Evaluation of New Monoclonal Anti-MyoD1 (MX049) for the Diagnosis of Rhabdomyosarcoma: Comparison with 5.8A, EP212, Anti-Desmin, Anti-Myogenin, and Fluorescence in situ Hybridization.

Rhabdomyosarcoma (RMS) is a primitive embryonal mesenchymal neoplasm demonstrating skeletal muscle differentiation. Diagnosis of RMS remains difficult due to the diversity of clinical features, pathological forms, and lesion's locations. Immunohistochemistry and Fluorescence in Situ Hybridization are common methods used to aid RMS diagnosis. In this research we tested protein expression of Desmin (Clone MX046), MyoD1 (Clone MX049), MyoD1 (Clone 5.8A), MyoD1 (Clone EP212), Myogenin (Clone F5D), and cytogenetic features in 21 RMS cases, with following results: positive rates of Desmin (Clone MX046), MyoD1 (Clone MX049), MyoD1 (Clone 5.8A), MyoD1 (Clone EP212) and Myogenin (Clone F5D) were 100.00%, 100.00%, 90.48%, 95.24% and 85.71%, respectively, with cytoplasmic stains of MyoD1 (Clone 5.8A) in 38.10% (8/21) cases and only nuclear stains of MyoD1 (Clone EP212), MyoD1 (Clone MX049) in all positive cases. FOXO1 gene was detected apart in 9 alveolar RMS samples, where MyoD1 (Clone MX049), MyoD1 (Clone 5.8A) and MyoD1 (Clone EP212) were 100% positive but MyoD1 (Clone 5.8A) only 44.44% (4/9). Thus we believe MyoD1 (Clone MX049) performs more sensitive and specific than MyoD1 (Clone 5.8A) and MyoD1 (Clone EP212).

Rhabdomyosarcoma and Wilms tumors contain a subpopulation of noggin producing, myogenic cells immunoreactive for lens beaded filament proteins.

Myo/Nog cells are identified by their expression of the skeletal muscle specific transcription factor MyoD and the bone morphogenetic protein inhibitor noggin, and binding of the G8 monoclonal antibody. Their release of noggin is critical for morphogenesis and skeletal myogenesis. In the adult, Myo/Nog cells are present in normal tissues, wounds and skin tumors. Myo/Nog cells in the lens give rise to myofibroblasts that synthesize skeletal muscle proteins. The purpose of this study was to screen human lens tissue, rhabdomyosarcoma cell lines, and tissue sections from rhabdomyosarcoma, Wilms and tumors lacking features of skeletal muscle for co-localization of antibodies to Myo/Nog cell markers and the lens beaded filament proteins filensin and CP49. Immunofluorescence localization experiments revealed that Myo/Nog cells of the lens bind antibodies to beaded filament proteins. Co-localization of antibodies to G8, noggin, filensin and CP49 was observed in most RC13 and a subpopulation of RD human rhabdomyosarcoma cell lines. Western blotting with beaded filament antibodies revealed bands of similar molecular weights in RC13 and murine lens cells. Human alveolar, embryonal, pleomorphic and spindle cell rhabdomyosarcomas and Wilms tumors contained a subpopulation of cells immunoreactive for G8, noggin, MyoD and beaded filaments. G8 was also co-localized with filensin mRNA. Staining for beaded filament proteins was not detected in G8 positive cells in leiomyosarcomas, squamous and basal cell carcinomas, syringocarciomas and malignant melanomas. Lens beaded filament proteins were thought to be present only in the lens. Myo/Nog-like cells immunoreactive for beaded filaments may be diagnostic of tumors related to the skeletal muscle lineage.

Isolation and Culture of Individual Myofibers and Their Adjacent Muscle Stem Cells from Aged and Adult Skeletal Muscle.

The isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the analysis and comparison of muscle stem cells from aged and young mice. This method has the advantage that muscle stem cells are cultured on the myofiber, thereby culturing them in conditions as close to their endogenous niche as possible. Here we describe the isolation, culture, transfection with siRNA, and subsequent immunostaining for muscle stem cells on their adjacent myofibers from aged and young mice.

