Rhabdomyosarcoma (RMS) is the most common sarcoma of childhood and adolescence. Approximately 10% arise in the orbit, where the embryonal type is most common variant. The alveolar variant is less frequent and has a worse prognosis. Cytogenetic studies have revealed that most alveolar rhabdomyosarcomas have translocations involving the PAX and the FOX01 genes, giving rise to fusion genes that contribute to lack of differentiation and proliferation of the tumor cells. However, approximately 20% of alveolar rhabdomyosarcomas lack translocations and have been found to behave more similarly to embryonal cases. Histopathology remains the basis of diagnosis, but cytogenetic features and molecular signatures are becoming part of the routine analysis of RMS, since they determine not only prognosis, but also management and treatment regimens. A comprehensive review of the recent published literature in relation to orbital rhabdomyosarcomas and their cytogenetic features as well as clinical and therapeutic implications will be discussed.

Rhabdomyosarcoma (RMS) represents the most common soft tissue sarcoma in the pediatric age group. While RMS has been traditionally classified on the basis of its histological appearance (with embryonal and alveolar being most common), it is now clear that the PAX-FOXO1 fusion product drives prognosis. We report here a case of pelvic embryonal RMS in a 3-month-old male who was subsequently found to have developed brain metastases during the course of chemotherapy. Cytogenetic analysis of the brain metastases at the time of autopsy as well as next-generation sequencing analysis revealed a reciprocal translocation involving the SH3 domain containing ring finger 3 gene (SH3RF3, on chromosome 2q13) and the Lipase C gene (LIPC, on chromosome 15q21.3). Due to the poor quality of the pretreatment and postresection samples, cytogenetics and NGS analysis looking for the presence of this balanced translocation in these specimens could not be performed. To the authors' knowledge, this translocation has never been described in RMS. Further studies are needed to determine the biological and clinical implications of this novel translocation.

AIMS: Spindle cell/sclerosing rhabdomyosarcomas (SC/SRMS) feature spindled and/or rounded rhabdomyosarcomatous cells within variably hyalinised stroma. Only 30-67% of SC/SRMSs harbour neomorphic MYOD1 p.L122R mutations, indicating heterogeneity in this RMS type. We compared MYOD1-mutant and non-mutant cases to characterise the histological and genetic spectrum of mutated SC/SRMS.
METHODS AND RESULTS: Seventeen RMSs with spindled, sclerosing or hybrid histology were sequenced to identify MYOD1 and PIK3CA mutations and reappraised to assess histological features and myogenic immunophenotypes. Twelve SC/SRMSs harboured MYOD1 mutations, including homozygous p.L122R (n = 8), heterozygous p.L122R (n = 3) and heterozygous p.E118K (n = 1). MYOD1-mutant tumours affected nine females and three males aged 8-64 years (median = 22.5), had a median size of 4.2 cm (range = 2-22) and involved the head and neck (n = 7), extremities (n = 4) and mediastinum (n = 1). Fascicular/spindle histology was predominant in four cases, including one with heterologous lipoblasts in focally myxoid stroma. Four sclerosing cases mainly comprised rounded cells, including one with multinucleated tumour cells. Four cases were histologically hybrid. The only PIK3CA (p.H1047R) mutation was detected in a predominantly spindled MYOD1-p.L122R-mutated case, but not in its laser-microdissected lipoblast-containing area. All MYOD1-mutant cases exhibited diffuse MYOD1 expression but patchy myogenin reactivity. At final follow-up (median = 13.5 months), recurrences (n = 4), metastases (n = 2) or both (n = 1) occurred in seven MYOD1-mutant cases; one had died of disease. Five non-mutated cases were reclassified as spindle embryonal (n = 3), dense embryonal (n = 1) and unclassifiable (n = 1) RMSs.
CONCLUSION: MYOD1-mutant RMSs are uncommonly mutated with PIK3CA and behave aggressively with an expanded morphological and genetic spectrum, including lipoblastic differentiation, multinucleated cells and the alternative p.E118K mutation.

