Soft Tissue Sarcoma In Children Research Articles

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Fusion-positive RMS (FPRMS), expressing the PAX3/7-FOXO1, has a worse prognosis compared to the more common fusion-negative RMS (FNRMS). Although several studies reported hierarchical organization for FNRMS with the identification of cancer stem cells, the cellular organization of FPRMS is not yet clear. In this study we investigated the expression of key stem cell markers, developed a sphere assay, and investigated the seven most common FPRMS cell lines for subpopulations of tumor propagating cancer stem-like cells, also called cancer stem cells (CSCs). Moreover, loss- and gain-of-functions of the stem cell genes SOX2, OCT4, and NANOG were investigated in the same cells. Single-cell clonal analysis was performed in vitro as well as in vivo. We found that no stable CSC subpopulation could be enriched in FPRMS. Unlike depletion of PAX3-FOXO1, neither overexpression nor siRNA-mediated downregulation of SOX2, OCT4, and NANOG affected physiology of RMS cells. Every single subclone-derived cell clone initiated tumor growth in mice, despite displaying considerable heterogeneity in gene expression. FPRMS appears to contain a high frequency of tumor propagating stem-like cells, which could explain their higher propensity for metastasis and relapse. Their dependency on PAX3-FOXO1 activity reinforces the importance of the fusion protein as the key therapeutic target.

Surgical Challenges in a Case of a Recurrent Giant Pleomorphic Liposarcoma of Neck And Mediastinum in a Child: A Rare Occurrence.

Liposarcomas are rare in childhood, representing < 1% of all childhood soft tissue sarcomas. The most frequent site of involvement are the extremities with the involvement of head and neck being an absolute rarity. Pleomorphic subtype of liposarcoma is pretty aggressive and hence portends a poor prognosis. Here, we present the case of a child who presented with a recurrence of liposarcoma in the neck and mediastinum within 3 months of the index surgery. Supplementary InformationThe online version contains supplementary material available at 10.1007/s12070-021-02837-w.

Single-cell transcriptional profiling reveals the heterogeneity in embryonal rhabdomyosarcoma.

Rhabdomyosarcoma is the most common soft tissue sarcoma in children, and embryonal rhabdomyosarcoma is the most typical type of rhabdomyosarcoma. The heterogeneity, etiology, and origin of embryonal rhabdomyosarcoma remain unknown.After obtaining the gene expression data of every cell in the tumor tissue by single-cell RNA sequencing, we used the Seurat package in R studio for quality control, analysis, and exploration of the data. All cells are divided into tumor cells and non-tumor cells, and we chose tumor cells by marker genes. Then, we repeated the process to cluster the tumor cells and divided the subgroups by their differentially expressed genes and gene ontology/Kyoto Encyclopedia of Genes and Genomes analysis. Additionally, Monocle 2 was used for pseudo-time analysis to obtain the evolution trajectory of cells in tumor tissues.Tumor cells were divided into 5 subgroups according to their functions, which were characterized by high proliferation, sensing and adaptation to oxygen availability, enhanced epigenetic modification, enhanced nucleoside phosphonic acid metabolism, and ossification. Evolution trajectory of cells in tumor tissues is obtained.We used pseudo-time analysis to distinguish between mesenchymal stem cells and fibroblasts, proved that embryonal rhabdomyosarcoma in the pelvic originated from skeletal muscle progenitor cells, showed the evolutionary trajectory of embryonal rhabdomyosarcoma, and improved the method of evaluating the degree of malignancy of embryonal rhabdomyosarcoma.

Upregulation of miR181a/miR212 Improves Myogenic Commitment in Murine Fusion-Negative Rhabdomyosarcoma.

