Sources/Clones
Accurate/Novocastra (5.8A) and Dako (5.8A).
Fixation/Preparation
Anti-MyoD1 can be used on formalin-fixed, paraffin-embedded tissue sections. Deparaffinized tissue sections require heat pretreatment in citrate buffer prior to immunohistochemical staining procedure. Sialinized slides are recommended to improve adherence of tissue sections to glass slides. Ideally, this antibody requires fresh-frozen tissue for optimum results.
Background
The differentiation of skeletal muscle at the molecular level requires activation and transcription of genes encoding muscle-specific proteins and enzymes such as desmin and creatine kinase. These activities are controlled by a set of genes includingMyoD1, myogenin, myf-5 and myf-6 (Funk et al, 1991; Tonin et al, 1991). It is thought thatMyoD1 activation is an early event that commits the cell to skeletal muscle lineage (Hosoi et al, 1992). Transfection of theMyoD1 gene into non-muscle cells has been shown to induce conversion of fibroblasts into myoblasts (Davis et al, 1987). Similarly, muscle-specific genes in tumor cell lines may be activated by forced expression of exogenously introducedMyoD1 (Weintraub et al, 1989). The MyoD1 gene has been localized to the short arm of chromosome 11 (Gressler et al, 1990). Its activation, as reflected in the detection of mRNA or protein product, represents a stage of skeletal muscle differentiation that is earlier than that of currently available immunohistochemical markers, such as desmin and myoglobin.
The MyoD1 protein is a 45 kD nuclear phosphoprotein (5.8A reacts with an epitope between amino acid residues 170 and 209), with nuclear expression restricted to skeletal muscle tissue. Monoclonal anti-MyoD1 strongly stains nuclei of myoblasts in developing skeletal muscle whilst the majority of adult skeletal muscle has been found to be negative (Wang et al, 1995), including a wide variety of normal tissue. However, weak cytoplasmic staining has been observed in non-muscle tissue, including glandular epithelium (Wang et al, 1995).
Applications
MyoD1 nuclear immunostaining has been demonstrated in the majority of rhabdomyosarcomas of various histological subtypes (Appendix 1.24). In fact, it has been shown that the MyoD1 expression in rhabdomyosarcomas is inversely related to the degree of cellular differentiation of tumor cells (Wang et al, 1995). This phenomenon is useful to distinguish embryonal rhabdomyosarcomas from other small blue round cell tumors of childhood, such as, Ewing's sarcoma/peripheral primitive neuroectodermal tumor, neuroblastoma and childhood lymphomas. Wilm's tumors and ectomesenchymoma with rhabdomyosarcomatous foci also show nuclear expression of MyoD1 (Dias et al, 1992) (Appendix 1.3). It has also been shown that the sensitivity and specificity of the MyoD1 antibody in the differential diagnosis of adult pleomorphic soft tissue sarcomas approaches that of pediatric rhabdomyosarcomas (Wesche et al, 1995). The demonstration of MyoD1 protein in four cases of alveolar soft part sarcoma has provided good evidence for its rhabdomyosarcomatous differentiation (Rosai et al, 1991).
Comments
Caution is necessary in that cytoplasmic immunoreactivity with antibody to MyoD1 has been demonstrated in most neuroblastomas and occasional Ewing's sarcomas/PNETs (Wang et al, 1995). Hence, only nuclear staining should be considered as evidence of skeletal myogenic differentiation, although our own experience has been that nuclear expression occurs in the primitive skeletal tumors, whilst tumors with cytoplasmic/myogenic differentiation have demonstrated cytoplasmic immunopositivity. The cytoplasmic immunostaining with anti-MyoD1 (clone 5.8A) has been suggested to represent crossreactivity with an unknown cytoplasmic antigen. In their study of 12 cases of alveolar soft part sarcoma, Wang et al (1996) found positivity for desmin in six tumors but no specimen showed nuclear expression of MyoD1 or myogenin. However, there was considerable cytoplasmic staining with the anti-MyoD1, a phenomenon observed with various non-muscle and neoplastic tissues with this antibody. Biochemical analysis of fresh-frozen tumor tissue showed no specific band corresponding to the 45 kD MyoD1.
