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Background: CWS/RMS-96 was an international multicenter trial with randomization between two therapy arms of the standard four-drug therapy (vincristine, ifosfamide, adriamycin, dactinomycin [VAIA]) versus an intensified six-drug regimen (carboplatin, epirubicin, vincristine, dactinomycin, ifosfamide, and etoposide [CEVAIE]) for high-risk rhabdomyosarcoma (RMS), extraskeletal Ewing sarcoma (EES), and undifferentiated sarcoma (UDS) in children, adolescents, and young adults aiming to improve their survival. Intensified chemotherapy with CEVAIE did not improve outcome. Methods: Patients younger than 21 years with a previously untreated localized HR-RMS, EES, and UDS were enrolled from Cooperative Weichteilsarkom Studiengruppe (CWS) centers in Germany, Austria, Poland, Switzerland, and from Italian Soft Tissue Sarcoma Committee (STSC) centers. Randomization (1:1) to receive either 9 x 21 days cycles of VAIA or CEVAIE was performed separately in CWS and STSC. Hyperfractionated accelerated radiotherapy (32-44.8 Gy) was added at week 9-12 according to histology and response to chemotherapy. A secondary microscopically complete nonmutilating resection was performed if possible. Primary endpoints were response to chemotherapy, event-free (EFS) and overall survival (OS). Results: Five hundred fifty-seven patients (HR-RMS: n = 416, EES and UDS: n = 141) underwent randomization: VAIA (n = 273) or CEVAIE (n = 284). Radiotherapy was given to 70% of patients in both groups. A secondary resection was performed in 47% and 48% patients, respectively. The 5-year EFS and OS for the VAIA and CEVAIE treatment arms were 59.8% and 60.8% (p = .89), and 74.2% and 68.3% (p = .16), respectively. No differences in response, toxicity, or second malignancies emerged in the two groups. Conclusion: The use of an intensified regimen failed to show a significant improvement in tumor response and outcome of patients with localized HR-RMS, EES, and UDS.

Liquid biopsy analysis represents a powerful and noninvasive tool to uncover biomarkers for disseminated disease assessment and longitudinal monitoring of patients. Herein, we explored the value of circulating and disseminated tumor cells (CTC and DTC, respectively) and cell-free DNA (cfDNA) in pediatric rhabdomyosarcoma (RMS). Peripheral blood and bone marrow samples were analyzed to detect and enumerate CTC and DTC, respectively. We used the epithelial cellular adhesion molecule (EpCAM)-based CellSearch platform coupled with an automatic device to collect both EpCAM-positive and EpCAM-low/negative CTCs. The standard assay was implemented, including the mesenchymal marker desmin. For selected cases, we molecularly profiled primary tumors and liquid biopsy biomarkers using whole-exome sequencing and droplet digital PCR, respectively. RMS patients with metastatic disease had a significantly higher number of CTCs compared to those with localized disease, whereas DTCs were detected independently of disease presentation. The use of the desmin marker remarkably increased the identification of CTCs and DTCs in RMS samples. Of note, CTC clusters were detected in RMS patients with disseminated disease. Further, cfDNA and CTC molecular features closely reflected the molecular makeup of primary tumors and informed of disease course.