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 (RMS) is a family of soft tissue cancers that are related to the skeletal muscle lineage and predominantly occur in children and young adults. A specific chromosomal translocation t(2;13)(q35;q14) that gives rise to the chimeric oncogenic transcription factor PAX3-FOXO1 has been identified as a hallmark of the aggressive alveolar subtype of RMS. PAX3-FOXO1 cooperates with additional molecular changes to promote oncogenic transformation and tumorigenesis in various human and murine models. Its expression is generally restricted to RMS tumor cells, thus providing a very specific target for therapeutic approaches for these RMS tumors. In this article, we review the recent understanding of PAX3-FOXO1 as a transcription factor in the pathogenesis of this cancer and discuss recent developments to target this oncoprotein for treatment of RMS.

MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.

Embryonal rhabdomyosarcoma is a rare cancer that often requires multimodality therapy to treat; however, these therapies can cause changes in the biology of the tumor. Several reports have documented pathologic changes but only recently have genetic changes been mapped. We present case of two separate synchronous primary rhabdomyosarcomas in a 17-month-old patient and discuss the pathophysiology and genetic changes that occur with treatment. We hypothesize that a genetic field defect arising in development of the urogenital sinus caused the tumors, but that treatment modalities may have caused genetic alterations changing clinical behavior of the tumors and responses to treatment.

The last 25 years have brought significant progress in the treatment of sarcomas in children, especially rhabdomyosarcoma (RMS). Nevertheless, treatment failure in some patients results from considerable biological heterogeneity noted in these tumours. RMS, the most common malignant soft tissue neoplasm in children, includes two main subtypes: embryonal (ERMS) and alveolar (ARMS). Due to greater aggressiveness and worse prognosis of ARMS in comparison to ERMS, discrimination between different rhabdomyosarcoma subtypes is of crucial clinical importance. This paper presents the current histological classification of RMS, up-to-date immunohistochemical and biological research regarding RMS, and its associated clinical and prognostic significance.

Vincristine (VCR) is a common chemotherapeutic agent used in the treatment of multiple types of pediatric tumors. VCR's adverse effects are well documented and commonly involve peripheral neuropathy via axonal degeneration. Neuropathic severity is dose-dependent, with sensory deficits occurring with as little as 4 mg cumulative dose. Severe peripheral neuropathy is generally rare, but its effects become additive when given to patients with undiagnosed hereditary peripheral neuropathy such as Charcot-Marie-Tooth. We report a case of an effect of VCR administration given to a patient who developed grade 4 neuropathy and was found to be a carrier of Charcot-Marie-Tooth disease type 4.

Splice-switching oligonucleotides (SSOs) that can modulate RNA splicing are used for the treatment of many genetic disorders. To enhance the efficacy of modulating splicing, it is important to optimize SSOs with regard to target sites, GC content, melting temperature (Tm value), chemistries, and lengths. Thus, in vitro assay systems that allow for the rapid and simple screening of SSOs are essential for optimizing SSO design. In this study, we established a novel tri-chromatic reporter cell line for SSO screening. This reporter cell line is designed to express three different fluorescent proteins (blue, green, and red) and was employed for high content screening (HCS, also known as high content analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the expression levels of fluorescent proteins. The blue fluorescent protein is stably expressed throughout the cell and is useful for data normalization using cell numbers. Furthermore, both the green and red fluorescent proteins were used for monitoring the splicing patterns of target genes. Indeed, we demonstrated that this novel reporter cell line involving HCS leads to a more rapid and simple approach for the evaluation of exon skipping than widely used methods, such as RT-PCR, western blotting, and quantitative RT-PCR. Additionally, a brief screening of Locked nucleic acids (LNA)-based SSOs targeting exon 51 in DMD was performed using the reporter cell line. The LNA-based SSO cocktail shows high exon 51 skipping in a dose-dependent manner. Furthermore, the LNA-based SSO cocktails display high exon 51 skipping activities on endogenous DMD mRNA in human rhabdomyosarcoma cells.