References
•Davis RL, Weinbtraub H, Lassar AB 1987 Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.
•Dias P, Parham DM, Shapiro DN et al 1992 Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility. Cancer Research 52: 6431-6439.
•Funk WD, Ouellette M, Wright WE 1991 Molecular biology of myogenic regulatory factors. Molecular Biology Medicine 8: 185-193.
•Gressler M, Hameister H, Henry I et al 1990 The human MyoD1 (MYF3) gene maps on the short arm of chromosome 11 but is not associated with the WAGR locus on the region for the B-W syndrome. Human Genetics 86: 135-138.
•Hosoi H, Sugimoto T, Hayashi Y et al 1992 Differential expression of myogenic regulatory genes, MyoD1 and myogenin in human rhabdomyosarcoma sublines. International Journal of Cancer 50: 977-983.
•Rosai J, Dias P, Parham DM, Shapiro DN, Houghton P 1991 MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature. American Journal of Surgical Pathology 15: 974-981.
•Tonin PN, Scrable H, Shimada H, Cavence WK 1991 Muscle-specific gene expression in rhabdomyosarcomas and stages of human fetal skeletal muscle development. Cancer Research 51: 100-106.
•Wang NP, Marx J, McNutt MA et al 1995 Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood. American Journal of Pathology 147: 1799-1810.
•Wang NP, Bacchi CE, Jiang JJ et al 1996 Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunohistochemical and biochemical study of myogenic regulatory protein expression. Modern Pathology 9: 495-506.
•Weintraub H, Tapscott SJ, Davis RL et al 1989 Activation of muscle-specific gene in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD. Proceedings of the National Academy of Science USA 86: 5434-5438.
•Wesche WA, Fletcher CDM, Dias E et al 1995 Immunohistochemistry of MyoD1 in adult pleomorphic soft tissue sarcomas. American Journal of Surgical Pathology 19: 261-269.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Accurate/Novocastra (5.8A) and Dako (5.8A).
Fixation/Preparation
Anti-MyoD1 can be used on formalin-fixed, paraffin-embedded tissue sections. Deparaffinized tissue sections require heat pretreatment in citrate buffer prior to immunohistochemical staining procedure. Sialinized slides are recommended to improve adherence of tissue sections to glass slides. Ideally, this antibody requires fresh-frozen tissue for optimum results.
Background
The differentiation of skeletal muscle at the molecular level requires activation and transcription of genes encoding muscle-specific proteins and enzymes such as desmin and creatine kinase. These activities are controlled by a set of genes includingMyoD1, myogenin, myf-5 and myf-6 (Funk et al, 1991; Tonin et al, 1991). It is thought thatMyoD1 activation is an early event that commits the cell to skeletal muscle lineage (Hosoi et al, 1992). Transfection of theMyoD1 gene into non-muscle cells has been shown to induce conversion of fibroblasts into myoblasts (Davis et al, 1987). Similarly, muscle-specific genes in tumor cell lines may be activated by forced expression of exogenously introducedMyoD1 (Weintraub et al, 1989). The MyoD1 gene has been localized to the short arm of chromosome 11 (Gressler et al, 1990). Its activation, as reflected in the detection of mRNA or protein product, represents a stage of skeletal muscle differentiation that is earlier than that of currently available immunohistochemical markers, such as desmin and myoglobin.
The MyoD1 protein is a 45 kD nuclear phosphoprotein (5.8A reacts with an epitope between amino acid residues 170 and 209), with nuclear expression restricted to skeletal muscle tissue. Monoclonal anti-MyoD1 strongly stains nuclei of myoblasts in developing skeletal muscle whilst the majority of adult skeletal muscle has been found to be negative (Wang et al, 1995), including a wide variety of normal tissue. However, weak cytoplasmic staining has been observed in non-muscle tissue, including glandular epithelium (Wang et al, 1995).