BACKGROUND: Spindle cell rhabdomyosarcoma (RMS) is a rare variant of RMS accounting for up to 10% of cases in infants. In older children and adults, spindle cell RMS is associated with MYOD1 mutations and a poor prognosis. In infants, it is associated with recurring fusions involving NCOA2 and VGLL2. Reports in the literature suggest a favorable prognosis for this subset, however, little is known about treatment and outcome data of infants with spindle cell RMS. METHODS: Characteristics, treatment, and outcome of an international cohort of 40 patients aged ≤12 months with spindle cell RMS treated from 1997–2018 were evaluated. RESULTS: Localized disease (LD) was diagnosed in 39 patients. The median age at diagnosis was 2.5 months (range 0–12 months). Expert pathologic review confirmed the diagnosis of spindle cell RMS in all patients. Among 26 tumors that had molecular evaluation, 13 had rearrangements of NCOA and/or VGLL. Multimodal treatment of infants with LD included conventional (age adjusted) chemotherapy (n=37), resection (n=31) and radiotherapy (RT) (n=5, brachytherapy in 3). Complete remission was achieved in 37/39 patients. Progressive disease occurred in two infants, relapsed disease in three. Microscopically complete surgical resection was associated with 5-year event-free survival (EFS) and overall survival (OS) of 100%. Two patients with tumors ≤ 5cm were treated with microscopically complete resection only and were alive 1 and 4.2 years after diagnosis. The 5-year EFS and OS for infants with LD were 86 % (±11; CI 95%) and 91% (±9; CI 95%), respectively. One patient had metastatic disease (NCOA fusion positive) with primary tumor in head and neck and brain metastases. This patient died despite chemotherapy and delayed resection of the primary tumor due to respiratory failure secondary to cytomegalovirus infection 1.2 years after diagnosis. CONCLUSION: Infants with spindle cell RMS have an excellent prognosis. Multimodal treatment including microscopically complete resection of the tumor is strongly recommended.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma characterized by a very poor prognosis when relapses occur after front-line therapy. Therefore, a major challenge for patients' management remains the identification of markers associated with refractory and progressive disease. In this context, cancer autoantibodies are natural markers of disease onset and progression, useful to unveil novel therapeutic targets. Herein, we matched autoantibody profiling of alveolar RMS (ARMS) patients with genes under regulatory control of PAX3-FOXO1 transcription factor and revealed fibroblast growth factor 8 (FGF8) as a novel ARMS tumor antigen of diagnostic, prognostic, and therapeutic potential. We demonstrated that high levels of FGF8 autoantibodies distinguished ARMS patients from healthy subjects and represented an independent prognostic factor of better event-free survival. FGF8 was overexpressed in ARMS tumors compared to other types of pediatric soft tissue sarcomas, acting as a positive regulator of cell signaling. Indeed, FGF8 was capable of stimulating ARMS cells migration and expression of pro-angiogenic and metastasis-related factors, throughout MAPK signaling activation. Of note, FGF8 was found to increase in recurrent tumors, independently of PAX3-FOXO1 expression dynamics. Risk of recurrence correlated positively with FGF8 expression levels at diagnosis and reduced FGF8 autoantibodies titer, almost as if to suggest a failure of the immune response to control tumor growth in recurring patients. This study provides evidence about the crucial role of FGF8 in ARMS and the protective function of natural autoantibodies, giving new insights into ARMS biology and laying the foundations for the development of new therapeutic strategies.

Procedure Congenital rhabdomyosarcoma (RMS) represents a challenging disease due to its characteristics and the difficulties in delivering treatment in this immature population. Methods We analyzed treatment and outcome of patients with congenital RMS, defined as tumor diagnosed in the first 2 months of life, enrolled in the European paediatric Soft tissue sarcoma Study Group protocols. Results Twenty-four patients with congenital RMS were registered. All, except one patient (PAX3-FOXO1-positive metastatic RMS), had favorable histology and localized disease. Three patients had VGLL2-CITED2/NCOA2 fusion. Complete tumor resection was achieved in 10 patients. No radiotherapy was given. Chemotherapy doses were adjusted to age and weight. Only two patients required further dose reduction for toxicity. The 5-year event-free survival (EFS) and overall survival (OS) were 75.0% (95% confidence interval [CI] 52.6-87.9) and 87.3% (95% CI 65.6-95.7), respectively. Progressive disease was the main cause of treatment failure. Conclusion Patients with congenital RMS presented with a favorable disease, allowing weight- and age-adjusted doses of chemotherapy and avoidance of irradiation, without compromising the outcome.

