Genomic profiling of pleomorphic rhabdomyosarcoma reveals a genomic signature distinct from that of embryonal rhabdomyosarcoma.

Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive sarcoma, occurring mostly in the deep soft tissues of middle-aged adults and showing a variable degree of skeletal muscle differentiation. The diagnosis is challenging as pathologic features overlap with embryonal rhabdomyosarcoma (ERMS), malignant Triton tumor, and other pleomorphic sarcomas. As recurrent genetic alterations underlying PRMS have not been described to date, ancillary molecular diagnostic testing is not useful in subclassification. Herein, we perform genomic profiling of a well-characterized cohort of 14 PRMS, compared to a control group of 23 ERMS and other pleomorphic sarcomas (undifferentiated pleomorphic sarcoma and pleomorphic liposarcoma) using clinically validated DNA-targeted Next generation sequencing (NGS) panels (MSK-IMPACT). The PRMS cohort included eight males and six females, with a median age of 53 years (range 31-76 years). Despite similar tumor mutation burdens, the genomic landscape of PRMS, with a high frequency of TP53 (79%) and RB1 (43%) alterations, stood in stark contrast to ERMS, with 4% and 0%, respectively. CDKN2A deletions were more common in PRMS (43%), compared to ERMS (13%). In contrast, ERMS harbored somatic driver mutations in the RAS pathway and loss of function mutations in BCOR, which were absent in PRMS. Copy number variations in PRMS showed multiple chromosomal arm-level changes, most commonly gains of chr17p and chr22q and loss of chr6q. Notably, gain of chr8, commonly seen in ERMS (61%) was conspicuously absent in PRMS. The genomic profiles of other pleomorphic sarcomas were overall analogous to PRMS, showing shared alterations in TP53, RB1, and CDKN2A. Overall survival and progression-free survival of PRMS were significantly worse (p < 0.0005) than that of ERMS. Our findings revealed that the molecular landscape of PRMS aligns with other adult pleomorphic sarcomas and is distinct from that of ERMS. Thus, NGS assays may be applied in select challenging cases toward a refined classification. Finally, our data corroborate the inclusion of PRMS in the therapeutic bracket of pleomorphic sarcomas, given that their clinical outcomes are comparable.

FGFR1 fusions as a novel molecular driver in rhabdomyosarcoma.

The wide application of RNA sequencing in clinical practice has allowed the discovery of novel fusion genes, which have contributed to a refined molecular classification of rhabdomyosarcoma (RMS). Most fusions in RMS result in aberrant transcription factors, such as PAX3/7::FOXO1 in alveolar RMS (ARMS) and fusions involving VGLL2 or NCOA2 in infantile spindle cell RMS. However, recurrent fusions driving oncogenic kinase activation have not been reported in RMS. Triggered by an index case of an unclassified RMS (overlapping features between ARMS and sclerosing RMS) with a novel FGFR1::ANK1 fusion, we reviewed our molecular files for cases harboring FGFR1-related fusions. One additional case with an FGFR1::TACC1 fusion was identified in a tumor resembling embryonal RMS (ERMS) with anaplasia, but with no pathogenic variants in TP53 or DICER1 on germline testing. Both cases occurred in males, aged 7 and 24, and in the pelvis. The 2nd case also harbored additional alterations, including somatic TP53 and TET2 mutations. Two additional RMS cases (one unclassified, one ERMS) with FGFR1 overexpression but lacking FGFR1 fusions were identified by RNA sequencing. These two cases and the FGFR1::TACC1-positive case clustered together with the ERMS group by RNAseq. This is the first report of RMS harboring recurrent FGFR1 fusions. However, it remains unclear if FGFR1 fusions define a novel subset of RMS or alternatively, whether this alteration can sporadically drive the pathogenesis of known RMS subtypes, such as ERMS. Additional larger series with integrated genomic and epigenetic datasets are needed for better subclassification, as the resulting oncogenic kinase activation underscores the potential for targeted therapy.