AIMS: Rhabdomyosarcomas of bone are extremely rare, with fewer than 10 reported cases. A very rare subtype of spindle cell/sclerosing rhabdomyosarcoma harbouring a FUS-TFCP2 fusion and involving both soft tissue and bone locations has been reported very recently. We report only the fourth case of this unusual, clinically aggressive rhabdomyosarcoma.
MATERIAL AND RESULTS: A previously well 72-year-old male presented with a destructive lesion of the mandible. Morphological and immunohistochemical study of a needle biopsy and the subsequent resection showed a spindle cell rhabdomyosarcoma. RNA-seq, RT-PCR and FISH confirmed the presence of the FUS-TFCP2 fusion.
CONCLUSIONS: Spindle cell rhabdomyosarcomas carrying the FUS-TFCP2 fusion are very rare rhabdomyosarcoma variants with osseous predilection. The classification and differential diagnosis of this unusual molecular variant of spindle cell/sclerosing rhabdomyosarcoma are discussed.

The TP53 gene is fundamental to genomic integrity, cell cycle regulation, and apoptosis; it is the most commonly mutated gene in human cancer. Heterozygous germline mutations cause the autosomal dominant cancer predisposition syndrome, Li-Fraumeni Syndrome. Homozygous germline TP53 mutations in humans are rare. We report an infant from a consanguineous family who presented with synchronous malignancies. Remarkably, he carries a homozygous germline TP53 mutation (NM_000546.4:c.52delA), predicted to cause protein truncation. The family history is consistent with Li-Fraumeni syndrome.

Different types of malignancies can be seen in patients with neurofibromatosis type 1 (NF-1). Herein we present a rare combination of NF-1 and biliary rhabdomyosarcoma in a male infant. An 11-month-old boy, who was recently diagnosed with NF-1, presented to the outpatient clinic with a 3-month history of prolonged jaundice, and failure to thrive. Clinical examination showed >20 café au let spots distributed mainly over the abdominal trunk. Hepatomegaly (4 cm below the costal margin) was additionally observed. His father was diagnosed with NF-1. Radiologic imaging studies showed a 6×5×5 cm in diameter cystic mass with multiple septations in the segment 4A of the liver. Surgical excision of the left hepatic lobe followed by hepatojejunostomy was further performed. Histopathology examination showed embryonal type rhabdomyosarcoma originating from the biliary duct. Chemotherapy regimen consisting of cyclophosphamide, actinomycin D, and vincristine, and radiotherapy were then initiated. This treatment led to a significant improvement in the patient's clinical status, and radiologic finding portrayed attainment of complete resolution. He is still in complete remission without any sequelae for 8 years.

BACKGROUND: Alveolar rhabdomyosarcomas (ARMSs) can harbour MET and anaplastic lymphoma kinase (ALK) alterations. We prospectively assessed crizotinib in patients with advanced/metastatic ARMS.
METHODS: Eligible patients with a central diagnosis of ARMS received oral crizotinib 250 mg twice daily. Patients were attributed to MET/ALK+ or MET/ALK- subcohorts by assessing the presence or absence of the forkhead box O1 (FOXO1; a marker of MET upregulation) and/or ALK gene rearrangement. The primary end-point was the objective response rate (ORR). Secondary end-points included duration of response (DOR), disease control rate (DCR), progression-free survival (PFS), progression-free rate (PFR), overall survival (OS) and safety.
FINDINGS: Nineteen of 20 consenting patients had centrally confirmed ARMS. Molecular assessment revealed rearrangement of FOXO1 in 17 tumours and ALK in none. Thirteen eligible patients were treated, but only eight were evaluable for the primary end-point because of the observed aggressiveness of the disease. Among seven evaluable MET+/ALK- patients, only one achieved a confirmed partial response (ORR: 14.3%; 95% confidence interval [CI]: 0.3-57.8) with a DOR of 52 d. Further MET+/ALK- efficacy end-points were DCR: 14.3% (95% CI: 0.3-57.8), median PFS: 1.3 months (95% CI: 0.5-1.5) and median OS: 5.6 months (95% CI: 0.7-7.0). The remaining MET+/ALK- and MET-/ALK- patients had early progression as best response. Common treatment-related adverse events were fatigue (5/13 [38.5%]), nausea (4/13 [30.8%]), anorexia (4/13 [30.8%]), vomiting (2/13 [15.4%]) and constipation (2/13 [15.4%]). All 13 treated patients died early because of progressive disease.
INTERPRETATION: Crizotinib is well tolerated but lacks clinically meaningful activity as a single agent in patients with advanced metastatic ARMS. Assessing single agents in aggressive, chemotherapy-refractory ARMS is challenging, and future trials should explore established chemotherapy ± investigational compounds in earlier lines of treatment.
CLINICAL TRIAL NUMBER: EORTC 90101, ClinicalTrials.gov NCT01524926.