PROCEDURE The survival of children with rhabdomyosarcoma (RMS) has gradually improved as a result of the adoption of multidisciplinary treatments. Dedicated skills and facilities are indispensable and more readily available at reference centers. In this study, we examined the role of centers' experience (based on the number of patients treated) in their management of patients with RMS. METHODS We analyzed 342 patients with localized RMS enrolled in the European RMS 2005 protocol from October 2005 to December 2016 at 31 Italian centers that are part of the Soft Tissue Sarcoma Committee (STSC). We grouped the centers by the number of patients each one enrolled (Group 1: >40; Group 2: 10; and Group 3

Background: Spindle cell rhabdomyosarcoma (S-RMS) is a rare tumor that was previously considered as an uncommon variant of embryonal RMS (ERMS) and recently reclassified as a distinct RMS subtype with NCOA2, NCOA1, and VGLL2 fusion genes. In this study, we established a cell line (S-RMS1) derived from a four-month-old boy with infantile spindle cell RMS harboring SRF-NCOA2 gene fusion. Methods: Morphological and molecular characteristics of S-RMS1 were analyzed and compared with two RMS cell lines, RH30 and RD18. Whole genome sequencing of S-RMS1 and clinical exome sequencing of genomic DNA were performed. Results: S-RMS1 showed cells small in size, with a fibroblast-like morphology and positivity for MyoD-1, myogenin, desmin, and smooth muscle actin. The population doubling time was 3.7 days. Whole genome sequencing demonstrated that S-RMS1 retained the same genetic profile of the tumor at diagnosis. A Western blot analysis showed downregulation of AKT-p and YAP-p while RT-qPCR showed upregulation of endoglin and GATA6 as well as downregulation of TGFßR1 and Mef2C transcripts. Conclusion: This is the first report of the establishment of a cell line from an infantile spindle cell RMS with SRF-NCOA2 gene fusion. S-RMS1 should represent a useful tool for the molecular characterization of this rare and almost unknown tumor.

BACKGROUND As dermatofibrosarcoma protuberans (DFSP) are rare with no prospective series within paediatric sarcoma trials, the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) examined the clinical data and outcomes of DFSP enrolled in a multinational study of non-rhabdomyosarcoma soft tissue sarcomas (NRSTS). PATIENTS AND METHODS Forty-six patients with confirmed DFSP were enrolled into the EpSSG NRSTS 2005 study. All had surgical resection and none had any further therapy at diagnosis. RESULTS The median age at diagnosis was 6.9 years (range 0.4-17.5). All patients had localised disease, and the majority had small

Insulin-like growth factor-binding protein 2 (IGFBP2) is a tumor-associated protein measurable in patients&rsquo, biopsies and blood samples. Increased IGFBP2 expression correlates with tumor severity in rhabdomyosarcoma (RMS). Thus, we examined the plasmatic IGFBP2 levels in 114 RMS patients and 15 healthy controls by ELISA assay in order to evaluate its value as a plasma biomarker for RMS. Additionally, we looked for the presence of a humoral response against IGBFP2 protein measurable by the production of anti-IGFBP2 autoantibodies. We demonstrated that both circulating IGFBP2 protein and autoantibodies were significantly higher in RMS patients with respect to controls and their combination showed a better discriminative capacity. IGFBP2 protein identified metastatic patients with worse event-free survival, whereas both IGFBP2 and anti-IGFBP2 antibodies negatively correlated with overall survival. Our study suggests that IGFBP2 and anti-IGFBP2 antibodies are useful for diagnostic and prognostic purposes, mainly as independent negative prognostic markers in metastatic patients. This is the first study that reports a specific humoral response in RMS plasma samples and proves the value of blood-based biomarkers in improving risk assessment and outcome of metastatic RMS patients.

EpCAM is a transmembrane glycoprotein typically overexpressed in cancer of epithelial origin and mainly involved in the epithelial-to–mesenchymal transition (EMT) of tumor cells that spread and disseminate. Strategies for the targeting and capture of EpCAM-expressing tumor cells are showing promise in cancers prone to metastatize, both as diagnostic tools and potential therapies. Sarcomas are among the most aggressive tumors in children, with a common mesenchymal origin that comprises both soft tissue sarcomas (STS) and bone sarcomas. The aim of this study was to assess EpCAM expression in pediatric sarcomas and correlate its expression with disease progression. To do so, we analyzed a set of cell lines and primary tumor tissues from rhabdomyosarcoma (RMS), Ewing sarcoma (ES), synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT) STS, or osteosarcoma (OS) bone cancer. We demonstrated that EpCAM was variably expressed in pediatric sarcomas, with DSRCT, a rare, aggressive and almost fatal tumor type, characterized by the highest EpCAM expression levels. Interestingly, although EpCAM expression was lower in RMS tumors, high levels at diagnosis correlated with reduced patients' overall survival (p

