had been earlier irradiated. This antiserum was therefore a specific but not a very sensitive tumor marker. Its rate of staining of rhabdomyosarcomas is compared with the results in the literature. A great disparity is found and the reasons for this are discussed.

A transplantable tumor strain designated YNnu and a cultured cell line designated YN were established from the human paratesticular rhabdomyosarcoma of a 15-year-old boy. In vitro the cells showed ultrastructural features of immature mesenchymal cells, and a few cells were labeled by antibodies to myoglobin and/or desmin. In nude mice, the cells became to contain numerous myofibrils with Z-bands, and considerable number of cells reacted with anti-myoglobin and/or anti-desmin antibodies. We conclude that spindle-shaped or small round cells are the most immature rhabdomyoblast with multiplicity for cellular differentiation.

Heterogeneity of cell and clone populations in various histogenesis of rat tumours has been studied by the content of the endogenic SH-groups. The distribution of the ovary tumour cells and their clone lines, as well as lung clones of the sublines of rat rhabdomyosarcoma RA-2 as to the content of endogenic SH-groups is of persistent and monopeak character. Modal classes are well expressed, in several clone lines variability of the cell composition is higher than in the initial population. In this case the distribution of cells as to the SH-group content differs from the normal (asymmetrically). The level of SH-groups in the RA-2 rat subclone in 71 lung clones positively correlates with the level in hypodermic transplants. So, the heterogeneity of the clone populations as to the amount of SH-groups depends on both the genetic interclonal differences and fluctuations of the environmental factors.

In a review of a national series of malignant tumors in middle-aged and elderly individuals (over 40 years of age), in all 107 cases primarily diagnosed and reported to the Swedish Cancer Registry as rhabdomyosarcomas during the period 1972-1981, 4 cases were accepted as botryoid, embryonal or alveolar rhabdomyosarcoma, using light-microscopic criteria for the diagnosis. An electron-microscopic and immunohistochemical analysis was performed on the 4 cases along with 7 cases of botryoid, embryonal and alveolar rhabdomyosarcoma in patients of over 40 years of age obtained from our own files. Rhabdomyoblastic differentiation was established ultrastructurally by the presence of myofilaments and Z-like densities in 10 of these 11 cases. There were tumor cells in the formaldehyde-fixed, paraffin-embedded material which were positively stained for desmin in all cases, for myoglobin in 7/11 cases, for vimentin in 5/11 cases and for actin in all cases, using monoclonal antibodies. The demonstration of desmin by the monoclonal antibody which was used on the formaldehyde-fixed, paraffin-embedded material is of particular value in the diagnosis of rhabdomyosarcoma. Another tumor, located in the minor pelvis, lacked the light-microscopic features of botryoid, embryonal or alveolar rhabdomyosarcoma, but presented ultrastructural and immunohistochemical evidence of a rhabdomyoblastic differentiation. This tumor was epithelioid in appearance and shared features with alveolar soft part sarcoma. The label epithelioid rhabdomyosarcoma is proposed for this tumor. Nine pleomorphic sarcomas were selected from the national series as possible pleomorphic rhabdomyosarcomas because of the presence of ribbon-shaped tumor cells with an eosiniphilic cytoplasm. There was no electron-microscopic or immunohistochemical evidence of a myogenic differentiation in any of these 9 tumors. The present investigation indicates that a pleomorphic type of rhabdomyosarcoma, indistinguishable from embryonal, botryoid and alveolar rhabdomyosarcoma, is extremely rare or non-existent.

16 spontaneous tumors of the peripheral nerves and 18 spontaneous tumors of mesenchymal origin in BDVI rats were studied by peroxidase-antiperoxidase method using anti-serum (DAKOPATT) against protein S-100. The majority of spontaneous peripheral nerve tumors were of cystic histological structure identical to that of cystic neurinomas induced in rats by ethylnitrosourea and almost all of these tumors were S-100 protein positive. The incidence of spontaneous neurinomas in BDVI rats was in some experiments as high as 5%. All tumors of mesenchymal origin (except one lipoma) were S-100 protein negative: 2 fibromas, 6 fibrosarcomas, 3 malignant fibrous histiocytomas, one rhabdomyosarcoma and one hemangioendothelioma. S-100 protein is found, as in human pathology, useful for distinguishing tumors of schwann cell and mesenchymal origin in rats.