Fusion-negative rhabdomyosarcoma (FN-RMS) is the most common soft tissue sarcoma of childhood arising from undifferentiated skeletal muscle cells from uncertain origin. Currently used therapies are poorly tumor-specific and fail to tackle the molecular machinery underlying the tumorigenicity and uncontrolled proliferation of FN-RMS. We and other groups recently found that microRNAs (miRNA) network contributes to myogenic epigenetic memory and can influence pluripotent stem cell commitments. Here, we used the previously identified promyogenic miRNAs and tailored it to the murine FN-RMS. Subsequently, we addressed the effects of miRNAs in vivo by performing syngeneic transplant of pre-treated FN-RMS cell line in C57Bl/6 mice. miRNA pre-treatment affects murine FN-RMS cell proliferation in vivo as showed by bioluminescence imaging analysis, resulting in better muscle performances as highlighted by treadmill exhaustion tests. In conclusion, in our study we identified a novel miRNA combination tackling the anti-myogenic features of FN-RMS by reducing proliferation and described novel antitumorigenic therapeutic targets that can be further explored for future pre-clinical applications.

Immunohistochemical expression of cyclin-dependent kinase inhibitors p16 and p57 in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a highly malignant cancer and is the most common soft tissue sarcoma in children and adolescents, but it is rare in adults (<1% of all adult malignancies). Altered expression and molecular abnormalities of cell-cycle-regulatory proteins are one of the most prominent features in RMS. Therefore, we evaluated the expression of cyclin-dependent kinase inhibitors p57 and p16, as well as p16 methylation status, along with clinicopathological characteristics and overall survival (OS) in RMS patients. This analysis was conducted on 23 pediatric and 44 adult patients. There was a male predominance in both groups and extremities were the most frequent tumor site. In adults, alveolar and pleomorphic types were almost equally represented. The majority of pediatric tumors were low grade, whereas, in adults, only one patient had a low-grade tumor. Seven pediatric (30.43%) and eight adult (18.18%) patients had a low p16 expression. The analysis of methylation status of the p16 promoter showed the presence of methylated allele only in one sample with pleomorphic histology. Six (26.1%) pediatric and 15 (34.1%) adult patients had low p57 expression, while in 17 (73.9%) pediatric and 29 (65.9%) adult patients it was assessed as high. Ninetyone percent of the pediatric patients and 32.6% of adults were alive at the end of the observational period. In adults, significant associations were found between OS and age (P = 0.020), gender (P = 0.027), tumor size (P < 0.001), lymph node status (P < 0.001), presence of metastases (P = 0.015), and p57 expression (P = 0.039). Stratification by histological type showed the correlation of low p57 expression (P = 0.030) and worse OS of patients with alveolar RMS. Univariate analysis identified age > 50 yrs. (HR 2.447), tumors > 5 cm (HR 21.31), involvement of regional lymph nodes (HR 3.96), the presence of metastases (HR 2.53), and low p57 expression (HR 2.11) as predictors of lower OS. Tumor size, regional lymph nodes involvement, and metastases were the independent predictors after multivariate analysis, while p57 did not predict OS in an independent way. In summary, although p57 was not confirmed to be an independent predictor of OS, our results indicate that its low expression may be the marker of aggressive phenotype and poor prognosis in adult RMS patients. Also, our findings suggest that epigenetic inactivation of p16 is not important in the pathogenesis of rhabdomyosarcoma.

Cancer predisposition genes in Japanese children with rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children. Germline mutations in cancer-predisposition genes have been detected in approximately 10% of pediatric cancers. However, the genetic background of RMS is still unclear, especially in Asian children. DNA was extracted from the peripheral blood of children with RMS and cancer-associated genes analyzed using targeted re-sequencing. Twenty patients participated in this study. There were three deaths due to RMS. One patient developed a second neoplasm. Nine patients had long-term co-morbidities. Six pathogenic variants were found in five patients: one nonsense variant of DICER1, one exon deletion of TP53, and three missense variants of BUB1B, LIG4, and MEN1. Two of the five patients had a family history of cancer. Two patients with missense variants of LIG4 had long-term co-morbidities of drug-induced cardiomyopathy. The missense variants of LIG4, essential for DNA double-strand break repair, were detected in two unrelated patients. While this is the first report of the germline genetic analysis of Japanese children with RMS with detailed clinical information, the frequency of the variant was almost equivalent to that of previous reports from western countries. Unbiased exon sequencing may be useful to clarify the pathogenesis of RMS in children and in predicting the clinical course of these patients.

