Sarcoma In Children Research Articles

Synovial Sarcoma in Children, Adolescents, and Young Adults: A Report From the Children's Oncology Group ARST0332 Study.

Synovial sarcoma (SS) is the second most common malignant soft tissue tumor in children. ARST0332 evaluated a risk-based treatment strategy for young patients with soft tissue sarcoma designed to limit therapy for low-risk (LR) disease and to test neoadjuvant chemoradiotherapy for unresected higher-risk disease. Newly diagnosed patients with SS age < 30 years were assigned to four treatment arms based on disease features: A (surgery only), B (55.8 Gy radiotherapy [RT]), C (ifosfamide and doxorubicin [ID] chemotherapy plus 55.8 Gy RT), and D (neoadjuvant ID and 45 Gy RT, then surgery and RT boost based on margins followed by adjuvant ID). Patients treated in Arms A and B were considered LR, arms C and D without metastases as intermediate-risk (IR), and those with metastases as high-risk (HR). Of the 146 patients with SS enrolled, 138 were eligible and evaluable: LR (46), IR (71), and HR (21). Tumors were 80% extremity, 70% > 5 cm, 70% high-grade, 62% invasive, 95% deep, and 15% metastatic. Treatment was on arm A (29.7%), B (3.6%), C (16.7%), and D (50%). There were no toxic deaths and four unexpected grade 4 adverse events. By risk group, at a median follow-up of 6.8 years, estimated 5-year event-free survival was LR 82%, IR 70%, and HR 8%, and overall survival was LR 98%, IR 89%, and HR 13%. After accounting for the features that defined risk category, none of the other patient or disease characteristics (age, sex, tumor site, tumor invasiveness, and depth) improved the risk stratification model. The risk-based treatment strategy used in ARST0332 produced favorable outcomes in patients with nonmetastatic SS relative to historical controls despite using RT less frequently and at lower doses. The outcome for metastatic SS remains unsatisfactory and new therapies are urgently needed.

The Role of BiP and the IRE1α-XBP1 Axis in Rhabdomyosarcoma Pathology.

Simple SummaryThe expression of BiP (GRP78), spliced XBP1 (sXBP1, nuclear XBP1), and IRE1α, were significantly associated with Rhabdomyosarcoma (RMS) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS; (n = 192) compared to normal skeletal muscle tissues (n = 16). There was a significant correlation between BiP expression and the lymph node score, and between IRE1α, cytosolic XBP1 and sXBP1 expression and the stage score in all of the types of RMS. BiP and sXBP1 expression were significantly associated with all of the subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. There were correlations between BiP expression and the lymph node score in ARMS, and sXBP1 expression and the tumor score in PRMS.Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and is associated with a poor prognosis in patients presenting with recurrent or metastatic disease. The unfolded protein response (UPR) plays pivotal roles in tumor development and resistance to therapy, including RMS. Methods: In this study, we used immunohistochemistry and a tissue microarray (TMA) on human RMS and normal skeletal muscle to evaluate the expression of key UPR proteins (GRP78/BiP, IRE1α and cytosolic/nuclear XBP1 (spliced XBP1-sXBP1)) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS. We also investigated the correlation of these proteins with the risk of RMS and several clinicopathological indices, such as lymph node involvement, distant metastasis, tumor stage and tumor scores. Results: Our results revealed that the expression of BiP, sXBP1, and IRE1α, but not cytosolic XBP1, are significantly associated with RMS (BiP and sXBP1 p-value = 0.0001, IRE1 p-value = 0.001) in all of the studied types of RMS tumors (n = 192) compared to normal skeletal muscle tissues (n = 16). In addition, significant correlations of BiP with the lymph node score (p = 0.05), and of IRE1α (p value = 0.004), cytosolic XBP1 (p = 0.001) and sXBP1 (p value = 0.001) with the stage score were observed. At the subtype level, BiP and sXBP1 expression were significantly associated with all subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. Importantly, the expression levels of IRE1α and sXBP1 were more pronounced in ARMS than in any of the other subtypes. The results also showed correlations of BiP with the lymph node score in ARMS (p value = 0.05), and of sXBP1 with the tumor score in PRMS (p value = 0.002). Conclusions: In summary, this study demonstrates that the overall UPR is upregulated and, more specifically, that the IRE1/sXBP1 axis is active in RMS. The subtype and stage-specific dependency on the UPR machinery in RMS may open new avenues for the development of novel targeted therapeutic strategies and the identification of specific tumor markers in this rare but deadly childhood and young-adult disease.