The RRAS2 pathogenic variant (c.67G>T; p. Gly23Cys) produces Noonan syndrome with embryonal rhabdomyosarcoma.

BACKGROUND: Noonan syndrome (NS) due to the RRAS2 gene, the pathogenic variant is an extremely rare RASopathies. Our objective was to identify the potential site of RRAS2, combined with the literature review, to find the correlation between clinical phenotype and genotype. De novo missense mutations affect different aspects of the RRAS2 function, leading to hyperactivation of the RAS-MAPK signaling cascade. METHODS: Conventional G-banding was used to analyze the chromosome karyotype of the patient. Copy number variation sequencing (CNV-seq) was used to detect the chromosomal gene microstructure of the patient and her parents. The exomes of the patient and her parents were sequenced using trio-based whole exome sequencing (trio-WES) technology. The candidate variant was verified by Sanger sequencing. The pathogenicity of the variant was predicted with a variety of bioinformatics tools. RESULTS: Chromosome analysis of the proband revealed 46, XX, and no abnormality was found by CNV-seq. After sequencing and bioinformatics filtering, the variant of RRAS2(c.67G>T; p. Gly23Cys) was found in the proband, while the mutation was absent in her parents. To the best of our knowledge, our patient was with the typical Noonan syndrome, such as short stature, facial dysmorphism, and developmental delay. Furthermore, our study is the first case of NS with embryonal rhabdomyosarcoma (ERMS) caused by the RRAS2 gene mutation reported in China. CONCLUSIONS: Our investigations suggested that the heterozygous missense of RRAS2 may be a potential causal variant in a rare cause of Noonan syndrome, expanding our understanding of the causally relevant mutations for this disorder.

[Diagnosis of uterine sarcomas and rare uterine mesenchymal tumours with malignant potential. Guidelines of the French Sarcoma Group and Rare Gynaecological Tumours]. / Diagnostic des sarcomes utérins et tumeurs mésenchymateuses utérines rares à potentiel de malignité. Référentiels du Groupe Sarcome Français et des Tumeurs Rares Gynécologiques.

The landscape of uterine sarcomas is becoming more complex with the description of new entities associated with recurrent driver molecular alterations. Uterine sarcomas, in analogy with soft tissue sarcomas, are distinguished into complex genomic and simple genomic sarcomas. Leiomyosarcomas and undifferentiated uterine sarcomas belong to complex genomic sarcomas group. Low-grade and high-grade endometrial stromal sarcomas, other rare tumors associated with fusion transcripts (such as NTRK, PDGFB, ALK, RET ROS1) and SMARCA4-deficient uterine sarcoma are considered simple genomic sarcomas. The most common uterine sarcoma are first leiomyosarcoma and secondly endometrial stromal sarcomas. Three different histological subtypes of leiomyosarcoma (fusiform, myxoid, epithelioid) are identified, myxoid and epithelioid leiomyosarcoma being more aggressive than fusiform leiomyosarcoma. The distinction between low-grade and high-grade endometrial stromal sarcoma is primarily morphological and immunohistochemical and the detection of fusion transcripts can help the diagnosis. Uterine PEComa is a rare tumor, which is distinguished into borderline and malignant, according to a risk assessment algorithm. Embryonal rhabdomyosarcoma of the uterine cervix is more common in children but can also occur in adult women. Embryonal rhabdomyosarcoma of the uterine cervix is almost always DICER1 mutated, unlike that of the vagina which is wild-type DICER1, and adenosarcoma which can be DICER1 mutated but with less frequency. Among the emerging entities, sarcomas associated with fusion transcripts involving the NTRK, ALK, PDGFB genes benefit from targeted therapy. The integration of molecular data with histology and clinical data allows better identification of uterine sarcomas in order to better treat them.

Staufen1 controls mitochondrial metabolism via HIF2α in embryonal rhabdomyosarcoma and promotes tumorigenesis.

