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Undifferentiated small round cell sarcoma in a young male: a case report

CIC-rearranged sarcomas (CRSs) have recently been characterized as a distinct sarcoma subgroup with a less favorable prognosis compared to other small round cell sarcomas. CRSs share morphologic features with Ewing's sarcoma and prior to 2013 were grouped under undifferentiated sarcomas with round cell phenotype by the WHO classification. In this report, whole-genome sequencing and RNA sequencing were performed for an adolescent male patient with CRS who was diagnosed with undifferentiated pleomorphic sarcoma (UPS) by three contemporary institutions. Somatic mutation analysis identified mutations in IQGAP1, CCNC, and ATXN1L in pre- and post-treatment tissue samples, as well as a CIC–DUX4 fusion that was confirmed by qPCR and DUX4 immunohistochemistry. Of particular interest was the overexpression of the translation factor eEF1A1, which has oncogenic properties and has recently been identified as a target of the investigational agent plitidepsin. This case may provide a valuable waypoint in the understanding and classification of CRSs and may provide a rationale for targeting eEF1A1 in similar soft tissue sarcoma cases.


INTRODUCTION
Because of the rarity of each non-rhabdomyosarcoma soft tissue sarcoma (NRSTS) subtype, clinical investigation of NRSTS aggregates children with many diagnoses into the same clinical trial. Synovial sarcoma, malignant peripheral nerve sheath tumor (MPNST), fibrosarcoma, undifferentiated pleomorphic sarcoma (UPS), and CIC-rearranged sarcoma (CRS) are just a few of more than 50 NRSTSs grouped together for pediatric and adult clinical trials. Survival rates for NRSTSs can be poor when tumors are high-grade, large (>5 cm), difficult to resect in their entirety, and/or metastatic (Spunt et al. 2008;Ferrari et al. 2011).
Until 2013, CRS was classified as an undifferentiated sarcoma by the World Health Organization (WHO) (Doyle 2014). Because CRSs share similar morphological features to Ewing's sarcoma, these NRSTSs are often considered "Ewing's sarcoma-like" and historically may have followed similar treatment protocols (Doyle 2014). Morphologically, CRSs are characterized by their medium round or ovoid cells and lack of differentiation, with rare cases having an epithelioid appearance and mild nuclear pleomorphism (Italiano et al. 2012;Antonescu et al. 2017). However, further clinical and molecular evidence has distinguished CRSs as a distinct new subgroup because of their clinicopathologic and molecular features (Antonescu et al. 2017).
Here, we present the case of a young adolescent male patient initially diagnosed with undifferentiated epithelioid and pleomorphic sarcoma posthumously confirmed to be a high-grade CRS. Prior to the identification of the CIC-DUX4 rearrangement, the patient was treated as an UPS, which has varying clinical treatment approaches that are minimally effective. Broadly, the treatment for CRS is poorly defined. Previous studies suggest that patients with the CIC-DUX4 gene fusion from either a t(4:19) or t(10:19) translocation tend to have an aggressive clinical course with metastatic relapse in 61% (11/18) of cases at follow-up (Italiano et al. 2012). Other CRS translocations include CIC-FOXO4 or CIC-NUTM1 fusions, but these alternative fusions account for only ∼5% of CRSs (Watson et al. 2018). Although these new findings suggest that the CIC translocation is a driver of tumor growth and metastasis in CRS, the translocation is currently not targetable. To further advance translational insights for CRS, we have investigated the whole-genome and RNA sequencing of a young adolescent with CRS.

Clinical Presentation
An 11-yr-old male first presented with a soft tissue mass in the proximal left lower leg, which was initially diagnosed as a ganglion cyst. Because the mass continued to grow, a second opinion was sought ∼3 mo later. After magnetic resonance imaging (MRI) confirmed the presence of a solid tumor ( Fig. 1; Supplemental Fig. 1), he underwent excisional biopsy. Histologic examination of the mass demonstrated epithelioid-to-spindled cells distributed in sheets with alternating cellular and hypocellular fibrotic areas ( Fig. 2A-C). Large scattered cells with abundant eosinophilic cytoplasm were present, whereas other areas demonstrated sheets of smaller tumor cells with a more primitive appearance within a looser, myxoid * * Figure 1. MRI scans of the left knee. A well-defined elliptical lesion (red asterisk) in the subcutaneous fat anteromedial to the proximal tibia (right) was found with the following multiplanar sequences: survey (left) and lateral tibia axial proton density with fat saturation (right). matrix. The tumor nuclei were polygonal with round to oval nuclei, vacuolated chromatin, and prominent nucleoli. Immunostaining revealed cells that were positive for CD99 and vimentin and negative for SMA, desmin S100, CD34, MyoD1, AE1/AE3, and CD31. Thus, no evidence was found to support any line of differentiation, including leiomyosarcoma, rhabdomyosarcoma, epithelioid sarcoma, perivascular epithelial cell tumor, nor myoepithelial neoplasm. The tumor was ultimately classified as an undifferentiated epithelioid and pleomorphic sarcoma, and the patient underwent treatment for a UPS.