Objectives: Multinodular goiter (MNG) and well-differentiated thyroid carcinoma (WDTC) are emerging phenotypes of DICER1 syndrome.
Methods: Histologic and molecular findings of botryoid-type embryonal rhabdomyosarcoma (bERMS) and thyroid nodules from a 12-year-old DICER1 mutation carrier (p.Arg1060Ilefs*7) were investigated, providing interesting clues for understanding thyroid carcinogenesis.
Results: The patient had bERMS at age 7 years. The thyroid was enlarged and multinodular (61 g). Histologically, some nodules were classified as adenomatous and others as tumors with "intermediate" nuclei. One displayed vascular invasion and was classified as WDTC not otherwise specified (NOS). Somatic DICER1 mutations were identified in bERMS, two tumors with "intermediate" nuclei and WDTC. No somatic DICER1 mutations were found in adenomatous nodules. No molecular alterations were detected in BRAF600, NRAS61, HRAS12/61, KRAS12/61, TERT promoter, RET/PTC1, RET/PTC3, and PAX8/PPARγ.
Conclusions: The findings obtained from this single case support the assumption that DICER1 syndrome-related WDTC NOS may develop on a background of MNG, via a stepwise process, involving DICER1 somatic mutations and additional molecular events, distinct from the classic pathways of papillary/follicular carcinoma.

Sequencing studies have revealed recurrent mutations in the RAS pathway in rhabdomyosarcoma (RMS). However, RAS effector pathways in RMS are poorly defined. Here, we report that coinhibition of NRAS or MEK plus PI3Kα triggers widespread apoptosis in

Rhabdomyosarcoma is the most common soft-tissue sarcoma in childhood and histologically resembles developing skeletal muscle. Alveolar rhabdomyosarcoma (ARMS) is an aggressive subtype with a higher rate of metastasis and poorer prognosis. The majority of ARMS tumors (80%) harbor a PAX3-FOXO1 or less commonly a PAX7-FOXO1 fusion gene. The presence of either the PAX3-FOXO1 or PAX7-FOXO1 fusion gene foretells a poorer prognosis resulting in clinical re-classification as either fusion-positive (FP-RMS) or fusion-negative RMS (FN-RMS). The PAX3/7-FOXO1 fusion genes result in the production of a rogue transcription factors that drive FP-RMS pathogenesis and block myogenic differentiation. Despite knowing the molecular driver of FP-RMS, targeted therapies have yet to make an impact for patients, highlighting the need for a greater understanding of the molecular consequences of PAX3-FOXO1 and its target genes including microRNAs. Here we show FP-RMS patient-derived xenografts and cell lines display a distinct microRNA expression pattern. We utilized both loss- and gain-of function approaches in human cell lines with knockdown of PAX3-FOXO1 in FP-RMS cell lines and expression of PAX3-FOXO1 in human myoblasts and identified microRNAs both positively and negatively regulated by the PAX3-FOXO1 fusion protein. We demonstrate PAX3-FOXO1 represses miR-221/222 that functions as a tumor suppressing microRNA through the negative regulation of CCND2, CDK6, and ERBB3. In contrast, miR-486-5p is transcriptionally activated by PAX3-FOXO1 and promotes FP-RMS proliferation, invasion, and clonogenic growth. Inhibition of miR-486-5p in FP-RMS xenografts decreased tumor growth, illustrating a proof of principle for future therapeutic intervention. Therefore, PAX3-FOXO1 regulates key microRNAs that may represent novel therapeutic vulnerabilities in FP-RMS.