Applications
MyoD1 nuclear immunostaining has been demonstrated in the majority of rhabdomyosarcomas of various histological subtypes (Appendix 1.24). In fact, it has been shown that the MyoD1 expression in rhabdomyosarcomas is inversely related to the degree of cellular differentiation of tumor cells (Wang et al, 1995). This phenomenon is useful to distinguish embryonal rhabdomyosarcomas from other small blue round cell tumors of childhood, such as, Ewing's sarcoma/peripheral primitive neuroectodermal tumor, neuroblastoma and childhood lymphomas. Wilm's tumors and ectomesenchymoma with rhabdomyosarcomatous foci also show nuclear expression of MyoD1 (Dias et al, 1992) (Appendix 1.3). It has also been shown that the sensitivity and specificity of the MyoD1 antibody in the differential diagnosis of adult pleomorphic soft tissue sarcomas approaches that of pediatric rhabdomyosarcomas (Wesche et al, 1995). The demonstration of MyoD1 protein in four cases of alveolar soft part sarcoma has provided good evidence for its rhabdomyosarcomatous differentiation (Rosai et al, 1991).
Comments
Caution is necessary in that cytoplasmic immunoreactivity with antibody to MyoD1 has been demonstrated in most neuroblastomas and occasional Ewing's sarcomas/PNETs (Wang et al, 1995). Hence, only nuclear staining should be considered as evidence of skeletal myogenic differentiation, although our own experience has been that nuclear expression occurs in the primitive skeletal tumors, whilst tumors with cytoplasmic/myogenic differentiation have demonstrated cytoplasmic immunopositivity. The cytoplasmic immunostaining with anti-MyoD1 (clone 5.8A) has been suggested to represent crossreactivity with an unknown cytoplasmic antigen. In their study of 12 cases of alveolar soft part sarcoma, Wang et al (1996) found positivity for desmin in six tumors but no specimen showed nuclear expression of MyoD1 or myogenin. However, there was considerable cytoplasmic staining with the anti-MyoD1, a phenomenon observed with various non-muscle and neoplastic tissues with this antibody. Biochemical analysis of fresh-frozen tumor tissue showed no specific band corresponding to the 45 kD MyoD1.
References
•Davis RL, Weinbtraub H, Lassar AB 1987 Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.
•Dias P, Parham DM, Shapiro DN et al 1992 Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility. Cancer Research 52: 6431-6439.
•Funk WD, Ouellette M, Wright WE 1991 Molecular biology of myogenic regulatory factors. Molecular Biology Medicine 8: 185-193.
•Gressler M, Hameister H, Henry I et al 1990 The human MyoD1 (MYF3) gene maps on the short arm of chromosome 11 but is not associated with the WAGR locus on the region for the B-W syndrome. Human Genetics 86: 135-138.
•Hosoi H, Sugimoto T, Hayashi Y et al 1992 Differential expression of myogenic regulatory genes, MyoD1 and myogenin in human rhabdomyosarcoma sublines. International Journal of Cancer 50: 977-983.
•Rosai J, Dias P, Parham DM, Shapiro DN, Houghton P 1991 MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature. American Journal of Surgical Pathology 15: 974-981.
•Tonin PN, Scrable H, Shimada H, Cavence WK 1991 Muscle-specific gene expression in rhabdomyosarcomas and stages of human fetal skeletal muscle development. Cancer Research 51: 100-106.
•Wang NP, Marx J, McNutt MA et al 1995 Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood. American Journal of Pathology 147: 1799-1810.
•Wang NP, Bacchi CE, Jiang JJ et al 1996 Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunohistochemical and biochemical study of myogenic regulatory protein expression. Modern Pathology 9: 495-506.
•Weintraub H, Tapscott SJ, Davis RL et al 1989 Activation of muscle-specific gene in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD. Proceedings of the National Academy of Science USA 86: 5434-5438.
•Wesche WA, Fletcher CDM, Dias E et al 1995 Immunohistochemistry of MyoD1 in adult pleomorphic soft tissue sarcomas. American Journal of Surgical Pathology 19: 261-269.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