Methylcholanthrene-induced murine rhabdomyosarcomas and skeletal muscle of 10 and 18 d old murine embryos were investigated by lectin histochemistry (WGA, RCA-I, LCA, Con-A, PSA, UEA-I, PNA) and by immunohistochemistry (vimentin, desmin, myoglobulin). In rhabdomyosarcomas as well as in the developing skeletal muscle a clear trend was visible. A decrease of vimentin positivity and an increase of desmin positivity were associated with a diminution of binding sites for WGA, RCA, and LCA. No binding moieties for these lectin could be demonstrated in myoglobin positive normal and neoplastic rhabdomyomatous cells at all. The homologous expression or absence of markers reflected the cellular variability in rhabdomyosarcomas and may be explained as a phenomenon of different tumor cell maturation. The results show that rhabdomyosarcomatous cells are imitating the normal skeletal muscle development.

Using an immunoperoxidase method, myoglobin is localized in the cytoplasm of normal and neoplastic human skeletal muscle. The staining intensity is variable in individual cells. This can be explained by the variable concentration of myoglobin in the different fiber types of normal muscle or the different degree of differentiation in neoplastic muscle cells, respectively. A potential application of this method in tumor pathology is discussed in connection with myoglobin as a tissue-specific marker for skeletal muscle neoplasms.

Intracellular myoglobin represents an excellent marker for specific characterization of normal (adult and fetal) and malignant skeletal muscle cells in paraffin sections. With an immunoperoxidase indirect sandwich technique for detection of intracellular myoglobin, positive staining was observed in 13 of 17 rhabdomyosarcoma specimens including 5 of 7 of the alveolar type, 5 of 5 of the embryonal type, and 3 of 5 of the pleomorphic type. Initial fixation in Zenker's-acetic acid solution gave optimal staining, but satisfactory results were obtained with fixation in formalin, Bouin's, and B5 solutions. Other types of sarcomas (13 cases) and other types of tumors (24 cases) that sometimes mimic rhabdomyosarcoma on histologic examination gave negative results. The immunoperoxidase method affords a sensitive and specific method for identifying rhabdomyoblasts in tissue on the basis of intracellular myoglobin and is of use in distinguishing rhabdomyosarcomas from other sarcomas and from malignant tumors of other types.

Pulmonary blastoma is a rare lung tumor composed of epithelial and mesenchymal elements; the latter element may show various patterns of differentiation toward mature tissue, such as cartilage, smooth muscle, and bone. Rhabdomyoblastic differentiation in pulmonary blastoma is quite rare; only five such cases have been reported. We report two cases of pulmonary blastoma with rhabdomyoblastic differentiation documented for the first time by electron microscopy and immunohistochemistry including documentation for myoglobin, actin, vimentin and desmin. The diffuse and prominent rhabdomyoblastic differentiation in one case is most unusual.

Histologic examination was carried out in 65 cases of childhood rhabdomyosarcoma (RMS), 53 embryonal, and 12 alveolar. Cross-striations were seen on light microscopy in 12 (23%) embryonal and 4 (33%) alveolar tumors. The capacity of immunohistochemical staining (PAP technique) to increase diagnostic accuracy was assessed, using antibodies against myoglobin, the MM isoenzyme of creatine kinase, desmin, calcium magnesium-dependent ATPase of sarcoplasmic reticulum and calsequestrin. Myoglobin was detected in 16 (30%) embryonal and eight (67%) alveolar RMS, higher numbers than obtained by viewing cross-striations on light microscopy. The creatine kinase antibody was slightly better than the antibody to myoglobin and 15 of 25 (60%) embryonal RMS were positive when both specificities were used. The remaining three antibodies were less useful. Of 13 (two alveolar and 11 embryonal) RMS studied by electron microscopy, four showed cross-striations, contained late myoblasts, and were positive for myoglobin. Three additional cases showed only late myoblasts and one of these was positive for myoglobin. Thus, 16 of 25 (64%) of the embryonal and seven of nine (78%) of the alveolar RMS showed either positive immunostaining or ultrastructural features of RMS. This study indicates that a combination of immunohistochemical staining, using antimyoglobin and anticreatine kinase (MM isoenzyme) antibodies, and electron microscopy are useful markers in the diagnosis of childhood RMS.