Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents a high-grade neoplasm of skeletal myoblast-like cells. Decades of clinical and basic research have gradually improved our understanding of the pathophysiology of RMS and helped to optimize clinical care. The two major subtypes of RMS, originally characterized on the basis of light microscopic features, are driven by fundamentally different molecular mechanisms and pose distinct clinical challenges. Curative therapy depends on control of the primary tumour, which can arise at many distinct anatomical sites, as well as controlling disseminated disease that is known or assumed to be present in every case. Sophisticated risk stratification for children with RMS incorporates various clinical, pathological and molecular features, and that information is used to guide the application of multifaceted therapy. Such therapy has historically included cytotoxic chemotherapy as well as surgery, ionizing radiation or both. This Primer describes our current understanding of RMS epidemiology, disease susceptibility factors, disease mechanisms and elements of clinical care, including diagnostics, risk-based care of newly diagnosed and relapsed disease and the prevention and management of late effects in survivors. We also outline potential opportunities to further translate new biological insights into improved clinical outcomes.

The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.

BACKGROUND: MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01. METHODS: MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively. RESULTS: MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed. CONCLUSIONS: An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.

Rhabdomyosarcoma of the orbit in a four months old infant in Zimbabwe: A case report.

Infants younger than one year of age with Rhabdomyosarcoma appear to have worse prognosis compared to older children due partly to high rates of local failure. We report a 4 months old infant with orbital rhabdomyosarcoma with poor outcome. Reluctance to use aggressive local control measures and suboptimal chemotherapy dosing are significant contributory factors. Call is made for need for more studies to determine appropriate local therapy in infants with rhabdomyosarcoma.

A case of rhabdomyosarcoma masquerading as acute leukemia at presentation: a case report.

Non-hematopoietic malignancies infiltrating bone marrow have always been a source of erroneous diagnosis. Among these, the small round cell tumors like neuroblastomas and rhabdomyosarcomas mimick the hematopoietic blasts. Several case reports of rhabdomyosarcoma mimicking acute leukemia, clinically and morphologically at presentation have been reported in the literature. To the best of our knowledge such an entity has not been reported in Indian literature. We report here one such case of alveolar rhabdomyosarcoma masquerading as acute leukemia. A thorough clinical examination with high degree of suspicion on bone marrow morphology and judicious use of appropriate immunohistochemistry markers will solve many of these cases.

The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas.

The receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF), have been implicated in the genesis of the paediatric tumour rhabdomyosarcoma (RMS). Addition of exogenous HGF to RH30 RMS cells enhanced non-chemotactic migration. Stable transfection of dominant negative MET into RH30 cells attenuated Matrigel invasion and in vivo tumour growth. To assess the role of a putative HGF-MET pathway in human RMS, we measured their expression in a panel of 68 human primary tumours. All tumours expressed MET but with a three orders of magnitude variation of expression and 62% of tumours co-expressed HGF. In contrast with other tumour types, neither high-MET expression nor HGF/MET coexpression correlated with metastatic disease. In a microarray screen, we identified CCN1 as being 7.8-fold up regulated following addition of HGF to RH30 cells and in RMS tumours, CCN1 expression correlated with HGF expression. Surprisingly, we identified MET as a consistent feature of embryonal and not alveolar RMS.

Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3-FKHR positive and negative tumors.

We analyzed the expression signatures of 14 tumor biopsies from children affected by alveolar rhabdomyosarcoma (ARMS) to identify genes correlating to biological features of this tumor. Seven of these patients were positive for the PAX3-FKHR fusion gene and 7 were negative. We used a cDNA platform containing a large majority of probes derived from muscle tissues. The comparison of transcription profiles of tumor samples with fetal skeletal muscle identified 171 differentially expressed genes common to all ARMS patients. The functional classification analysis of altered genes led to the identification of a group of transcripts (LGALS1, BIN1) that may be relevant for the tumorigenic processes. The muscle-specific microarray platform was able to distinguish PAX3-FKHR positive and negative ARMS through the expression pattern of a limited number of genes (RAC1, CFL1, CCND1, IGFBP2) that might be biologically relevant for the different clinical behavior and aggressiveness of the 2 ARMS subtypes. Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR.

Proliferative and apoptotic differences between alveolar rhabdomyosarcoma subtypes: a comparative study of tumors containing PAX3-FKHR or PAX7-FKHR gene fusions.

BACKGROUND: Most alveolar rhabdomyosarcomas (ARMS) have chromosome translocations and resultant gene fusion products. The more common translocation fuses the PAX3 and FKHR genes; patients who have PAX3-FKHR-positive ARMS have reduced event-free survival compared to patients with ARMS containing the less common translocation that fuses the PAX7 and FKHR genes. PROCEDURE: We examined histology, immunohistochemical markers of differentiation, and cell cycle characteristics of a panel of ARMS containing either PAX3-FKHR or PAX7-FKHR transcript to determine if these features differ between the ARMS subsets. RESULTS: Cell cycle parameters varied significantly: the number of nuclei that stained with either an immunohistochemical marker of proliferation (MIB1), or a TUNEL-based assay for apoptosis was significantly greater in tumors that expressed PAX3-FKHR compared to tumors that expressed PAX7-FKHR transcript. CONCLUSIONS: We conclude that compared to PAX7-FKHR-containing tumors, ARMS that contain PAX3-FKHR transcript have (1) increased cell proliferation, consistent with greater loss of cell cycle regulation, and (2) apoptosis that is increased but insufficient to prevent tumor formation. More marked cell cycle dysregulation may contribute to poorer prognosis for patients with ARMS that have PAX3-FKHR fusion. Med Pediatr Oncol 2001;37:83-89.