Rhabdomyosarcoma is an aggressive solid tumor that may disseminate hematogenously giving metastasis which represents the most important prognostic factor. Chances of an effective cure in childhood cancer rely on the capacity to make an early and accurate diagnosis, detect metastatic disease or relapse, and predict the response to treatment.Liquid biopsy is a very promising blood test for cancer detection and noninvasive disease monitoring. This method has a great advantage to use blood and plasma, a more accessible biological material, quick and easy to obtain with minimal pain and risk for patients. In particular, circulating free DNA (cfDNA) represents a tumor biomarker detected in plasma that gives information on biology and genetic background of tumor.Moreover, cfDNA mutation detection could be a reliable method to monitor the efficacy of treatment and to discover novel targets for a personalized treatment in pediatric solid tumor. Here, we describe an optimized protocol to cfDNA isolation from small amount of plasma, as well as a method to assess the quantity and quality of cfDNA. Finally, we propose ddPCR as a reliable method to detect mutations at low frequency in cfDNA obtained from pediatric rhabdomyosarcoma samples.

Rhabdomyosarcoma is an aggressive solid tumor that may disseminate hematogenously giving metastasis which represents the most important prognostic factor. Chances of an effective cure in childhood cancer rely on the capacity to make an early and accurate diagnosis, detect metastatic disease or relapse, and predict the response to treatment.Liquid biopsy is a very promising blood test for cancer detection and noninvasive disease monitoring. This method has a great advantage to use blood and plasma, a more accessible biological material, quick and easy to obtain with minimal pain and risk for patients. In particular, circulating free DNA (cfDNA) represents a tumor biomarker detected in plasma that gives information on biology and genetic background of tumor.Moreover, cfDNA mutation detection could be a reliable method to monitor the efficacy of treatment and to discover novel targets for a personalized treatment in pediatric solid tumor. Here, we describe an optimized protocol to cfDNA isolation from small amount of plasma, as well as a method to assess the quantity and quality of cfDNA. Finally, we propose ddPCR as a reliable method to detect mutations at low frequency in cfDNA obtained from pediatric rhabdomyosarcoma samples.

Distinguishing between alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) is crucial because treatment and prognosis are different. We describe a case of paratesticular rhabdomyosarcoma (RMS), which was classified as mixed ERMS/ARMS. Fluorescence in situ hybridization (FISH) detected losses of 3'PAX3 and 5'FOXO1, suggesting they had undergone an unbalanced rearrangement that probably produced the PAX3-FOXO1 fusion. Double-color FISH and reverse transcription-polymerase chain reaction (RT-PCR) revealed PAX3-FOXO1, which is characteristic of high-risk RMS. This finding highlights the importance of supplementing histology with genetics so that atypical RMS is appropriately classified and patients are correctly stratified and treated.

Background Rhabdomyosarcoma (RMS) is a highly malignant tumor typically affecting children and adolescents. Central nervous system (CNS) dissemination is rare in RMS patients, but seems to have a particularly negative impact. The aim of this study was to analyze treatment and outcome of patients with RMS and evidence of CNS disease who were registered in the protocols coordinated by the Italian Soft Tissue Sarcoma Committee from March 1979 to December 2016. Methods We analyzed 39 patients with CNS disease. Depending on when their CNS disease was identified, we grouped patients as: Group A, at diagnosis; Group B, progression during treatment; Group C, at first relapse. Results Six patients were in Group A (2.7% of metastatic RMS patients at diagnosis); 24 were in Group B and 9 in Group C (6.5% of patients with tumor progression/relapse included in the protocols). Only 5 patients (4 in Group A, 1 in Group B) survived the event and are alive in complete remission with a median follow-up of 17.5 years. These 5 patients received systemic chemotherapy and craniospinal radiotherapy, and 2 of them also received intrathecal therapy with topotecan. Conclusions CNS involvement at diagnosis is a rare and prognostically negative event in RMS patients, but not always fatal when it is found at diagnosis. It is more frequent during or shortly after treatment, and the more dismal prognosis in these cases underscores the need to improve our ability to identify patients at risk of CNS dissemination in order to attempt more effective treatments that can sterilize the meninges.