Rhabdomyosarcoma is a mesenchymal malignancy associated with the skeletal muscle lineage and is also the most common pediatric soft tissue cancer. Between the two pediatric subtypes, embryonal and alveolar rhabdomyosarcoma, the alveolar subtype is generally more aggressive and high-risk. Despite intensive multimodal therapy, patients with high-risk rhabdomyosarcoma continue to have poor prognosis. In this chapter we address the mechanisms underlying the dysregulation of myogenesis in rhabdomyosarcoma. We specifically focus on recently identified signaling pathways that function to inhibit myogenesis and how similar functions have been shown to overlap in rhabdomyosarcoma, potentially contributing to the disease.

BACKGROUND: The Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31.
METHODS: Paraffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients.
RESULTS: We found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%-97%) compared to patients with higher values (40%, 95%CI, 12%-67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio.
CONCLUSION: CD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.

BACKGROUND/AIM: Rhabdomyosarcoma is the most common type of pediatric soft-tissue sarcoma. Among the subsets of this disease, alveolar rhabdomyosarcoma (ARMS) expressing paired box 3 (PAX3) and forkhead box O1 (PAX3-FOXO1) fusion oncoprotein has the worst prognosis. The goal of this study was to investigate the chemotherapeutic effects of sphingosine on PAX3-FOXO1-positive ARMS cells [tumor protein p53 (TP53)-mutated RH30 and TP53 wild-type RH18 cells].
MATERIALS AND METHODS: The proliferation, cell death, apoptosis, cell cycle, and MYCN proto-oncogene (MYCN) expression of RH30 and RH18 cells were determined.
RESULTS: Sphingosine inhibited the growth and caused cell death in a dose-dependent manner in both cell lines. Sphingosine triggered cell death by inducing apoptosis without affecting the cell cycle. MYCN expression was down-regulated within 2 and 4 h of sphingosine treatment in both RH30 and RH18 cells.
CONCLUSION: Sphingosine exerts antiproliferative and pro-apoptotic effects via MYCN down-regulation independently of TP53 mutation status in PAX3-FOXO1-positive ARMS cells.

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome, with the characteristics of early onset of cancer and high cancer incidence. TP53 is widely accepted as a pathogenic gene of LFS. A 2 years and 6 months old boy is reported in this article, who was diagnosed with embryonal rhabdomyosarcoma (RMS) in the left submandibular region. His brother died of RMS, and his grandmother was diagnosed with breast cancer. TP53 gene mutation detection was performed in this patient and some family members, indicating a missense mutation in exon 8 of the patient: c.844C>T (p.Arg282Trp, heterozygous). TP53 mutation was also found in his mother and sister. The boy met the diagnostic criteria for LFS. Among pediatric patients, the most common LFS diseases include osteosarcoma, adrenocortical cancer, central nervous system tumor, and soft tissue tumor. Additionally, leukemia and lymphoma are also involved. LFS patients have a high risk to suffer secondary or even multiple cancers. Therefore, it is necessary to perform genetic detection for pediatric cancer patients, especially those with hereditary predisposition cancers. TP53 mutation often indicates poor prognosis, so it is important to take active treatment and systematic monitoring for LFS family.