Genetic Characterization, Current Model Systems and Prognostic Stratification in PAX Fusion-Negative vs. PAX Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents and accounts for approximately 2% of soft tissue sarcomas in adults. It is subcategorized into distinct subtypes based on histological features and fusion status (PAX-FOXO1/VGLL2/NCOA2). Despite advances in our understanding of the pathobiological and molecular landscape of RMS, the prognosis of these tumors has not significantly improved in recent years. Developing a better understanding of genetic abnormalities and risk stratification beyond the fusion status are crucial to developing better therapeutic strategies. Herein, we aim to highlight the genetic pathways/abnormalities involved, specifically in fusion-negative RMS, assess the currently available model systems to study RMS pathogenesis, and discuss available prognostic factors as well as their importance for risk stratification to achieve optimal therapeutic management.

Pediatric liposarcoma: A case series and literature review.

Liposarcoma is arare soft tissue sarcoma in children. While prognosis, clinical behavior, and response to therapy among the various histologic subtypes are well described in adults, data in children are limited. Here, we describe our experience treating 14 children with liposarcoma at a large, academic pediatric center and review the available pediatric literature. This comprehensive report adds treatment, survival, and genomic data to pediatric liposarcoma literature.

Locally aggressive rarely metastazing tumors and low-grade sarcoma in children, adolescents and young adults: The benefits of a national network

IntroductionComplete surgical resection constitutes the mainstay of treatment for locally aggressive, rarely metastazing tumor and low-grade soft tissue sarcomas (LAS). Local relapse is the most common tumor event, especially in the presence of positive margins (R1 margins). The aims of this study are to assess the impact of the national network on patient care and to evaluate the role of immediate re-excision in children, adolescents and young adults with incompletely resected LAS. MethodsNational retrospective multicenter study of all young patients (≤25 years) included in the Sarcoma “ConticaBase” treated for LAS between 2005 and 2017 for whom pathology/biology review was available via the national NETSARC + network. ResultsA total of 96 patients were identified (median age: 16 years). Tumors were localized in 99% of cases (1 N+ tumor). With a median follow-up of 4.7 years (range: 0.1–11.9), eight local relapses and two distant metastases were observed. No patient died. Overall 5-year event-free survival (EFS) was 90.4% [95%CI, 84.3–97]. Five year EFS for R1 patients (n = 51) with (n = 24) and without (n = 27) immediate re-excision was 90.5% [95%CI, 78.8–100.0] and 80.3% [95%CI, 64.7–99.9], respectively (p = 0.34). The 37 patients directly treated in a reference center more commonly had a diagnostic biopsy (78% vs. 21%; p < 0.001), more complete surgery (R0: 65% vs. 14%; p < 0.001) and less commonly underwent re-excision (16% vs. 54%; p < 0.001). ConclusionsThis large series indicates that LAS are rare in young patients and have a favorable prognosis. Immediate management in reference centers is associated with better standard of care. The main tumor events are local relapses.

Surfaceome Profiling of Rhabdomyosarcoma Reveals B7-H3 as a Mediator of Immune Evasion.

Simple SummaryRhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and there is a critical need to develop efficacious and tolerable anticancer therapies against this aggressive disease. To uncover druggable RMS-associated tumor antigens, we analyzed the cell surface protein repertoire in RMS tumor cells and normal tissue. We identified several surface proteins highly enriched in RMS, including the immune checkpoint molecule B7-H3. A further analysis using patient specimens showed that B7-H3 is overexpressed in most of RMS tumors and weakly or not detected in normal organs. Interestingly, we found that B7-H3 depletion was associated with higher immune cell killing activity against tumor cells. In line with this, high B7-H3 tumor expression was associated with lower CD8 T-cell density. Our study reveals novel RMS-associated proteins for the development of targeted therapies. In addition, we demonstrate that targeting B7-H3 function can pave the way for the design of new immunotherapies in the treatment of RMS.Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.

Periorbital Mass in a 2-month-old Girl.

Periorbital Mass in a 2-month-old Girl.

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.

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.

Magnetic resonance imaging findings of synovial sarcoma in children: location-dependent differences.