Elevated mitochondrial metabolism promotes tumorigenesis of Embryonal Rhabdomyosarcomas (ERMS). Accordingly, targeting oxidative phosphorylation (OXPHOS) could represent a therapeutic strategy for ERMS. We previously demonstrated that genetic reduction of Staufen1 (STAU1) levels results in the inhibition of ERMS tumorigenicity. Here, we examined STAU1-mediated mechanisms in ERMS and focused on its potential involvement in regulating OXPHOS. We report the novel and differential role of STAU1 in mitochondrial metabolism in cancerous versus non-malignant skeletal muscle cells (NMSkMCs). Specifically, our data show that STAU1 depletion reduces OXPHOS and inhibits proliferation of ERMS cells. Our findings further reveal the binding of STAU1 to several OXPHOS mRNAs which affects their stability. Indeed, STAU1 depletion reduced the stability of OXPHOS mRNAs, causing inhibition of mitochondrial metabolism. In parallel, STAU1 depletion impacted negatively the HIF2α pathway which further modulates mitochondrial metabolism. Exogenous expression of HIF2α in STAU1-depleted cells reversed the mitochondrial inhibition and induced cell proliferation. However, opposite effects were observed in NMSkMCs. Altogether, these findings revealed the impact of STAU1 in the regulation of mitochondrial OXPHOS in cancer cells as well as its differential role in NMSkMCs. Overall, our results highlight the therapeutic potential of targeting STAU1 as a novel approach for inhibiting mitochondrial metabolism in ERMS.

Complete loss of TP53 and RB1 is associated with complex genome and low immune infiltrate in pleomorphic rhabdomyosarcoma.

Rhabdomyosarcoma accounts for roughly 1% of adult sarcomas, with pleomorphic rhabdomyosarcoma (PRMS) as the most common subtype. Survival outcomes remain poor for patients with PRMS, and little is known about the molecular drivers of this disease. To better characterize PRMS, we performed a broad array of genomic and immunostaining analyses on 25 patient samples. In terms of gene expression and methylation, PRMS clustered more closely with other complex karyotype sarcomas than with pediatric alveolar and embryonal rhabdomyosarcoma. Immune infiltrate levels in PRMS were among the highest observed in multiple sarcoma types and contrasted with low levels in other rhabdomyosarcoma subtypes. Lower immune infiltrate was associated with complete loss of both TP53 and RB1. This comprehensive characterization of the genetic, epigenetic, and immune landscape of PRMS provides a roadmap for improved prognostications and therapeutic exploration.

Changes on chromosome 11p15.5 as specific marker for embryonal rhabdomyosarcoma?

Rhabdomyosarcomas (RMS) constitute a heterogeneous spectrum of tumors with respect to clinical behavior and tumor morphology. The paternal uniparental disomy (pUPD) of 11p15.5 is a molecular change described mainly in embryonal RMS. In addition to LOH, UPD, the MLPA technique (ME030kit) also determines copy number variants and methylation of H19 and KCNQ1OT1 genes, which have not been systematically investigated in RMS. All 127 RMS tumors were divided by histology and PAX status into four groups, pleomorphic histology (n = 2); alveolar RMS PAX fusion-positive (PAX+; n = 39); embryonal RMS (n = 70) and fusion-negative RMS with alveolar pattern (PAX-RMS-AP; n = 16). The following changes were detected; negative (n = 21), pUPD (n = 75), gain of paternal allele (n = 9), loss of maternal allele (n = 9), hypermethylation of H19 (n = 6), hypomethylation of KCNQ1OT1 (n = 6), and deletion of CDKN1C (n = 1). We have shown no difference in the frequency of pUPD 11p15.5 in all groups. Thus, we have proven that changes in the 11p15.5 are not only specific to the embryonal RMS (ERMS), but are often also present in alveolar RMS (ARMS). We have found changes that have not yet been described in RMS. We also demonstrated new potential diagnostic markers for ERMS (paternal duplication and UPD of whole chromosome 11) and for ARMS PAX+ (hypomethylation KCNQ1OT1).