Five weeks after the initial surgery, the patient underwent primary re-excision that achieved complete margins. A metastatic workup including chest computed tomography (CT) and bone scan was negative. The patient was subsequently enrolled in Children's Oncology Group study ARST0332, in which he was observed without adjuvant therapy. Tumor surveillance included an MRI of the primary tumor region every 3 mo and a chest X ray every 6 mo. At month 11 after diagnosis, the patient developed left shoulder pain and a persistent cough and was diagnosed with pneumonia. Subsequent imaging due to symptom persistence demonstrated bilateral pulmonary and pleural metastases with a right pleural effusion. After short-term pleural fluid drainage with a chest tube, the patient received ifosfamide/doxorubicin chemotherapy for a total of six cycles (cumulative 54 g/m 2 ifosfamide, 375 mg/m 2 doxorubicin). A minor tumor response was documented after three cycles of chemotherapy, and right thoracotomy with complete resection of the right-sided lung nodules was performed. Pathology showed an extensive treatment effect with a <5% viable tumor.
Tumor tissue from the right thoracotomy was sent for next-generation panel sequencing using the FoundationOne Heme test, which identifies genomic alterations in 405 genes (median exon coverage of ∼500×). Alterations were found in CHD2, FOXO1, HDAC7, RPTOR, Translation factor eEF1A1 overexpression in a CIC-rearranged sarcoma case

Molecular Case Studies
TSC2, and WDR90, all with unknown significance (Supplemental Table 1). Thus, no actionable genomic alterations were found. Tissue samples from the first excision of the right lung metastatic tumor were also sent to Human Longevity Inc. (HLI) for whole-genome sequencing (WGS) (113× coverage) and RNA sequencing together revealed no cancer-relevant somatic alterations or copy-number variations (CNVs). Overall WGS mutation rate was <1 mutation per megabase (MB). Of note, because of the low tumor purity observed in the samples, the sensitivity to capture low allele fractions variants was reported to be suboptimal; thus, some low-frequency alterations may not be called. In addition, copy-number alterations could not be assessed because of the tumor purity. Quantitative polymerase chain reaction (qPCR) studies at HLI raised the question of a CIC-DUX4 fusion and the suggestion of FISH studies, which is discussed in a later section below. After completion of the remaining chemotherapy, a left thoracotomy and resection of the remaining lung nodules were performed. Pathology reported microscopic residual disease at the margins of two resected lung nodules. Fresh frozen tumor samples were sent from the left thoracotomy to HLI for WGS (127× coverage), whole-exome sequencing (WES, 574× coverage), RNA sequencing (80 M reads), Oncomine panel, and qPCR. Again, no cancer-relevant single-nucleotide variants (SNVs), indels, or CNVs were reported. However, the samples each had low tumor purities based on pathological assessments of the second sample tested and computational assessments performed on all three samples. An orthogonal sequencing method using Oncomine Comprehensive Assay (OCAv2) with Ion Torrent technology (ThermoFisher Scientific) was also performed and did not detect any SNVs, indels, or fusions. The samples were found to have a low mutational burden with two mutations per megabase (Mb). Evidence of a potential CIC-DUX4 fusion, described as translocation t(4;19)(q35;q13.1), was identified in the FFPE sample and the fresh frozen sample by WGS (four reads/sample) and confirmed by qPCR in the fresh frozen RNA sample: Two out of the three sets of primers designed to span the predicted fusion junctions produced a positive result amplifying a fusion junction in CIC-DUX4. Blood was also sent as a normal control for WGS with 31× and WES with 227×. No pathogenic variants associated with inherited cancer risks and no variants associated with adverse drug reaction or drug metabolism were reported. HLI reported relative quantities of PD-1, CTLA4, and NYESO-1 from qPCR and provided the patient's Human Leukocyte Antigen (HLA) typing (Tables 1 and 2). Treatment with pazopanib was initiated two months later upon adequate postoperative recovery. A CT scan two months after initiation of pazopanib revealed tumor regrowth in the patient's chest wall/diaphragm and superior mediastinum leading to discontinuation of pazopanib and initiation of pembrolizumab, with palliative radiation therapy (RT) to the right chest wall and right superior mediastinal tumors. The rationale for this therapeutic approach was a report of objective responses to pembrolizumab in adults with UPS (Tawbi et al. 2016), along with early evidence of abscopal effects from combining radiotherapy with PD-1 antibody. Further, immunotherapy was given off-trial because previous studies have shown a rich immune infiltrate in adults with UPS; however, such an infiltrate was not seen in this case.
