4654

MYOD1

MYF3|MYOD|PUM|bHLHc1;myogenic differentiation 1;MYOD1;myogenic differentiation 1

class C basic helix-loop-helix protein 1|myf-3|myoblast determination protein 1|myogenic factor 3

11p15.4

protein-coding

childhood rhabdomyosarcoma (RMS) has two major histological subtypes: alveolar (aRMS) and embryonal. The aim of the study was to monitor minimal disseminated disease (MDD) using real-time quantitative reverse-transcription PCR (RQ-RT-PCR)of the PAX3-FKHR, PAX7-FKHR fusion genes and myoD1 gene. We prepared an assay using RQ-RT-PCR for a quantitative assessment of MDD in aRMS by using hydrolysisprobe for quantification of PAX3-FKHR, PAX7-FKHR and myoD1 genes and beta-2-microglobulin housekeeping gene. Primary tumor samples (44), samples of local recurrences (26) from 48 patients with aRMS were examined by nested RT-PCRand RQ-RT-PCR techniques. Additionally, bone marrow samples (115), peripheral blood progenitor cell samples (27), and peripheral blood samples (25) from 33 aRMS patients were tested. PAX3/7-FKHR and myoD1 transcripts proved to be a sensitive tool for detection of MDD in RMS. We were able to identify 15/25 patients with bone marrow (BM) involvement at the time of presentation using RQ-RT-PCR. We analyzed PAX3-FKHR or PAX7-FKHR expression during the course of the disease. The RQ-RT-PCR results correlated well with nested RT-PCR results (p < 0.0001). The presence of metastases is the most adverse prognostic factor in RMS, and bone marrow is a frequent site of the tumor dissemination in RMS, especiallyin aRMS. Our results detecting the fusion transcripts or myoD1 transcript in theBM or peripheral blood suggest that patients with positive findings are at high risk of the tumor progression.

Transcripts for the muscle regulatory gene MyoD1 are expressed during normal skeletal muscle myogenesis and in rhabdomyosarcomas but not in other tissues or in soft-tissue sarcomas. Here we report the distribution of MyoD1 protein, determined by reactivity with anti-MyoD1 polyclonal sera in normal tissues, rhabdomyosarcoma cell lines, and in a variety of pediatric solid tumors. The distribution of MyoD1 protein was highly restricted in normal tissues and was detected only in fetal skeletal muscle and more faintly in adult skeletal muscle. ALL six human rhabdomyosarcoma cell lines analyzed expressed MyoD1 mRNA transcripts as well as immunoreactive protein. The immunohistochemical expression of MyoD1 protein was then examined in 49 surgical specimens from a variety of pediatric solid tumors. Each of 16 rhabdomyosarcoma specimens was positive for MyoD1, including four that did not express the intermediate filament protein desmin. Two of five specimens originally designated sarcoma type indeterminate (STI) and two of three specimens originally designated extraosseous Ewing's sarcoma (EOE) were positive for MyoD1, suggesting commitment to myogenic differentiation. Three of eight Wilms' tumors, which also expressed desmin and had clearly evident myogenic elements, also were positive for MyoD1. tumors thatfailed to express MyoD1 protein included neuroblastoma, primitive neuroectodermal tumor, non-Hodgkins lymphoma, embryonal sarcoma of the liver, malignant fibrous histiocytoma, malignant rhabdoid tumor, and Ewing's sarcoma of the bone. These results indicate that expression of MyoD1 protein is highly restricted in normalhuman tissues and that expression of this gene product in malignant tissue may be diagnostic for rhabdomyosarcoma. Furthermore MyoD1 staining may be a valuable adjunct in the classification of pediatric soft-tissue sarcomas.

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereasJDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, withpresumed skeletal muscle origins, because of its myogenic phenotype. RMS is composed of two main subtypes, embryonal RMS (eRMS) and alveolar RMS (aRMS). Whereas eRMS histologically resembles embryonic skeletal muscle, the aRMS subtype is more aggressive and has a poorer prognosis. In addition, whereas the genetic profile of eRMS is not well established, aRMS is commonly associated with distinct chromosome translocations that fuse domains of the transcription factors Pax3 and Pax7 to the forkhead family member FOXO1A. Both eRMS and aRMS tumor cells express myogenic markers such as MyoD, but their ability to complete differentiation is impaired. How this impairment occurs is the subject of this review, which will focus on several themes, including signaling pathways that converge on Pax-forkhead gene targets, alterations in MyoD function, epigenetic modifications of myogenic promoters, and microRNAs whose expression patterns in RMS alter key regulatory circuits to help maintain tumor cells in an opportunistically less differentiated state.#CI- (c) 2013 FEBS.

Cytogenetic and molecular analysis of soft tissue tumors has yielded a wealth ofinformation over the past decade. Some of the genetic aberrations that have beenidentified appear to be fairly specific for individual tumor types. It is because of this specificity that these findings harbor the promise to become useful as diagnostic and/or prognostic markers. Technical advances that allow the application of cytogenetic and molecular techniques to archival material have been crucial in this respect. Molecular genetics has already become an integral part of the work-up of some tumors, e.g., small cell sarcomas of childhood, which demonstrate fairly characteristic translocations, often involving the Ewing's sarcoma gene. Some genetic abnormalities have become established as prognostic markers, such as the deletion of the short arm of chromosome 1 for neuroblastomas. Soft tissue tumor pathology has also benefitted from major advances in identifying genes that are critical in mesenchymal differentiation or cell cycle control. MyoD is a good example of a such a gene, that has become useful as a diagnostic tool in rhabdomyosarcomas. Beyond potential practical applications of cytogenetic and molecular analyses in the diagnosis of these tumors, we also review their impact on several philosophical concepts concerningsoft tissue neoplasia.

Previous findings that the intermediate filament nestin is expressed in immatureskeletal muscle cells prompted us to compare the staining patterns of nestin anddesmin in rhabdomyosarcomas (RMSs) and in other small cell tumors of infancy. Wefound that nestin immunoreactivity was present in ALL of 29 examined typical RMSs, which also expressed desmin. Two undifferentiated tumors, primarily suspected to be RMSs, expressed nestin, but not desmin. One of these nestin-positive, desmin-negative tumors was positive for the expression of the myogenic regulatory gene MyoD and is considered to represent an undifferentiatedRMS. The other, a paratesticular tumor, did not contain transcripts for MyoD, and most likely does not represent a RMS. In several RMSs and nonmuscle tumors, a z-disc-associated nestin immunoreactivity occurred as a paramalignant phenomenonin cross-striated muscle fibers adjacent to the tumor cells. Our findings indicate that nestin, although present also in tumors of the central and peripheral nervous systems, as well as in endothelial cells and in some muscle cells adjacent to tumors, is a useful complementary marker for RMS, particularlyin very undifferentiated desmin-negative tumors.