A dysfunctional miR-1-TRPS1-MYOG axis drives ERMS by suppressing terminal myogenic differentiation.

Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1 fusion-positive alveolar subtype. Here, we demonstrate that the expression levels of the transcriptional repressor TRPS1 are specifically enhanced in the embryonal subtype, resulting in impaired terminal myogenic differentiation and tumor growth. During normal myogenesis, expression levels of TRPS1 have to decrease to allow myogenic progression, as demonstrated by overexpression of TRPS1 in myoblasts impairing myotube formation. Consequentially, myogenic differentiation in embryonal rhabdomyosarcoma in vitro as well as in vivo can be achieved by reducing TRPS1 levels. Furthermore, we show that TRPS1 levels in RD cells, the bona fide model cell line for embryonal rhabdomyosarcoma, are regulated by miR-1 and that TRPS1 and MYOD1 share common genomic binding sites. The myogenin (MYOG) promoter is one of the critical targets of TRPS1 and MYOD1; we demonstrate that TRPS1 restricts MYOG expression and thereby inhibits terminal myogenic differentiation. Therefore, reduction of TRPS1 levels in embryonal rhabdomyosarcoma might be a therapeutic approach to drive embryonal rhabdomyosarcoma cells into myogenic differentiation, thereby generating postmitotic myotubes.

Cervical Pleuropulmonary Blastoma-like Tumor Associated With DICER1 and TP53 Mutations.

We describe a very unusual cervical tumor in a 12-yr-old patient with a clinical history indicative of DICER1 syndrome. Morphologic, immunohistochemical, and molecular genetic analysis together helped to diagnose this lesion as a cervical pleuropulmonary blastoma-like tumor, associated with TP53 and DICER1 mutations. The tumor displayed usual histologic features including mixtures of embryonal rhabdomyosarcoma, sarcomatous cartilage, compact blastema, primitive spindle cells and anaplasia, akin to type III pleuropulmonary blastoma, and trabecular and retiform patterns. In addition to expanding the phenotypic spectrum of DICER1 -associated conditions, we draw attention to genotype-phenotype correlations in DICER1 -associated tumors, particularly as they relate to the discovery of a heritable tumor predisposition syndrome.

Molecular characterization of an embryonal rhabdomyosarcoma occurring in a patient with Kabuki syndrome: report and literature review in the light of tumor predisposition syndromes.

Kabuki syndrome is a well-recognized syndrome characterized by facial dysmorphism and developmental delay/intellectual disability and in the majority of patients a germline variant in KMT2D is found. As somatic KMT2D variants can be found in 5-10% of tumors a tumor predisposition in Kabuki syndrome is discussed. So far less than 20 patients with Kabuki syndrome and a concomitant malignancy have been published. Here we report on a female patient with Kabuki syndrome and a c.2558_2559delCT germline variant in KMT2D who developed an embryonal rhabdomyosarcoma (ERMS) at 10 years. On tumor tissue we performed DNA-methylation profiling and exome sequencing (ES). Copy number analyses revealed aneuploidies typical for ERMS including (partial) gains of chromosomes 2, 3, 7, 8, 12, 15, and 20 and 3 focal deletions of chromosome 11p. DNA methylation profiling mapped the case to ERMS by a DNA methylation-based sarcoma classifier. Sequencing suggested gain of the wild-type KMT2D allele in the trisomy 12. Including our patient literature review identified 18 patients with Kabuki syndrome and a malignancy. Overall, the landscape of malignancies in patients with Kabuki syndrome was reminiscent of that of the pediatric population in general. Histopathological and molecular data were only infrequently reported and no report included next generation sequencing and/or DNA-methylation profiling. Although we found no strong arguments pointing towards KS as a tumor predisposition syndrome, based on the small numbers any relation cannot be fully excluded. Further planned studies including profiling of additional tumors and long term follow-up of KS-patients into adulthood could provide further insights.