While overall survival for children is greater than that for adults, synovial sarcoma remains an aggressive neoplasm with a potentially poor prognosis, and its magnetic resonance imaging (MRI) findings in children are not well described. We aimed to characterize the spectrum of MRI findings of synovial sarcoma in children with respect to anatomical location and outcome. Children with histologically confirmed synovial sarcoma and preoperative MRI performed within the past 11years (2009-2020) were included. Two radiologists retrospectively reviewed each MRI to categorize location, signal characteristics and associated findings. Chi-square and Fisher exact tests were used to assess associations with locations and outcomes. This study included 23 children (13 girls, 10 boys; mean age: 12.7±4.2years) with 7 axial, 8 proximal and 8 distal appendicular lesions. Kappa ranged from 0.53 to 1. MRI findings differed significantly between locations with axial lesions measuring larger (P=0.01) and more likely to contain fluid levels (P=0.02), triple sign (P=0.02), inhomogeneous signal (T1-weighted images, P=0.003; T2-weighted images, P=0.02, contrast-enhanced images, P=0.03) with all lesions containing partially solid composition (P=0.03). At a median follow-up of 14months (interquartile range: 7-33months), 39% relapsed. Predictors of relapse (P<0.05) included metastasis at presentation, larger lesions, axial lesions and MRI findings of fluid level, T1-weighted hyperintensity, inhomogeneous signal (T1- and T2-weighted images) and poorly circumscribed margins. A significant association was found between location and MRI findings in our cohort of children with synovial sarcoma. Axial lesions were more likely to be larger, appear heterogeneous and be associated with a worse outcome.

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.

A child with Ewing's sarcoma in scapula: A rare case report

IntroductionEwing's sarcoma is considered to be the second most frequent primary sarcoma in children. It affects children and young adults with a male predominance. Ewing's sarcoma is usually found in the femur, humerus, ilium and tibia; and in extremely rare cases Ewing's sarcoma might affect the scapula. There are only 15 papers in PubMed database regarding scapular Ewing's sarcoma.Case presentationA 14-year-old male, with no significant medical or surgical history, presented with a swelling in the right scapular area for two months. The patient also experienced mild pain and fever. On physical examination, there was a tender mass and restriction in the right shoulder joint movements. MRI showed a large scapular mass with the characteristics of a sarcoma. The final diagnosis was made based on pathologic findings. Eventually, the patient was subjected to neoadjuvant chemotherapy.DiscussionThe most common tumors affecting scapula are chondrosarcoma and osteosarcoma. In a cohort study about patients diagnosed with ES between 1988 and 2018, only 29 cases were involved in the scapula. There are 12 cases of congenital ES have been reported in the medical literature, 3 of them were in the shoulder girdle. Surgery with neoadjuvant chemotherapy is considered better for total survival in ES of scapula in comparison with chemotherapy and/or radiotherapy alone.ConclusionEwing sarcoma is extremely rare in the scapula and should be considered as a differential diagnosis for any patient with scapular tumor.

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 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.

Vincristine, Irinotecan, and Temozolomide as a Salvage Regimen for Relapsed or Refractory Sarcoma in Children and Young Adults.

PurposeNo standard salvage regimen is available for relapsed or refractory sarcoma. We investigated the efficacy and toxicity of the vincristine, irinotecan, and temozolomide combination (VIT) for relapsed or refractory sarcomas of variable histology in children and young adults.Materials and MethodsWe retrospectively reviewed data from the relapsed or refractory sarcoma patients who were treated with VIT. The VIT protocol was given every 3 weeks as follows: vincristine, 1.5 mg/m2 intravenously on day 1, irinotecan, 50 mg/m2/day intravenously on days 1–5, and temozolomide, 100 mg/m2/day orally on days 1–5.ResultsA total of 26 patients (12 males) with various sarcoma histology were included in the study. Most common diagnosis was rhabdomyosarcoma (n=8) followed by osteosarcoma (n=7). Median age at the start of VIT was 18.5 years (range, 2.0 to 39.9). VIT was delivered as 2nd to 7th line of treatment, with 4th line most common (9/26, 34.6%). Median number of VIT courses given was 3 (range, 1 to 18). Of the 25 evaluable patients, there was two partial response (PR) and 11 stable disease (SD) with an overall control rate (complete remission+PR+SD) of 52%. PR was seen in one (50%) of the two evaluable patients with Ewing sarcoma and one (14.3%) of the seven patients with osteosarcoma. Overall survival and progression-free survival rates were 79.3% and 33.9% at 1 year, and 45.5% and 25.4% at 2 years, respectively. There was no treatment-related mortality.ConclusionThe VIT regimen was effective and relatively safe in our cohort of sarcoma patients.

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.