Two months after initiation of pembrolizumab, following radiation and two doses of pembrolizumab, imaging showed a mixed response with shrinkage of the right chest tumor (within the radiotherapy field) and emergence of a new right lower lobe pulmonary metastasis. The patient underwent resection of the mediastinal, right lower lobe, and right chest wall tumors with en bloc resection of ribs 5-8 and the anterior diaphragm with bioprosthetic mesh reconstruction. Pathology showed complete but close margins, and pembrolizumab was resumed. Two months later, a CT scan showed a new soft tissue mass lateral to the liver near the surgical bed. Pembrolizumab was discontinued and the tumor was treated with 40 Gy of stereotactic body radiation therapy in six fractions. Over the subsequent 7 mo, slowly progressive lung metastases developed, and the patient subsequently was enrolled on a phase I trial of the B7H3-targeted antibody MGA-271. He developed multiple brain and leptomeningeal metastases prompting discontinuation of MGA-271 and initiation of steroids and whole-brain radiotherapy. Imaging confirmed systemic disease progression in the lungs, chest wall, and intrathoracic lymph nodes. Despite improvement in neurologic function, death occurred as a result of respiratory failure related to intrathoracic disease progression.
After the patient's death, autopsy noted extensive UPS metastatic disease involving the heart, diaphragm, right chest wall, small bowel, both kidneys, liver, spleen, and pulmonary hilar lymph nodes. Posthumously, a CIC-rearranged sarcoma diagnosis was later rendered based on the presence of diffuse DUX4 nuclear staining by immunohistochemistry (Fig.  2D), which confirmed the CIC-DUX4 rearrangement identified by NGS and qPCR.

Genomic Analyses
To identify possible genomic mutations and investigate the relationship between pre-and post-treatment tumors, WGS was performed on the tumor samples obtained from the Translation factor eEF1A1 overexpression in a CIC-rearranged sarcoma case C O L D S P R I N G H A R B O R patient. We analyzed tumor and matched normal genome sequencing data for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions/deletions, and gene CNVs. After filtering the list against germline sequence variants, 75 somatic mutations and 16 potential germline mutations were identified. Fourteen pretreatment mutations of interest were identified, and 17 post-treatment genes of interest were identified, with 11 mutations in common (Table 3; Figs. 3 and 4). IQGAP1, CCNC, and ATXN1L were identified as genes bearing high-or medium-impact mutations in the pretreatment sample that were not found in the matched normal genome. RNA sequencing revealed significant up-regulation of VGF, AP1M2, and ETV4 from pretreatment to post-treatment. ETV4 is often up-regulated in CRS and is a frequent diagnostic immunohistochemistry (IHC) tool (Hung et al. 2016). Pretreatment and post-treatment were both 91 nonsynonymous mutations across all exonic regions (Table 4). Gene fusions were also identified using RNA sequencing from the pre-and post-treatment samples. Equivocal evidence was found for the EEF1A1-RPL32 fusion gene in the post-treatment, but not in the pretreatment tumor sample (Figs. 3 and 4; Supplemental Table 2). A CIC-DUX4 fusion could not be called by the standard calling parameters of STAR-Fusion. Raw chimeric read data also showed no evidence of CIC-DUX4 fusion reads (Supplemental Table 2) nor did manual aligned read extraction via "grep" (Panagopoulos et al. 2014). We expanded the search to any reads supporting a fusion between Chromosome 4 (location of DUX4) and Chromosome 19 (location of CIC). All t(4,19) translocation support reads were annotated with biomaRt and GeneLoc (Tables 5 and 6). GH04J119298, a gene promoter annotated in GeneLoc, was proximal to the right breakpoint in several reads. Most other annotated gene pairs bookend the right fusion breakpoint. For example, the breakpoint (190069535) is between GC04P190068 (190068071-190069067) and DUX4L7 (190071232-190072516). Additionally, all t(4,19) fusion reads included CIC as a fusion partner. Although the canonical CIC-DUX4 fusion was not found, support exists for potential CIC-DUX4L7 fusion. These findings point to the potential need to develop a FISH panel for CIC-DUX variant fusions.