Pax3 loss of function delays tumour progression in kRAS-induced zebrafish rhabdomyosarcoma models.

Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma.

Strongylopus grayii tadpole blastema extract exerts cytotoxic effects on embryonal rhabdomyosarcoma cells.

Amphibians have regenerative capacity and are resistant to developing cancer. This suggests that the developing blastema, located at the tissue regeneration site, may secrete anti-cancer factors. Here, we investigate the anti-cancer potential of tadpole tail blastema extracts (TAD) from the stream frog, Strongylopus grayii, in embryonal rhabdomyosarcoma (ERMS) cells. ERMS originates in skeletal muscle tissue and is a common pediatric soft tissue sarcoma. We show using MTT assays that TAD inhibited ERMS cell viability in a concentration-dependent manner, and phase contrast/fluorescent microscopy revealed that it induced morphological markers of senescence and apoptosis. Western blotting showed that this was associated with DNA damage (γH2AX) and activation of the p38/MAPK stress signaling pathway as well as molecular markers of senescence (p16INK4a), apoptosis (cleaved PARP), and inhibition of cell cycle promoters (cyclin A, CDK2, and cyclin B1). Furthermore, proteomics followed by gene ontology analyses showed that TAD treatment inhibited known tumor promoters and proteins required for cancer cell survival. Lastly, using the LINCS drug perturbation library, we show that there is an overlap between the proteomics signature induced by TAD and common anti-cancer drugs. Taken together, this study provides novel evidence that TAD exhibits cytotoxicity in ERMS cells.

Embryonal Rhabdomyosarcoma of the Uterine Cervix: A Clinicopathologic Study of 94 Cases Emphasizing Issues in Differential Diagnosis Staging, and Prognostic Factors.

Embryonal rhabdomyosarcoma of the uterine cervix (cERMS) is rare and frequently associated with DICER1 mutations. We report 94 tumors that arose in patients aged 7 to 59 (median=23) years and presented with vaginal bleeding (52), protruding vaginal mass (17), cervical polyp (8), or expelled tumor fragments per vagina (5). Nine had DICER1 syndrome, 8 of whom had other syndromic manifestations including ovarian Sertoli-Leydig cell tumor (7), multinodular goiter (3), pleuropulmonary blastoma (2), pineoblastoma (1), and osteosarcoma (1). Syndromic patients were younger than nonsyndromic patients (16 vs. 24 y). Tumor size ranged from 2 to 24 (median=4.5) cm. Ninety-two tumors were polypoid, most being grape-like (77 of 92). They were characterized by aggregates of primitive cells, almost always exhibiting a cambium layer, within a variably myxoedematous stroma and were hypocellular (63), moderately cellular (22), or hypercellular (9). Entrapped glands, typically scant, were present in 84 tumors. Primitive hyperchromatic ovoid to spindled cells with minimal cytoplasm predominated but differentiated rhabdomyoblasts with abundant eosinophilic cytoplasm (having cross-striations in 30) were seen in 83 tumors; they were often sparse but predominated in three. Nine tumors showed areas of intersecting fascicles and 4 zones with densely cellular (solid) growth. Cartilage was present in 38. Anaplasia was seen in 15 tumors, as was necrosis. Mitotic activity ranged from 1 to 58/10 high-power fields (median=8). The varied microscopic features resulted in a spectrum of differential diagnostic considerations, mainly typical and cellular forms of fibroepithelial polyps, Mullerian adenosarcoma, and other sarcomas. Follow-up was available for 79 patients ranging from 6 to 492 (median=90) months. Treatment information was available in 62 and included polypectomy in 6 patients (2 also received chemotherapy), limited resection in 26 (14 also received chemotherapy), hysterectomy in 29 (15 with adjuvant chemotherapy), and biopsies only in 1 (with chemotherapy). Staging was possible in 56 tumors; according to the "uterine sarcoma" system (tumor size and extent) they were: stage I (10/56; could not be further subclassified as size not available), IA (22/56), IB (18/56), IIA (2/56), IIB 3/56), IIIC (1/56). According to the "adenosarcoma" system (depth of invasion and extent) they were: stage IA (26/56), IB (14/56), IC (10/56), IIA (2/56), IIB (3/56), IIIC (1/56). Eight patients had local recurrence following incomplete excision (10%). Eleven of 79 patients had extrauterine recurrences (14%) and 9 died of disease (11%). Older age was associated with extrauterine recurrence (median 44 vs. 22; P =0.002) and decreased disease-specific survival (median 44 vs. 22; P =0.02). For patients with tumors initially confined to the cervix, the adenosarcoma staging system was superior to the uterine sarcoma staging system for predicting survival ( P =0.02). Three patients with DICER1 syndrome who underwent fertility-preserving surgery developed a second primary cERMS 7, 7, and 12 years after their primary tumor. All 9 patients with DICER1 syndrome had tumors confined to the cervix and none died of disease. This study highlights the intriguing clinical aspects of cERMS including its long-known tendency to occur in the young but also more recently appreciated association with DICER1 syndrome. Establishing the diagnosis may still be difficult because of the hazard of sampling a neoplasm which in areas may appear remarkably bland and also because of its potential confusion with other neoplasms. This study indicates that this tumor has a good prognosis at this site and in selected cases a conservative surgical approach is a realistic consideration.