Genomic analysis and preclinical xenograft model development identify potential therapeutic targets for MYOD1-mutant soft-tissue sarcoma of childhood.

The myogenic differentiation 1 gene (MYOD1) p.L122R somatic mutation was first discovered in a subset of clinically aggressive embryonal rhabdomyosarcomas and has since been described in both pediatric and adult spindle cell/sclerosing rhabdomyosarcomas. Relatively little is known about the clinical, molecular, and histopathological features of these tumors in children. In order to further characterize the genomic and clinical features of pediatric MYOD1-mutant sarcomas, we evaluated a cohort of soft-tissue sarcoma patients treated at Texas Children's Hospital. Tumor DNA was subjected to next-generation panel sequencing and/or Sanger sequencing of the MYOD1 hotspot mutation. The MYOD1 p.L122R mutation was identified in six tumors, with a variant allele fraction greater than 0.8 in three cases, suggestive of loss of heterozygosity. One sclerosing rhabdomyosarcoma lacking the MYOD1 hotspot mutation was observed to have a MYOD1 copy number gain, also with evidence of loss of heterozygosity. Cancer gene panel sequencing revealed potentially targetable alterations in six of seven (86%) patients with MYOD1 alterations, including four patients with an alteration in the PI3K-AKT pathway: two hotspot PIK3CA mutations and deletions in PTEN and TSC2. On histopathologic review, MYOD1-altered tumors exhibited spindle and/or round cells and varying degrees of hyaline sclerosis. At last follow-up, six patients had died of disease and the seventh progressed early and was subsequently lost to follow-up. Both pre- and post-therapy patient-derived xenograft models were generated from one patient's tumor. These models were confirmed to harbor the MYOD1 and PIK3CA mutations seen in the primary tumor and were shown to be sensitive to PI3K/mTOR inhibition in vitro and in vivo. In conclusion, this study adds to recent reports describing the clinicopathologic and genomic features of MYOD1-altered soft-tissue sarcomas in children, including dismal prognosis and potential molecular targets for therapy. The novel preclinical models developed will facilitate further biological and preclinical study of this rare and aggressive tumor. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 47: Identification of first-in-class KDM3B inhibitors that suppress PAX3-FOXO1 oncogene activity in fusion positive rhabdomyosarcoma