As we lacked RNA sequencing from normal adjacent or contralateral tissue, we used the median TPM values of a cohort of skeletal muscle tissues (n = 564) in the Genotype-Tissue Expression (GTEx) project as a comparison. Previous studies have seen up-regulation of genes in the PEA3 family in CIC-DUX4 sarcomas (Kawamura-Saito et al. 2006;Kao et al. 2017). In both the pretreatment and post-treatment samples, we found twofold higher expression levels of PEA3 family genes, such as BAX, CCND2, ETV1, ETV4, ETV5, FGF8, GBX2, ICAM1, MMP2/9, NOTCH1, PLAU, PTK2, SNAI1/2, SPRED2, SPRY2, SPRY4, and TWIST1, relative to the population of skeletal muscle tissues in the GTEx project (Table 7). In the pretreatment sample only, we found MMP1/7 and PTGS2 to have twofold higher expression levels and in the post-treatment sample we found NOTCH4 and SPP1 to have twofold higher expression levels (Table 7). We found twofold higher expression levels of eEF1A1 (13,778 TPM pretreatment and 12,775 TPM post-treatment) and RPL32 (4915 TPM pretreatment and 4168 TPM post-treatment) ( Table 8; Figs. 3 and 4). Moreover, eEF1A1 was found to have twofold higher expression compared to the median expression level of all nontumorous tissues sequenced in the GTEx project. Further, for most samples in our cohort, eEF1A1 expression of non-rhabdomyosarcomas soft tissue sarcomas (NRSTS) and rhabdomyosarcomas was found to be higher than that of nontumorous tissues (Fig. 5). Embryonal rhabdomyosarcoma (ERMS) cell lines Ruch2, Ruch3, SCMC-RMS, and RMS-YM, were found to have lower eEF1A1 expression potentially because of a difference in sequencing platforms used. Thus, further investigation into the role of eEF1A1 in multiple childhood cancers is warranted.
To validate the expression levels seen in RNA sequencing across several childhood cancers we immunoblotted for EEF1A1 in NRSTS (n = 4) and RMS (n = 7) biopsy samples (Fig. 6A) and cell lines (Fig. 6B). In the absence of CIC-rearranged sarcoma (CRS) biopsy samples, UPS Translation factor eEF1A1 overexpression in a CIC-rearranged sarcoma case   and epithelioid sarcoma (ES) biopsy samples and cell lines were used to represent NRSTS cases (Tables 9 and 10). RMS samples and the ES sample both had expression similar to or higher than normal skeletal muscle tissues and the human skeletal muscle myoblast (HSMM) cell line. The two UPS biopsy samples demonstrated lower expression than normal skeletal muscle, whereas the UPS cell line exhibited a similar or slightly lower expression level compared to HSMM. More NRSTS samples-in particular, more CRS samples-should be examined to provide a more accurate representation of the disease.
To further classify the molecular features of the pre-and post-treatment tumors in the context of sarcoma, we used unsupervised clustering to determine RMS and NRSTS endotypes (subgroups of samples with distinct pathobiological mechanisms). We used RNA and DNA from RMS and NRSTS patient samples (n = 53), patient-derived xenografts (n = 6), and cell lines (n = 16). In hierarchical clustering of both somatic mutations and gene expression, the patient clusters closest to CTG-1542, a PDX model of metastatic ERMS (Fig. 7). Most cell lines with both RNA-seq and WES available developed their own cluster, most likely because of a lack of a matched-normal sample resulting in several false positive mutations. Interestingly, in the hierarchical clustering of gene expression alone, EEF1A1 was found to have the highest gene expression level across the entire cohort (Fig. 8). Hierarchical clustering of gene expression alone identified CLF-PED-015-T (pediatric UPS) (Hong et al. 2016) to be in the endotype of the patient's pre-and post-treatment samples (Fig. 8). Parenthetically, if CRS cell lines had been available, CLF-PED-015-T may have been less closely related-pointing to the need to develop CRS cell lines. Given the resources available to us, we analyzed the tumor and matched normal sequencing data from CLF-PED-015-T using the same analysis pipeline and methods as the patient. We found eEF1A1 to also have twofold higher expression (9891 TPM) in the primary cell line compared to the population of skeletal muscle tissues from GTEx Translation factor eEF1A1 overexpression in a CIC-rearranged sarcoma case

Molecular Case Studies
Portal (n = 564, median TPM = 207) (Table 11). Additionally, we saw overexpression of ETV4 (1608 TPM), which is frequently used as a diagnostic tool for CRS cases (Hung et al. 2016). Given the biological similarities, immunohistochemistry to test for the presence of diffuse DUX4 nuclear staining in CLF-PED-015-T could be warranted. Because the patient's pre-and post-treatment samples were found to have similar molecular features to CLF-PED-015-T and a cell line has not been developed from the patient's tumor sample, CLF-PED-015-T might serve as a surrogate model in the absence of any CRS cell lines.