Pleuropulmonary blastoma (PPB) and other DICER1-associated high-grade malignancies are morphologically, genetically and epigenetically related - A comparative study of 4 PPBs and 6 sarcomas.

DICER1-related tumors occur hereditary or sporadically, with high-grade malignancies sharing clinicopathological and (epi)genetic features. We compared 4 pleuropulmonary blastomas (PPBs) and 6 sarcomas by mutation analysis, whole transcriptome sequencing and methylation profiling. 9/10 patients were female. PPB patients were 0-4 years. 3/4 were alive; 2 without disease. One patient died of metastatic disease (median follow-up, 16 months). Sarcoma patients were 16-56 years. Locations included: uterine cervix/corpus (3/1), soft tissue back/shoulder (1) and paravertebral (1). 5/6 patients were alive; 2 developed metastases: intracranial (1) and lung and kidney (1) (median follow-up, 17 months). The deceased patient previously had a PPB and a Sertoli-Leydig cell tumor. Histologically, tumors showed atypical primitive-looking cells with incomplete rhabdomyoblastic differentiation and cartilage (n = 5). Immunohistochemistry demonstrated desmin- (n = 9/10), myogenin- (n = 6/10) and keratin positivity (n = 1/1). Eight cases harbored biallelic DICER1 mutations with confirmed germline mutations in 4 cases. Two cases showed a monoallelic mutation. By RNA expression- and methylation profiling, distinct clustering of our cases was seen demonstrating a close relationship on (epi)genetic level and similarities to embryonal rhabdomyosarcoma. In conclusion, this study shows overlapping morphological, immunohistochemical and (epi)genetic features of PPBs and DICER1-associated high-grade sarcomas, arguing that these neoplasms form a spectrum with a broad clinicopathological range.

Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness.

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.

Anti­oncogenic and pro­myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma.