The sonic hedgehog (SHH) signaling pathway has been shown to play important roles in embryogenesis, cell proliferation as well as in cell differentiation. It is aberrantly activated in various common cancers in adults, but also in pediatric neoplasms, such as rhabdomyosarcoma (RMS) and atypical teratoid/rhabdoid tumors (AT/RTs). Dysregulation and germline mutation in PATCHED1 (PTCH1), a receptor for SHH, is responsible for the Gorlin Syndrome, a familial cancer predisposing syndrome including RMS. Here, we report a newborn diagnosed with congenital embryonal RMS. Whole-exome sequencing (WES) identified the presence of two heterozygous germline mutations in two target genes of the SHH signaling pathway. The PTCH1 mutation p.(Gly38Glu) is inherited from the mother, whereas the PTCH2 p.(His622Tyr) mutation is transmitted from the father. Quantitative RT-PCR expression analysis of GLI and SMO, key players of the SHH pathway, showed significantly increase in the tumor tissue of the patient and also enrichment in the germline sample in comparison to the parents indicating activation of the SHH pathway in the patient. These findings demonstrate that SHH pathway activity seems to play a role in eRMS as evidenced by high expression levels of GLI1 RNA transcripts. We speculate that PTCH2 modulates tumorigenesis linked to the PTCH1 mutation and is likely associated with the congenital onset of the RMS observed in our patient.

Driver oncogenes are prime targets for therapy in tumors many of which, including leukemias and sarcomas, express recurrent fusion transcription factors. One specific example for such a cancer type is alveolar rhabdomyosarcoma, which is associated in the majority of cases with the fusion protein PAX3-FOXO1. Since fusion transcription factors are challenging targets for development of small molecule inhibitors, indirect inhibitory strategies for this type of oncogenes represent a more promising approach. One can envision strategies at different molecular levels including upstream modifiers and activators, epigenetic and transcriptional co-regulators, and downstream effector targets. In this review, we will discuss the current knowledge regarding potential therapeutic targets that might contribute to indirect interference with PAX3-FOXO1 activity in alveolar rhabdomyosarcoma at the different molecular levels and extrapolate these findings to fusion transcription factors in general.

Soft tissue sarcomas (STS) and neuroblastomas (NBL), are childhood malignancies still associated with poor prognoses despite the overall improvement in childhood tumor survival of the past decades. Anaplastic lymphoma kinase (ALK) inhibition is promising new strategy to improve the outcome of these pediatric tumors. Eighteen histologic samples of pediatric STS and 19 NBL patients were analyzed for ALK abnormalities using fluorescent in situ hybridization (FISH) with break-apart probes and immunohistochemistry (IHC). ALK alterations were presented in 20 of the 37 sections. The presence of ALK alteration in NBL samples were detected using IHC in 84,2% of all cases compared to 21,1% FISH positivity. In STS cases the results were less different (IHC 16,7% vs FISH 22,2%). The difference can be explained by the different type of molecular alterations. FISH method detected translocation and amplification, but not the point mutation of ALK gene. IHC confirmed the diagnosis by detecting the expression of ALK protein.After ALK positivity was proven, the effectiveness and safety of the crizotinib therapy was examined in 4 patients (1 alveolar rhabdomyosarcoma (RMA), 1 embryonal rhabdomyosarcoma (RME), 1 inflammatory myofibroblastic tumor (IMT), 1 NBL). We observed continuous remission of the IMT patient, all other cases the inhibitor treatment was not curative.Our findings underline the importance of screening the ALK status parallel with both IHC and FISH. Crizotinib treatment had a long-term effect in ALK positive IMT patients, however itwas only temporary efficient in relapsed, progressive STS and NBL.

Rhabdomyosarcoma (RMS), a cancer characterized by skeletal muscle features, is the most common soft-tissue sarcoma of childhood. While low- and intermediate-risk groups have seen improved outcomes, high-risk patients still face a 5-year survival rate of <30%, a statistic that has not changed in over 40 years. Understanding the biologic underpinnings of RMS is critical. The developmental pathways of Notch and YAP have been identified as potent but independent oncogenic signals that support the embryonal variant of RMS (eRMS). Here, the cross-talk between these pathways and the impact on eRMS tumorigenesis is reported. Using human eRMS cells grown as three-dimensional (3D) rhabdospheres, which enriches in stem cells, it was found that Notch signaling transcriptionally upregulates