// Lucia Tombolan 1, 4 , Elena Poli 4 , Paolo Martini 1 , Angelica Zin 3 , Chiara Romualdi 1 , Gianni Bisogno 4 , Gerolamo Lanfranchi 1, 2 1 Department of Biology, University of Padova, Padova, Italy 2 Centro di Ricerca Interdipartimentale per le Biotecnologie Innovative, University of Padova, Padova, Italy 3 Institute of Pediatric Research, Padova, Italy 4 Department of Women’s and Children’s Health, Oncology Hematology Division, University of Padova, Padova, Italy Correspondence to: Lucia Tombolan, email: lucia.tombolan@unipd.it Keywords: DNA methylation, RRBS, sequencing, rhabdomyosarcoma, NELL1 Received: October 20, 2016 Accepted: March 14, 2017 Published: March 23, 2017 ABSTRACT Rhabdomyosarcoma (RMS), which represents the most frequent soft tissue sarcoma in pediatric populations, is classified into two major subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS subtype, which shows greater aggressiveness and proneness to metastasis with respect to ERMS, are characterized, in about 75% of cases, by specific chromosomal translocations that involve PAX and FOXO1 genes. Many findings have demonstrated that PAX/FOXO1 -positive ARMS have a worse prognosis than PAX/FOXO1 -negative ones and that distinct molecular features characterize RMS with different gene fusion statuses. DNA methylation, which presently represents a challenging research area, is involved in the modulation of gene expression. We performed a genome-wide DNA methylation analysis using reduced-representation bisulfite sequencing (RRBS) in RMS samples and we found that fusion-positive alveolar and embryonal subgroups have different DNA methylation signatures and that ARMS fusion-positive subtypes are characterized by overall hypomethylation levels. While NELL1 was found to be hypomethylated and transcriptionally enhanced in RMS alveolar subtypes, high NELL1 expression levels, which proved to be correlated with negative RMS prognostic factors such as fusion status and histology ( P < 0.0001), were found to discriminate between RMS patients with different outcomes ( P < 0.05). In conclusion, our results demonstrated that different DNA methylation patterns distinguish between different RMS subgroups and they suggest that epigenetic signatures could be useful for risk stratification of patients.

Background Rhabdomyosarcoma (RMS), which can be classified as embryonal RMS (ERMS) and alveolar RMS (ARMS), represents the most frequent soft tissue sarcoma in the pediatric population; the latter shows greater aggressiveness and metastatic potential with respect to the former. Epigenetic alterations in cancer include DNA methylation changes and histone modifications that influence overall gene expression patterns. Different tumor subtypes are characterized by distinct methylation signatures that could facilitate early disease detection and greater prognostic accuracy. Methods A genome-wide approach was used to examine methylation patterns associated with different prognoses, and DNA methylome analysis was carried out using the Agilent Human DNA Methylation platform. The results were validated using bisulfite sequencing and 5-aza-2′deoxycytidine treatment in RMS cell lines. Some in vitro functional studies were also performed to explore the involvement of a target gene in RMS tumor cells. Results In accordance with the Intergroup Rhabdomyosarcoma Study (IRS) grouping, study results showed that distinct methylation patterns distinguish RMS subgroups and that a cluster of protocadherin genes are hypermethylated in metastatic RMS. Among these, PCDHA4, whose expression was decreased by DNA methylation, emerged as a down-regulated gene in the metastatic samples. As PCDHA4-silenced cells have a significantly higher cell proliferation rate paralleled by higher cell invasiveness, PCDHA4 seems to behave as a tumor suppressor in metastatic RMS. Conclusion Study results demonstrated that DNA methylation patterns distinguish between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2936-3) contains supplementary material, which is available to authorized users.