Ultrastructural and immunohistochemical study of phenotypic switch in gastrointestinal smooth muscle cells.

Dedifferentiation is a loss of phenotypic specialization that converts differentiated cells into adult stem cells in order to proliferate and differentiate into replacement tissue. This occurs in several tissues from various organs, such as smooth muscle cells (SMCs) of the mammalian gastrointestinal tract. The aim of this study was to describe ultrastructural and immunohistochemical changes in SMCs which could be compatible with a dedifferentiation process in human and rabbit intestinal muscles. Ultrastructural study and immunohistochemical staining (SMemb and MyoD) on human and rabbit duodenum tissue sections were performed. In both species, this dedifferentiation process is characterized by a loss of intercellular junctions, increased intercellular spaces, cytoskeletal disorganization, perinuclear accumulation of large vacuoles that tend to fuse, rupture of the vacuole membrane and release of cytoplasmic fragments. Dedifferentiated cells show the characteristic phenotype of a mesenchymal cell with scarce perinuclear cytoplasm, long cytoplasmic prolongations and finely distributed granular chromatin in the nucleus. These morphological changes are accompanied by a modulation to a less mature phenotype showing immunoreactivity for the embryonic form of the myosin heavy chain and for the myogenic regulatory factor MyoD. We suggest that SMC dedifferentiation includes the elimination of the contractile apparatus, the activation of the nucleus and the re-expression of embryonic markers. We described an ultrastructural dedifferentiation process possible in intestinal SMCs. This dedifferentiation process seems to play a key role in the homeostasis of the intestinal muscle.

In vitro characterization of proliferation and differentiation of trout satellite cells.

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named "reserve" cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.

Rat monoclonal antibody specific for MyoD.

Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation.

[Immunomorphology in the diagnosis of orbital rhabdomyosarcoma].

Immunohistochemistry (IHC) using monoclonal antibodies is presently the most reliable method to verify tumor tissue belonging. The histological diagnosis of rhabdomyosarcoma (RMS) was immunohistochemically confirmed in 7 of 10 orbital neoplasms in children. In 3 cases the diagnosis of RMS was established after IHC study that revealed the atypical tumor cell expression of the muscular antigens--myoglobin, protein Myo D1, actin HHF-35, which is indicative of the rhabdomyogenic genesis of a tumor. IHC data indicated that, no matter what the histological pattern, orbital RMS is characterized by the expression of cytospecific proteins--myoglobin, Myo D1, actin HHF-35, and tissue-specific (intermediate filaments)--vimentin and desmin. The use of appropriate antibodies offers a means of making a histogenetic diagnosis even in cases of the atypical morphological pattern.

Pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal.

The paired-box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7-expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity.

Angiomyolipoma and PEComa are immunoreactive for MyoD1 in cell cytoplasmic staining pattern.

The family of tumors derived from mesenchymal perivascular epithelioid cells (so-called PEComas) includes angiomyolipoma, lymphangioleiomyomatosis, clear cell sugar tumor of the lung, clear cell myomelanocytic tumor of ligamentum teres/falciform ligament, and abdominopelvic sarcoma of perivascular epithelioid cells. These tumors were characterized by coexpression of melanocytic (HMB-45) and muscle markers. MyoD1 transcription factor has crucial role in commitment and differentiation of mesenchymal progenitor cells to myogenic lineage. Antibodies to MyoD1 protein (nuclear immunoreactivity) have been shown highly valuable adjuncts in the diagnosis of rhabdomyosarcomas. To evaluate expression of the transcription factor MyoD1 in PEComas, we performed immunohistochemistry. Monoclonal antibody 5.8A for MyoD1 was used on a series of cases of formalin-fixed, paraffin-embedded angiomyolipoma (n = 19), lymphangioleiomyomatosis (n = 3), clear cell sugar tumor of the lung (n = 1), and abdominopelvic sarcoma of perivascular epithelioid cells (n = 2). All cases showed strong granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 antibody. Cytoplasmic reactivity was noted in the spindle cells, fat cells, and epithelioid cells. Nuclei were negative in all tumors studied, and a clean background was obtained. Several normal and neoplastic human tissues have also been immunostained for MyoD1 without any positive cytoplasmic staining, with the exception of 2 alveolar soft part sarcomas. Cytoplasmic immunostaining with monoclonal antibody 5.8A for MyoD1 in PEComas may correspond to cross-reactivity with an undetermined cytoplasmic protein. Great caution should be exercised in interpreting the immunostaining results with anti-MyoD1 antibody 5.8A.