Abstract Background: PAX3/7-FOXO1 fusion gene is the major oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), a highly aggressive soft tissue sarcoma of childhood. The chimeric gene results from either t(2;13) or t(1;13) translocation and has been shown to drive FP-RMS carcinogenesis by activation of super enhancer driven transcription. Thus, this fusion gene represents a unique vulnerability in FP-RMS which can be targeted by small molecules. MATERIALS AND METHODS: Using luciferase assays simultaneously monitoring PAX3-FOXO1 super enhancer and general transcription activity in RMS cells, 62,643 compounds were screened for selective inhibitors of PAX3-FOXO1 activity. RNA-seq was performed on FP-RMS cell lines treated with the top hits and gene set enrichment analysis (GSEA) was performed. To characterize the functions of fusion gene inhibitors, histone lysine demethylase (KDM) enzyme inhibition assay and in vitro proliferation assay was performed. Western analysis was used to assess expression level of MYOG, PARP, and PAX3-FOXO1 in RMS cells after treatment with inhibitors. NMR techniques WaterLOGSY and CPMG were used to determine direct binding of compound to KDM3B. In vivo FP-RMS xenograft mouse model was used to determine tumor suppression by PAX3-FOXO1 inhibitors. RESULTS: PAX3-FOXO1 selective cell-based assay identified 64 compounds that inhibited PAX3-FOXO1 activity without general inhibition of transcription or cell death at 24 hours. Compound PFI-63 was identified as the top hit, and its analog PFI-90 was identified using a chemical similarity screen based on PFI-63. RNA-seq analysis on RMS cells treated with PFI-63 and PFI-90 indicated these compounds inhibit KDMs, resulting in apoptosis and myogenic differentiation while PAX3-FOXO1 downstream gene sets were repressed. Activation of apoptosis and myogenic differentiation was validated by Western analysis for PARP cleavage and increased expression of MYOG respectively. In vitro enzymatic inhibition assay confirmed activity against KDMs with highest specificity for KDM3B. Western analysis for methylation status of histone 3 lysines at positions K27, K4, and K9 showed increased histone 3 methylation at K4 and K9 sites after treatment. Furthermore, WaterLOGSY and CPMG NMR techniques showed direct binding of PFI-90 to KDM3B. Finally, PFI-90 delayed tumor progression in a FP-RMS xenograft mouse model. CONCLUSION: We identified two KDM3B selective inhibitors which directly bind to KDM3B and disrupt PAX3-FOXO1 downstream targets in FP-RMS. PFI-63 and PFI-90 induce apoptosis and cell differentiation resulting in delayed tumor progression in vivo. Thus, we describe here novel inhibitors of KDM3B that inhibit PAX3-FOXO1 action. These novel compounds represent a potential new therapy for FP-RMS. Citation Format: Yong Yean Kim, Robert G. Hawley, Berkley Gryder, Silvia Pomella, Joshua Kowalczyk, Joseph Barchi, Young Song, Javed Khan. Identification of first-in-class KDM3B inhibitors that suppress PAX3-FOXO1 oncogene activity in fusion positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 47.

Abstract 629: Flavonoids are NR4A1 antagonists and target PAX3-FOXO1 and G9a in alveolar rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma that is primarily observed in children and adolescent, and accounts for 50% of soft tissue sarcomas in children. There are two major forms of RMS, namely embryonal RMS (ERMS) and alveolar RMS (ARMS) and these tumors are readily distinguished by their histological and genomic characteristics. ERMS patients have a better prognosis than ARMS patients and both groups undergo radiation, surgical and chemotherapeutic regiments. Many ARMS patients express different oncogenes including the histone methyltransferase G9a and the PAX3-FOXO1 fusion gene that contribute to ARMS cell growth, survival and migration/invasion. Studies in this laboratory have demonstrated that both G9a and PAX3-FOXO1 are regulated by the orphan nuclear receptor 4A1 (NR4A1) and can be targeted by bis-indole derived (CDIMs) NR4A1 antagonists. A recent study showed that the flavonoid kaempferol decreased G9a expression in gastric cancer cells and we hypothesized that kaempferol may be an NR4A1 ligand. Kamepferol and quercetin were used as models and both compounds bound NR4A1 at low μM concentrations and decreased NR4A1-dependent transactivation in ARMS cells. Functional studies showed that like CDIM/NR4A1 ligands, kaempferol and quercetin decrease ARMS cell growth, survival and migration/invasion, consistent with their activity as antagonists and inhibitors of NR4A1 regulated pro-oncogenic functions. In addition, kaempferol and quercetin also decreased expression of G9a, PAX3-FOXO1 and downstream genes regulated by them in ARMS cells further demonstrating their activity as NR4A1 antagonists. These results suggest that NR4A1-active flavonoids could be readily incorporated into chemotherapies used for treatment of ARMS patients to improve their efficacy and decrease the toxic side-effects of currently used cytotoxic drugs. Citation Format: Rupesh Shrestha, Kumaravel Mohankumar, Gregory Martin, Stephen Safe. Flavonoids are NR4A1 antagonists and target PAX3-FOXO1 and G9a in alveolar rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 629.