Sclerosing rhabdomyosarcoma (ScRMS) and spindle cell rhabdomyosarcoma (SRMS) have been recently reclassified as a stand-alone pathologic entity, separate from embryonal RMS. Genetically, a subset of the congenital cases display NCOA2 gene rearrangements, whereas tumors occurring in older children or adults harbor MYOD1 gene mutations with or without coexisting PIK3CA mutations. Despite these recent advances, a significant number of tumors lack known genetic alterations. In this study we sought to investigate a large group of pediatric SRMS/ScRMS, spanning a diverse clinical and pathologic spectrum, by using a combined fluorescence in situ hybridization, targeted DNA, and whole-transcriptome sequencing methodology for a more definitive molecular classification. A total of 26 SRMS and ScRMS cases were selected from the 2 participating institutions for the molecular analysis. Ten of the 11 congenital/infantile SRMS showed recurrent fusion genes: with novel VGLL2 rearrangements seen in 7 (63%), including VGLL2-CITED2 fusion in 4 and VGLL2-NCOA2 in 2 cases. Three (27%) cases harbored the previously described NCOA2 gene fusions, including TEAD1-NCOA2 in 2 and SRF-NCOA2 in 1. All fusion-positive congenital/infantile SRMS patients with available long-term follow-up were alive and well, none developing distant metastases. Among the remaining 15 SRMS patients older than 1 year, 10 (67%) showed MYOD1 L122R mutations, most of them following a fatal outcome despite an aggressive multimodality treatment. All 4 cases harboring coexisting MYOD1/PIK3CA mutations shared sclerosing morphology. All 5 fusion/mutation-negative SRMS cases presented as intra-abdominal or paratesticular lesions.

// Patrizia Gasparini 1, * , Michela Casanova 2, * , Raffaella Villa 3 , Paola Collini 4 , Rita Alaggio 5 , Angelica Zin 6 , Paolo Bonvini 6 , Cristina R Antonescu 8 , Renata Boldrini 9 , Roberto Caserini 1 , Massimo Moro 1 , Giovanni Centonze 1 , Cristina Meazza 2 , Maura Massimino 2 , Luca Bergamaschi 2 , Roberto Luksch 2 , Stefano Chiaravalli 2 , Gianni Bisogno 10 , Nadia Zaffaroni 7 , MariaGrazia Daidone 3 , Gabriella Sozzi 1, # , Andrea Ferrari 2, # 1 Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2 Department of Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3 Biomarkers Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4 Soft Tissue and Bone Pathology, Histopathology and Pediatric Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5 Department of Pathology, University Hospital of Padova, Padova, Italy 6 Institute of Pediatric Research Citta della Speranza, Padova, Italy 7 Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA 9 Department of Pathology, Bambino Gesu Children Hospital-Research Institute, Rome, Italy 10 Hematology Oncology Division, Department of Mother and Child’s Health, University Hospital of Padova, Padova, Italy * These authors contributed equally to this work # Co-last authors Correspondence to: Gabriella Sozzi, email: gabriella.sozzi@istitutotumori.mi.it Keywords: anaplastic lymphoma kinase, rhabdomyosarcoma, EML4-ALK, chromosomal rearrangement, metastasis Received: February 11, 2016 Accepted: June 12, 2016 Published: July 01, 2016 ABSTRACT Rhabdomyosarcoma (RMS) is the most frequent soft tissue tumor in childhood and arises from immature mesenchymal cells committed to skeletal muscle differentiation. Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in several cancers. Moreover, ALK full-length receptor protein has been observed in RMS, although its clinical and functional significance is yet controversial. The role of ALK and its clinical relevance were investigated in a selected cohort of 74 FFPE pediatric RMS and a panel of RMS cell lines, evaluating its gene and protein status, utilizing Fluorescent In Situ Hybridization (FISH), immunohistochemistry (IHC) and Western blot approaches. Moreover, to get insight into its possible therapeutic relevance, effects of ALK silencing on cell proliferation, invasion and apoptosis were studied in RMS cells. ALK IHC positivity was significantly correlated with gene copy number gain, the alveolar subtype, PAX3/7-FOXO1 rearrangements, the presence of metastasis at diagnosis and a worse overall outcome. Furthermore, EML4-ALK fusion gene associated with higher protein expression was identified in an embryonal RMS. ALK silencing in RH30 ALK positive cells strongly inhibited invasion capability. Overall, our data suggest a potential role of ALK in pediatric RMS.

Background Rhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis. Methodology/Principal Findings The expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA). Conclusions Study results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.