MyoD1 and myogenin expression in human neoplasia: a review and update.

The MyoD1 family of myogenic nuclear regulatory proteins includes MyoD1/myf3 and myogenin/myf4. These genes and their proteins are critical for skeletal muscle development. Antibodies to MyoD1 and myogenin are relatively recent additions to the armamentarium of the surgical pathologist. This article reviews the biology of the myogenic nuclear regulatory proteins and their use in the diagnosis of rhabdomyosarcoma. Special attention is given to technical and interpretative issues critical to the use of these antibodies in diagnostic pathology.

MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts.

Isolated chicken myoblasts had previously been utilized in many studies aiming at understanding the emergence and regulation of the adult myogenic precursors (satellite cells). However, in recent years only a small number of chicken satellite cell studies have been published compared to the increasing number of studies with rodent satellite cells. In large part this is due to the lack of markers for tracing avian myogenic cells before they become terminally differentiated and express muscle-specific structural proteins. We previously demonstrated that myoblasts isolated from fetal and adult chicken muscle display distinct schedules of myosin heavy-chain isoform expression in culture. We further showed that myoblasts isolated from newly hatched and young chickens already possess the adult myoblast phenotype. In this article, we report on the use of polyclonal antibodies against the chicken myogenic regulatory factor proteins MyoD and myogenin for monitoring fetal and adult chicken myoblasts as they progress from proliferation to differentiation in culture. Fetal-type myoblasts were isolated from 11-day-old embryos and adult-type myoblasts were isolated from 3-week-old chickens. We conclude that fetal myoblasts express both MyoD and myogenin within the first day in culture and rapidly transit into the differentiated myosin-expressing state. In contrast, adult myoblasts are essentially negative for MyoD and myogenin by culture Day 1 and subsequently express first MyoD and then myogenin before expressing sarcomeric myosin. The delayed MyoD-to-myogenin transition in adult myoblasts is accompanied by a lag in the fusion into myotubes, compared to fetal myoblasts. We also report on the use of a commercial antibody against the myocyte enhancer factor 2A (MEF2A) to detect terminally differentiated chicken myoblasts by their MEF2+ nuclei. Collectively, the results support the hypothesis that fetal and adult myoblasts represent different phenotypic populations. The fetal myoblasts may already be destined for terminal differentiation at the time of their isolation, and the adult myoblasts may represent progenitors that reside in an earlier compartment of the myogenic lineage.

Evaluation of new monoclonal anti-MyoD1 and anti-myogenin antibodies for the diagnosis of rhabdomyosarcoma.

New monoclonal anti-MyoD1 and anti-myogenin antibodies were evaluated immunohistochemically to determine whether they are useful in discriminating rhabdomyosarcoma (RMS) from other soft tissue tumors in routinely processed sections. Neither MyoD1 nor myogenin was expressed in normal, mature striated muscle. In RMS, nuclear expression of MyoD1 and myogenin was found in 82 and 80% of non-overlapping cases, respectively. MyoD1 was generally expressed in small, primitive tumor cells, and larger cells exhibiting morphological evidence of skeletal muscle differentiation failed to express positive nuclear immunostaining. Positive nuclear staining for myogenin was stronger than that for MyoD1 in cases with abundant differentiated tumor cells, but was less prominent in cases in which small, primitive tumor cells predominated. No leiomyosarcomas, Ewing's sarcomas/peripheral primitive neuroectodermal tumors or other soft tissue tumors exhibited nuclear expression of MyoD1 or myogenin. In conclusion, both anti-MyoD1 and anti-myogenin antibodies are useful for diagnosing RMS and for discriminating RMS from other soft tissue tumors.