The role of chimeric paired box transcription factors in the pathogenesis of pediatric rhabdomysarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft tissue tumor with striated muscle differentiation. Chromosomal studies of these tumors identified 2;13 and 1;13 translocations. Using physical mapping and cloning strategies, we determined that t(2;13) and t(1;13) rearrange PAX3 and PAX7, which encode members of the paired box transcription factor family, and juxtapose these genes with FKHR, which encodes a novel member of the fork head transcription factor family. These translocations result in chimeric transcripts consisting of 5' PAX3 or PAX7 exons fused to 3' FKHR exons, which encode fusion proteins containing the PAX3 or PAX7 DNA-binding domain and the COOH-terminal FKHR transcriptional activation domain. In transfection studies, the PAX3-FKHR fusion activates transcription of reporter genes containing PAX DNA-binding sites, and is 10-100-fold more potent as a transcriptional activator than is wild-type PAX3. This increased function results from the insensitivity of the COOH-terminal FKHR activation domain to the inhibitory effects of NH2-terminal PAX3 domains. In addition to functional alterations, our studies demonstrated PAX3-FKHR and PAX7-FKHR overexpression resulting from two distinct mechanisms, increased transcription of PAX3-FKHR by a copy number-independent mechanism, and gene amplification of PAX7-FKHR. These findings indicate that the genetic changes in these tumors result in high levels of chimeric transcription factors that are hypothesized to inappropriately activate transcription of genes with PAX DNA-binding sites and thereby induce tumorigenic behavior. The differences in overexpression strategies suggest important differences between the mechanisms for regulating PAX3 and PAX7 expression. These differences extend to the phenotypic level, at which clinical differences have been found between the two ARMS subtypes: PAX7-FKHR tumors more often occur as localized lesions in the extremities of younger patients and are associated with longer event-free survival as compared to PAX3-FKHR tumors. Therefore, the clinical heterogeneity within the ARMS category is associated with genetic heterogeneity. Further analysis of the transcriptional function, regulation of expression, and phenotypic effects will help to elucidate the action of these fusion products and the biological basis of the clinical heterogeneity.

[Alveolar rhabdomyosarcoma presenting as acute leukemia]. / Alveoläres Rhabdomyosarkom unter dem Bild einer akuten Leukämie.

Rhabdomyosarcoma is the most common soft tissue sarcoma in adolescence and childhood, which manifests by the locally destructive growth of the primary tumor or its metastases. We report on a 29-year-old man with an alveolar rhabdomyosarcoma presenting with an unusual leukemia-like picture. On admission, the patient suffered from diffuse bone pain and renal insufficiency. Peripheral blood analysis showed anaemia, thrombocythaemia and blast-like cells. A bone marrow aspirate revealed extensive infiltration by atypical blast-like cells which were interpreted as acute lymphoblastic leukemia. Although confirmation of this diagnosis by immunophenotyping did not succeed chemotherapy was started immediately and led to partial remission. Histologic analysis of a bone marrow biopsy from the iliac crest, however, revealed an extensive solid tumor with alveolar spaces, lined by primitive round cells with positive PAS-reaction in the cytoplasm. Immunostaining demonstrated a positive reaction of the tumor cells for desmin and in a few tumor cells for smooth-muscle-actin. Chromosomal analysis showed a t(2;13) translocation typical for alveolar rhabdomyosarcoma. Although multiple lytic lesions of the skeletal system became evident during the further clinical course, the site of origin of the primary tumor could not be defined retrospectively. In conclusion, rhabdomyosarcoma should be included in the differential diagnosis of systemic diseases with extensive bone marrow infiltration by tumor cells that could otherwise be misinterpreted as a haematologic malignancy.

Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins.

The expression of a number of human paired box-containing (PAX) genes has been correlated with various types of tumors. Novel fusion genes encoding chimeric fusion proteins have been found in the pediatric malignant tumor alveolar rhabdomyosarcoma (RMS). They are generated by two chromosomal translocations t(2;13) and t(1;13) juxtaposing PAX3 or PAX7, respectively, with a forkhead domain gene FKHR. Here we describe that specific down-regulation of the t(2;13) translocation product in alveolar RMS cells by antisense oligonucleotides results in reduced cellular viability. Cells of embryonal RMS, the other major histiotype of this tumor, were found to express either wild type PAX3 or PAX7 at elevated levels when compared with primary human myoblasts. Treatment of corresponding embryonal RMS cells with antisense olignucleotides directed against the mRNA translational start site of either one of these two transcription factors similarly triggers cell death, which is most likely due to induction of apoptosis. Retroviral mediated ectopic expression of mouse Pax3 in a PAX7 expressing embryonal RMS cell line could partially rescue antisense induced apoptosis. These data suggest that the PAX3/FKHR fusion gene and wild-type PAX genes play a causative role in the formation of RMS and presumably other tumor types, possibly by suppressing the apoptotic program that would normally eliminate these cells.