Alternative splicing of the oncogene murine double minute 2 (MDM2) is induced in response to genotoxic stress. MDM2-ALT1, the major splice variant generated, is known to activate the p53 pathway and impede full-length MDM2's negative regulation of p53. Despite this perceptible tumor-suppressive role, MDM2-ALT1 is also associated with several cancers. Furthermore, expression of MDM2-ALT1 has been observed in aggressive metastatic disease in pediatric rhabdomyosarcoma (RMS), irrespective of histological subtype. Therefore, we generated a transgenic MDM2-ALT1 mouse model that would allow us to investigate the effects of this splice variant on the progression of tumorigenesis. Here we show that when MDM2-ALT1 is ubiquitously expressed in p53 null mice it leads to increased incidence of spindle cell sarcomas, including RMS. Our data provide evidence that constitutive MDM2-ALT1 expression is itself an oncogenic lesion that aggravates the tumorigenesis induced by p53 loss. On the contrary, when MDM2-ALT1 is expressed solely in B-cells in the presence of homozygous wild-type p53 it leads to significantly increased lymphomagenesis (56%) when compared with control mice (27%). However, this phenotype is observable only at later stages in life (⩾18 months). Moreover, flow cytometric analyses for B-cell markers revealed an MDM2-ALT1-associated decrease in the B-cell population of the spleens of these animals. Our data suggest that the B-cell loss is p53 dependent and is a response mounted to persistent MDM2-ALT1 expression in a wild-type p53 background. Overall, our findings highlight the importance of an MDM2 splice variant as a critical modifier of both p53-dependent and -independent tumorigenesis, underscoring the complexity of MDM2 posttranscriptional regulation in cancer. Furthermore, MDM2-ALT1-expressing p53 null mice represent a novel mouse model of fusion-negative RMS.

The morphologies of alveolar rhabdomyosarcoma (ARMS) are various. Some cases entirely lack an alveolar pattern and instead display a histological pattern that overlaps with embryonal rhabdomyosarcoma (ERMS). The method of pathological diagnosis of ARMS and ERMS has been updated in the 4th edition of the World Health Organization's guidelines for classification of skeletal muscle tumors. Under the new guidelines, there is still no molecular test to distinguish between these two subtypes of rhabdomyosarcoma (RMS). In the present study, we applied fluorescent in situ hybridization (FISH) and found that the Forkhead box O1 (FOXO1) gene broke apart, amplified, and displayed an aneuploid signal that was related to the RMS pathological subtype. Aside from the fact that FOXO1 break-apart and its amplification were correlated with atypical ARMS, aneuploidies were usually found in atypical ERMS. In conclusion, our results detail a potential biomarker to improve the accuracy of pathological diagnosis by discriminating between atypical ARMS and atypical ERMS.

BACKGROUND: The organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts.
RESULTS: We show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma.
CONCLUSIONS: The chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved.

BACKGROUND: Sarcomas are rare and heterogeneous cancers. We assessed the contribution of DICER1 mutations to sarcoma development.
METHODS: The coding region of DICER1 was sequenced in 67 sarcomas using a custom Fluidigm Access Array. The RNase III domains were Sanger sequenced in six additional sarcomas to identify hotspot DICER1 variants.
RESULTS: The median age of sarcoma diagnosis was 45.7 years (range: 3 months to 87.4 years). A recurrent embryonal rhabdomyosarcoma (ERMS) of the broad ligament, first diagnosed at age 23 years, harboured biallelic pathogenic somatic DICER1 variants (1 truncating and 1 RNase IIIb missense). We identified nine other DICER1 variants. One somatic variant (p.L1070V) identified in a pleomorphic sarcoma and one germline variant (c.2257-7A>G) may be pathogenic, but the others are considered to be benign.
CONCLUSIONS: We show that deleterious DICER1 mutations underlie the genetic basis of only a small fraction of sarcomas, in particular ERMS of the urogenital tract.