Abstract LB251: Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most commonly diagnosed soft tissue sarcoma in children. While the 5-year event-free survival for localized rhabdomyosarcoma is over 70%, it is less than 30% for patients presenting with metastatic disease. Given that the current treatment modalities for RMS patients remain cytotoxic therapies with profound life-long lasting complications, identification of less toxic and more effective therapies is highly desired. Whole-genome and RNA sequencing of human rhabdomyosarcoma have identified frequent RAS genes oncogenic mutations in fusion-negative rhabdomyosarcoma (FN-RMS), which comprises 65% of RMS. This high incidence of RAS mutations present opportunity for design of targeted therapeutic strategies for FN-RMS. By siRNA-mediated knock down of individual RAS isoforms we showed that H/NRAS Q61X-mutant FN-RMS cell lines exhibited oncogenic RAS dependency and that knockdown of the Q61X-mutant H/NRAS inhibited ERK MAPK, but not PI3K-AKT pathway activity. Knockdown of the Q61X-mutant H/NRAS phenocopied ERK inhibitor treatment by inducing myogenic differentiation. Further, as compared to RAS-wild type RMS cells, H/NRAS-Q61X FN-RMS cell lines were preferentially more sensitive to MEK or ERK, but not PI3K or AKT inhibitors. RNA sequencing analysis revealed significant overlap of genes signatures associated with NRAS-Q61H knockdown and ERK inhibitor treatment in the FN-RMS cell line RD. These results indicated a central role of the ERK MAPK pathway in mediating H/NRAS Q61X-dependency in FN-RMS. However, in vivo evaluation of clinically relevant exposers with the MEK inhibitor trametinib demonstrated poor response, which correlated with inefficient ERK inhibition in pharmacodynamic assays. A trametinib-sensitizing CRISPR screen pointed to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically blocked ERK activity and induced cell cycle arrest, myogenic differentiation and cell death. In vivo evaluation of low dose pan-RAFi + ERKi or MEKi + ERKi potently suppressed H/NRAS Q61X RMS tumor growth, with pan-RAFi + ERKi being more effective and better tolerated, suggesting wider therapeutic window. These results support the clinical translation of pan-RAFi + MEKi/ERKi for H/NRAS Q61X-mutant FN-RMS. Citation Format: Natalia Garcia, Vanessa Del-Pozo, Marielle Yohe, Craig Goodwin, Terry Shackleford, Long Wang, Kunal Baxi, Yidong Chen, Myron Ignatius, Kris Wood, Peter Houghton, Angelina Vaseva. Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB251.

Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium.

PURPOSERhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome.PATIENTS AND METHODSTumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed.RESULTSDNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data.CONCLUSIONThis is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.

DCX and CRABP2 are candidate genes for differential diagnosis between pre-chemotherapy embryonic and alveolar rhabdomyosarcoma in pediatric patients.

ABSTRACTImportanceRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. More than 90% of cases are classified as embryonic RMS (ERMS) or alveolar RMS (ARMS). ERMS has a worse prognosis than ARMS. Early differential diagnosis is of paramount importance for optimization of treatment.ObjectiveTo identify genes that are differentially expressed between ARMS and ERMS, which can be used for accurate rhabdomyosarcoma classification.MethodsThree Gene Expression Omnibus datasets composed of ARMS and ERMS samples were screened and 35 differentially expressed genes (DEGs) were identified. Receiver operating characteristic curve analysis and area under the curve analysis was performed for these 35 DEGs and seven candidate genes with the best differential expression scores between ARMS and ERMS were determined. The expression of these seven candidate genes was validated by immunohistochemical analysis of pre‐chemotherapy ARMS and ERMS specimens.ResultsThe levels of DCX and CRABP2 were confirmed to be remarkably different between paraffin‐embedded ARMS and ERMS tissues, while EGFR abundance was only marginally different between these two RMS subtypes.InterpretationDCX and CRABP2 are potential biomarkers for distinguishing ARMS from ERMS in pre‐chemotherapy pediatric patients.