Insights into the molecular and cellular biology of embryonal rhabdomyosarcoma (ERMS), an aggressive paediatric tumour, are required in order to identify new targets for novel treatments that may benefit patients with this disease. The present study examined the functional effects of MKK3 and MKK6, two upstream kinases of p38, and found that the ectopic expression of MKK6 led to rapid p38 activation and the myogenic differentiation of ERMS cells, whereas MKK3 failed to induce differentiation, while maintaining the proliferation state. Myogenin and myosin heavy chain were induced in MKK6­overexpressing ERMS cells and were inhibited by the p38 inhibitor, SB203580. The expression of Myc and ERK­PO4 increased under the effect of SB203580, whereas it decreased in MKK6­overexpressing cells. AKT activation was part of the myogenic program triggered by MKK6 overexpression alone. To the best of our knowledge, the present study demonstrates, for the first time, that the endogenous MKK6 pathway may be recovered by MEK/ERK inhibition (U0126 and trametinib) and that it concomitantly induces the reversal of the oncogenic pattern and the induction of the myogenic differentiation of ERMS cell lines. The effects of MEK/ERK inhibitors markedly increase the potential clinical applications in ERMS, particularly on account of the MEK inhibitor­induced early MKK6/p38 axis activation and of their anti­oncogenic effects. The findings presented herein lend further support to the antitumour effects of MKK6; MKK6 may thus represent a novel target for advanced personalised treatments against ERMS.

Molecular testing of rhabdomyosarcoma in clinical trials to improve risk stratification and outcome: A consensus view from European paediatric Soft tissue sarcoma Study Group, Children's Oncology Group and Cooperative Weichteilsarkom-Studiengruppe.

Rhabdomyosarcomas (RMSs) are the most common soft tissue sarcomas in children/adolescents less than 18 years of age with an annual incidence of 1-2/million. Inter/intra-tumour heterogeneity raise challenges in clinical, pathological and biological research studies. Risk stratification in European and North American clinical trials previously relied on clinico-pathological features, but now, incorporates PAX3/7-FOXO1-fusion gene status in the place of alveolar histology. International working groups propose a coordinated approach through the INternational Soft Tissue SaRcoma ConsorTium to evaluate the specific genetic abnormalities and generate and integrate molecular and clinical data related to patients with RMS across different trial settings. We review relevant data and present a consensus view on what molecular features should be assessed. In particular, we recommend the assessment of the MYOD1-LR122R mutation for risk escalation, as it has been associated with poor outcomes in spindle/sclerosing RMS and rare RMS with classic embryonal histopathology. The prospective analyses of rare fusion genes beyond PAX3/7-FOXO1 will generate new data linked to outcomes and assessment of TP53 mutations and CDK4 amplification may confirm their prognostic value. Pathogenic/likely pathogenic germline variants in TP53 and other cancer predisposition genes should also be assessed. DNA/RNA profiling of tumours at diagnosis/relapse and serial analyses of plasma samples is recommended where possible to validate potential molecular biomarkers, identify new biomarkers and assess how liquid biopsy analyses can have the greatest benefit. Together with the development of new molecularly-derived therapeutic strategies that we review, a synchronised international approach is expected to enhance progress towards improved treatment assignment, management and outcomes for patients with RMS.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma.

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

Importance of histopathological analysis and molecular genetics in a rare neonatal case of rhabdomyosarcoma.

We present a case of a neonate who presented with multiple cutaneous and subcutaneous nodules, which was found to be metastatic embryonal rhabdomyosarcoma. Rhabdomyosarcoma is a soft tissue malignancy that usually occurs in children aged one to five but is rare in neonates. The histopathological analysis and molecular genetics are important in the classification of subtype and in guiding treatment options and informing prognosis.

Spindle Cell/Sclerosing Rhabdomyosarcoma With PAX8::PPARG Fusion.

The spindle cell/sclerosing subtype of rhabdomyosarcoma is classified based on genetic features into the three categories of MYOD1-mutated, gene fusion-driven, and a subset without a currently identified genetic driver event. The gene fusion-driven spindle cell/sclerosing rhabdomyosarcomas are heterogenous and characterized by increasing numbers of gene fusions, the most common gene partners being VGLL2, NCOA2, and TFCP2. Here we report a spindle cell/sclerosing rhabdomyosarcoma that arose in the orbit of a 4-year-old male. This tumor harbored a unique PAX8::PPARG fusion. PAX8::PPARG fusions have previously only been described in follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. Our report adds to the growing number of gene fusions in spindle cell/sclerosing rhabdomyosarcomas.