Antibodies to desmin identify the blastemal component of nephroblastoma.

The blastemal component of Wilms' tumor (WT) might be indistinguishable histologically from other small, blue, round-cell tumors of childhood, including alveolar rhabdomyosarcoma (RMS), particularly in small biopsy specimens and in the setting of metastatic disease. Furthermore, there are currently no reliable blastemal markers. Deparaffinized sections of 9 formalin-fixed blastema-predominant WTs and 46 RMSs were immunostained with antibodies to desmin (D33), myogenin (F5D), MyoD1 (5.8A), and muscle-specific actins (HHF35), after heat-induced epitope retrieval. WE defined as positive those cases with more than 5% of cells immunostained (only nuclear staining was considered as positive for myogenin and MyoD1). Antibodies to desmin were positive in eight (89%) of nine cases of blastema-predominant WT; in contrast, no case was positive for any of the other muscle-associated proteins. Of the 46 cases of RMS, all were positive for desmin, 42 were positive for myogenin and MyoD1, and 43 were positive for muscle actins. Desmin immunoreactivity, of and by itself, cannot be considered specific for RMS, but when accompanied by immunoreactivity for other myogenic proteins, it is highly characteristic of RMS. Our data also suggest that desmin immunoreactivity, in the absence of other muscle-associated protein expression, might be considered a clue to the diagnosis of the blastemal WT. Particularly in the context of small biopsy specimens or in metastatic settings, the use of a panel of antibodies to desmin as well as to other myogenic proteins, such as MyoD1 or myogenin, can help to discriminate between WT and RMS. Additional studies are required to determine whether desmin immunoreactivity in the blastemal component of WT represents true desmin expression.

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD.

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression.

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor. Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma. Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as well as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin. Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin. In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody. To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed. Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1. They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen. Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood.

The distinction of rhabdomyosarcoma (RMS) from other small blue round cell tumors of childhood, such as Ewing's sarcoma/peripheral primitive neuroectodermal tumor (pPNET) and neuroblastoma, continues to present a diagnostic challenge to pathologists. The recent recognition of the master role of myogenic regulatory proteins in skeletal muscle commitment and differentiation, and the availability of monoclonal antibodies to two of them (myogenin and MyoD1), has prompted us to test their diagnostic utility in routinely processed, formalin-fixed, and deparaffinized tissue. Preliminary studies had demonstrated that, with the use of heat-induced epitope retrieval techniques, expression of myogenin and MyoD1 could be documented specifically in nuclei of fetal skeletal muscle by the respective antibodies. We performed a retrospective immunohistochemical analysis on 72 cases of small blue round cell tumors, including 33 RMSs, 1 metastatic myogenous Wilms' tumor, 26 Ewing's sarcomas/pPNETs, and 12 neuroblastomas. Nuclear expression of myogenin and MyoD1 were both found in 30/33 non-overlapping cases of RMS, with no significant differences in the sensitivity with respect to histological subtypes, and in 1/1 case of myogenous Wilms' tumor. None of the neuroblastomas or Ewing's sarcomas/pPNETs demonstrated positive nuclear staining with either antibody. However, most of the neuroblastomas, and occasional Ewing's sarcomas/pPNETs, showed variable fibrillary, cytoplasmic immunoreactivity with antibody to MyoD1. We conclude that, with the use of microwave-based epitope retrieval, antibodies to myogenin and MyoD1 are both useful markers for the identification of RMS among other small blue round cell tumors of childhood, but antibodies to myogenin have technical advantages over those to MyoD1, as the latter may cross-react with an unknown cytoplasmic antigen in non-muscle cells and tumors.