Clinical Characteristics and Treatment Outcome of Paediatric Rhabdomyosarcoma; A Retrospective Review.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. This paper aimed to assess the stage, site and treatment outcome among RMS patients. A retrospective chart review was completed from January 2011 to December 2017 of patients that presented to the Department of Paediatric Oncology, Shaukat Khanum Memorial Cancer Hospital, Lahore, Pakistan, for the management of RMS. Data collection included clinical characteristics, staging, grouping, risk stratification, treatment plan, radiotherapy doses and treatment outcome. Among 24 subjects, there were a total of 13 (54.2%) males and 11 (45.8%) females. The median age at the time of diagnosis was 2.5 years (range: 0.75-17 years). The majority of the subjects (91.7%) were <10 years of age. The median follow-up time was 0.6 years. According to the Children's Oncology Group Classification, 4 (16.7%) subjects were classified as low risk, 14 (58.3%) subjects were rated as intermediate risk and 6 (0.25%) subjects were stratified as high risk. The most common primary tumour site was genitourinary (62.5%) and abdomen/retroperitoneal (20.8%) regions. At the time of analysis, nine (37.5%) subjects had died because of the disease, 12 (50%) were alive with no evidence of disease and one subject had a recurrence of disease and was alive. One subject had abandoned the therapy and another was lost to follow-up. Patients with RMS presented at the late stages of the disease and it most frequently affected genitourinary and abdomen or retroperitoneal areas. Overall, RMS was found to have a poor outcome to therapy.

Pediatric Rhabdomyosarcoma: Epidemiology and Genetic Susceptibility.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, yet little is known about its etiology. Studies that examine either environmental exposures or germline genetic predisposition in RMS have begun to identify factors that contribute to this malignancy. Here, we summarize epidemiological reports of RMS incidence in terms of several factors, including age at diagnosis, biological sex, and geographic location. We then describe findings from association studies, which explore the role of parental exposures, birth and perinatal characteristics, and childhood exposures in RMS. Further, we discuss RMS predisposition syndromes and large-scale sequencing studies that have further identified RMS-associated genes. Finally, we propose future directions of study, which aim to advance our understanding of the origin of RMS and can provide knowledge for novel RMS therapies.

Rhabdomyosarcoma in the first year of life. Experience of Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, but is very rare in infants. RMS diagnosed during the first year of life is reported to have poor outcome. The aim of the study was to analyze the results of therapy of RMS in the first year of life treated in federal center in Russian Federation. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. All prospectively registered patients with RMS in infants treated during the period 02.2012–05.2018 (75 month) were included. Diagnosis was confirmed by histology. All patients were examined and stratified according to the GPOH Cooperative Weichteilsarkom Study (CWS) Group guidance 2009. 