Distinguishing between alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) is crucial because treatment and prognosis are different. We describe a case of paratesticular rhabdomyosarcoma (RMS), which was classified as mixed ERMS/ARMS. Fluorescence in situ hybridization (FISH) detected losses of 3'PAX3 and 5'FOXO1, suggesting they had undergone an unbalanced rearrangement that probably produced the PAX3-FOXO1 fusion. Double-color FISH and reverse transcription-polymerase chain reaction (RT-PCR) revealed PAX3-FOXO1, which is characteristic of high-risk RMS. This finding highlights the importance of supplementing histology with genetics so that atypical RMS is appropriately classified and patients are correctly stratified and treated.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and is mostly represented by the embryonal (ERMS) and alveolar (ARMS) histotypes. Whereas ERMS shows variable genetic alterations including TP53, RB1, and RAS mutations, ARMS carries a gene fusion between PAX3 or PAX7 and FOXO1. Epithelioid RMS is a morphologic variant of RMS recently described in adults. Five cases of epithelioid RMS were identified after histologic review of 85 cases of ARMS enrolled in Italian therapeutic protocols. Immunostaining analyses (muscle-specific actin, desmin, myogenin, AP-2β, EMA, cytokeratins, INI-1) and reverse transcription polymerase chain reaction assays to detect MyoD1, myogenin, and PAX3/7-FOXO1 transcripts were performed. In 4 cases DNA sequencing of TP53 was performed; and RB1 allelic imbalance and homozygous deletion were analyzed by quantitative real-time polymerase chain reaction. Histologically, epithelioid RMS displayed sheets of large cells without rhabdomyoblastic differentiation or anaplasia in 3 and prominent rhabdoid cells in 2; necrosis was evident in 4, often with a geographic pattern. Immunostainings for INI, desmin, myogenin (scattered cells in 4, diffuse in 1) were positive in all; EMA and MNF116 were positive in 2; AP-2β was negative. PAX3/7-FOXO1 transcripts were absent. In all cases RB1 was wild type, and a TP53 mutation at R273H codon was found in 1. All patients are in complete remission, with a median follow-up of 6 years. Epithelioid RMS may occur in children and is probably related to ERMS, as suggested by lack of fusion transcripts, weak staining for myogenin, negative AP-2β, evidence of TP53 mutation (although only in 1 case), and a favorable clinical course.

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (P

Expression of multidrug resistance (MDR) proteins is thought to significantly contribute to the different biological/clinical behaviour of soft tissue sarcomas (STS) of various histological types and clinicopathological stages, as they are responsible for active efflux of cytotoxic drugs from tumour cells. We investigated the expression of 3 MDR proteins, i.e., permeability glycoprotein 1 (P-gp), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance 3 (MDR3), in 43 STS specimens from newly-diagnosed paediatric patients, 31 with rhabdomyosarcoma (RMS) and 12 with non-RMS STS. To assess the influence of chemotherapy on STS drug resistance, the number of MDR-associated protein-positive cells was determined in 15 patients on both primary lesions before chemotherapy and on residual tumour after chemotherapy. At least one of the MDR-associated proteins tested was detected in 84% of primary untreated STS specimens. In these specimens, MRP1 was detected in a high percentage (70%) of the cases, followed by MDR3 in 58% and P-gp in 44%. Many specimens showed co-expression of two different MDR proteins. Interestingly, MDR3 was significantly associated with the presence of PAX3/PAX7-FKHR transcripts in RMS (p

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor–binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR -negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS.

Background Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-β-D-glucuronidase that cleaves heparan sulphate proteoglycans. Methods We determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable HPSE silencing has been performed by shRNA technique in RH30 and RD cell lines and their invasiveness has been evaluated by Matrigel-invasion assay. HPSE activity and mRNA expression have also been quantified in plasma and biopsies from RMS patients. Results HPSE expression and activity have been detected in all RMS cell lines. Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells. Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS. Conclusion In conclusion, our data demonstrate for the first time HPSE expression and activity in RMS and highlight its involvement in tumor cell invasion as revealed by shRNA silencing. Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects. Further studies are warranted to assess possible relationships between HPSE and clinical behaviour in RMS.