13 prospectively registered patients with RMS in infants treated during the period were included. Median age at initial diagnosis was 6.7 (range 0.23–11.9) months. The tumor was detected prenatally at 32 and 33 weeks of gestation in 2 (15%) patients. The age of these patients was ≤ 1 month in 2 of 13 patients. Tumor size was ≤ 5 cm in 8 (61,5%) of 13 patients. Median volume tumor was 24 (range 0.001–150) ml. The primary site was head and neck (n = 4; 31.5%), pelvis (n = 3; 23%), extremities (n = 3; 23%), genitourinary system (n = 2; 15%), other (n = 1; 7.5%). IRS stage distribution was stage III in 12 (92.5%) patients, stage IV – 1 (7.5%). Regional nodal metastasis (N1) occurred in 1 (7.5%) patient with tumor of the head and neck nonparameningeal. Distant metastasis occurred in 1 (7.5%) patient and the site of spread was subcutaneous fat (blueberry muffin syndrome), pleura, roots of both lungs, pancreas, bone marrow. Histology of these infants was RMA (n = 6; 46%), RME (n = 6; 46%) and spindle-cell RMS (n = 1; 7.5%). The FOXO1-fusion positive status was found in 4/6 patients RMA by fluorescence in situ hybridization (FISH). 11/13 (85%) patients were categorized as high risk, 1/13 (7.5%) as very high risk, 1/13 (7.5%) – treatment for stage IV patients with metastatic disease. All patients (n = 13; 100%) received chemotherapy according to CWS guidance 2009 protocol. All patients were administered at a reduced dose according to body weight. The distribution of treatment regimens was as follows: 11/13 (85%) – IVA, 1/13 (7.5%) – IVADO, 1/13 (7.5%) – CEVAIE. 3/13 patients aged less than 1 month was administered chemotherapy according to VAC. Initial surgery included biopsy in 8/13, surgery – 5/13 (R2 – resection). Local control was provided in 11/13 (85%) patients: only surgery in 7/13 (54%) patients, only radiotherapy in 1/13 (7.5%), radiotherapy and surgery 1/13 (7.5%), only brachytherapy 1/13 (7.5%), brachytherapy and surgery in 1/13 (7.5%). 2/13 patients were not local control: one patient with favorable site (vagina) and one patient with stage IV that showed complete response after chemotherapy in both cases. Radiotherapy including brachytherapy was administered of the all patients at the over 1 year. Radiotherapy was used in 2 of 13 patients (dose range 50.4 Gy and 51.2 Gy), brachytherapy – 36 Gy. At the start of the radiotherapy the age was 21.1 and 13.37 months, brachytherapy – 12,4 and 14,5 months. Second-look surgery was performed in 9/13 (69%) patients: R0 (n = 7), R1 (n = 1), R2 (n = 1). 2/9 patients were performed mutilating surgery: orbital exenteration and cystectomy. Induction therapy was completed in 12 (92.5%) of 13 patients. Median follow-up time was 42.7 (range 3.7–90) months. 8 patients were alive, 5 died. 8/13 patients whom alive were observation: 6/8 patients no relapse/progressive, 2/8 patients – remission after local relapse and progression. 3-year overall survival was 68,4% (95% confidence interval 42,6–94,1. 3-year event-free survival was 46,2% (95% confidence interval 19,1–73,3). Patients aged less than 1 year are particularly problematic. The relatively low event-free survival rate in this age group is associated with the impossibility of carrying out the entire volume of multimodal therapy and required a tailored therapeutic approach.

Complexity index in sarcoma and genomic grade index gene signatures in rhabdomyosarcoma of pediatric and adult ages.

Rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma in childhood, shows extensive heterogeneity in histology, site and age of onset, clinical course, and prognosis. Adolescents and young adults (AYA) with RMS form a subgroup of patients whose survival lacks behind that of children while diagnosed with histologically similar tumors. A 67-gene prognostic signature related to chromosome integrity, mitotic control, and genome complexity in sarcomas (CINSARC) is considered a powerful tool for identifying tumors with a highly metastatic potential. With this study, we investigated the prognostic value of CINSARC signature on a cohort of 48 pediatric (PEDs) and AYAs-RMS. CINSARC resulted not significantly correlated with age, suggesting other determinants to be responsible for that difference in survival. It remained a significant prognostic variable in both the groups of PEDs and AYAs. Also, genomic grade index signature was tested on the same cohort and showed very similar results with CINSARC. Our study showed that CINSARC correlated with outcome in RMS patients and may be potentially considered a tool to predict outcome, and so stratify RMS patients.

Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.

Embryonal rhabdomyosarcoma completely resected at diagnosis: The European paediatric Soft tissue sarcoma Study Group RMS2005 experience

BackgroundRhabdomyosarcoma (RMS) is the most common form of soft tissue sarcoma in children. We report the results of the European paediatric Soft tissue sarcoma Study Group (EpSSG) RMS 2005 study, which prospectively evaluated the reduction of chemotherapy in patients with embryonal RMS (ERMS) after initial surgery. MethodsBetween October 2005 and December 2016, all patients with localised ERMS with an initial microscopically complete resection (IRS group I) with lymph node-negative (N0) were prospectively enrolled in the low-risk (n = 70, subgroup A; age < 10 years and tumour size ≤ 5 cm) or standard-risk group (n = 108, subgroup B; age ≥ 10 years or tumour size > 5 cm. Subgroup A received 8 courses of vincristine and dactinomycin (VA) for 22 weeks; subgroup B received 4 courses of VA with ifosfamide (IVA) and 5 courses of VA for 25 weeks. ResultsThe 5-year event-free survival (EFS) and overall survival (OS) were 90.8% (95% confidence interval [CI]: 85.0–94.4) and 95.7% (95% CI: 90.5–98.1), respectively (n = 178). The EFS and OS were 95.5% (95% CI: 86.8–98.5) and 100% (subgroupA), and 87.8% (95% CI: 79.3–93.0) and 93.0% (95% CI: 84.8–96.8)(subgroup B), respectively. Bearman stage 2 veno-occlusive disease (VOD) occurred in 4 very young patients. ConclusionVA treatment for 8 courses was effective and well tolerated by the subgroup of patients with low-risk ERMS (group A). Four courses of IVA and 5 courses of VA instead of 9 courses of IVA also has very good results. Careful monitoring for liver toxicity is important in very young patients.European union drug regulating authorities clinical trials EUDRACT No. 2005-000217-35.

The Value of Early Tumor Size Response to Chemotherapy in Pediatric Rhabdomyosarcoma.

Simple SummaryRhabdomyosarcoma is the most common soft tissue sarcoma in childhood. At diagnosis, tumor and patient characteristics determine the prognosis and subsequent treatment stratification. There are currently no early biomarkers that identify good or poor responders to chemotherapy regimens, survival being the only valid endpoint. Early tumor size response, which is assessed by imaging, could be such a marker. We performed a systematic assessment of literature to November 2020. Six studies were included describing 2010 patients; quality assessment showed methodological limitations. We conclude that there is evidence that early progressive disease is associated with poorer survival compared to patients with non-progressive disease, being either stable disease, partial, or complete response. However, for the vast majority of patients with non-progressive disease, we found no evidence that the degree of response is prognostic for survival. Therefore, the value of early tumor size response as a prognostic marker, and its translation into treatment modifications on an individual patient or trial level should be reconsidered.Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood. Results of clinical trials, with three-year event-free and overall survival as primary outcomes, often take 7 to 10 years. Identification of an early surrogate biomarker, predictive for survival, is therefore crucial. We conducted a systematic review to define the prognostic value of early tumor size response in children with IRSG group III rhabdomyosarcoma. The search included MEDLINE/EMBASE from inception to 18 November 2020. In total, six studies were included, describing 2010 patients, and assessed by the Quality in Prognosis Studies (QUIPS) instrument. Four studies found no prognostic value for tumor size response, whereas two studies reported a prognostic effect. In these two studies, the survival rate of patients with progressive disease was not separately analyzed from patients with stable disease, potentially explaining the difference in study outcome. In conclusion, our findings support that early progression of disease is associated with poorer survival, justifying adaptation of therapy. However, in patients with non-progressive disease, there is no evidence that the degree of response is a prognostic marker for survival. Because the vast majority of patients do not have progressive disease, early tumor size response should be reconsidered for assessment of treatment efficacy. Therefore, at present, early surrogate biomarkers for survival are still lacking.

Rhabdomyosarcoma: From Obscurity to Clarity in Diagnosis … But With Ongoing Challenges in Management: The Farber-Landing Lecture of 2020.

Rhabdomyosarcoma, the most common soft tissue sarcoma in childhood, has challenged and intrigued soft tissue pathologists ever since the original descriptions. Once based on the identification of rhabdomyoblastic cells with elongate eosinophilic cytoplasm, the diagnosis has evolved to include tumors composed only of primitive mesenchymal cells but now relies heavily on immunohistochemical stains for desmin, myogenin, and MyoD. Rhabdomyosarcomas show a variety of histological patterns, giving rise to classifications that have included embryonal, alveolar, botryoid, pleomorphic, spindle cell, and sclerosing subtypes. These have been linked to prognosis and treatment assignment in the past, but that concept has been superseded by the identification of PAX3-FOXO1 or PAX7-FOXO1 fusions. Fusion testing results are more predictive of outcome and have become standard practice in clinical management. However, high risk tumors with alveolar histology or metastatic disease continue to resist oncologic treatment.