DISCUSSION
In this case study, pre-and post-treatment tumor DNA whole-genome samples were sequenced and analyzed against matched normal samples, as well as RNA sequencing of pre-and post-treatment tumor tissue. Sequencing revealed overexpression of the eEF1A1 translational factor, which is of special interest in this case. eEF1A1, an isoform of the α subunit of the eukaryotic Elongation Factor 1, is commonly overexpressed in human tumors and has oncogenic properties that favor tumor cell proliferation and inhibit apoptosis. eEF1A1 is reported to be a negative regulator p53 and p73 and leading to chemoresistance (Blanch et al. 2013), and the ubiquitin-like protein FAT10 has been shown to bind eEF1A1 which stabilizes the expression of eEF1A1 in favor of tumor cell proliferation (Liu et al. 2016). The isoform eEF1A1 shares 96% homology with the isoform eEF1A2, the primary target of plitidepsin (dehydrodidemnin B/Aplidin, PharmaMar), an antitumor agent of marine origin  (Losada et al. 2016). eEF1A isoforms were the only proteins detected as capable of binding plitidepsin, and sensitivity was dependent on eEF1A2 levels. Plitidepsin has completed phase III clinical trials for multiple myeloma, but more studies are necessary to validate the interaction between plitidepsin and eEF1A1 and the oncogenic properties and effectiveness of plitidepsin. A major limitation of this work is the absence of a patient-derived cell model for phenotypic screening of plitidepsin and other agents of interest. Coclinical trials allow for parallel or sequential screening of human primary tumors in mouse models. Particularly for cases where there is little clinically actionable molecular information, development of coclinical methods for phenotypic screening of primary tumors samples using "endotype" surrogate cell lines would help us understand the tumors' response to therapeutic agents. Our work presents evidence of a potential therapeutic target, advances our knowledge of an understudied Clinically, at least three of the four pathologists whom did not identify CRS in this patient had training in and are well-published in sarcomas. However, CRS is rare and has only recently been identified as their own distinct subgroup, making them challenging to identify. Given that CRS is a new entity that can go unrecognized, increased scrutiny of round cell histology undifferentiated sarcomas and development of confirmatory tools such as methylation profiling or FISH for variant CIC-DUX4-like fusions appears warranted.
Previous studies have also had difficulty identifying the CIC-DUX4 fusion gene using several fusion callers (Panagopoulos et al. 2014). A possible explanation for the failure in the fusion callers' ability to identify the CIC-DUX4 fusion gene is the large number of DUX4 pseudogenes with very similar sequences (e.g., concordance in 1283 of the first 1285 nucleotides in DUX4 and DUX4L7, which represents 1285 of the 1553 base pairs of DUX4 Exon 1, noted to be the location of most CIC-DUX4 fusions). Despite several fusion callers with multiple calling parameters failing to call CIC-DUX4, other fusion calling tools may be able to identify CIC-DUX4. A multifusion caller pipeline may be warranted to capture this challenging gene fusion, but consensus calling approaches will still be limited by the challenge in calling CIC-DUX4.
Here, we have demonstrated a case of an 11-yr-old male presented with a soft tissue mass that was later identified to be CRS. Using RNA sequencing from pretreatment and post-treatment tumor samples we have found an abundance of PEA3 family genes overexpressed, as well as eEF1A1 translational factor. Given the overexpression of eEF1A1 in this aggressive and severe NRSTS (CIC-rearranged sarcoma) the availability of plitidepsin as an eEF1A1/2 anti-tumor agent is potentially interesting. Our observations could warrant further in vivo preclinical studies to test whether eEF1A over-expression in CRS and other NRSTS implies sensitivity to plitidepsin. Such studies would likely be predicated on detection of eEF1A1/2 over-expression observed across a meaningfully large NRSTS subpopulation.

Molecular Case Studies
Whole-Genome Sequencing Tumor and matched normal genome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene CNVs. Sequence reads were aligned to the GRCh38 human reference genome and somatic mutations and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with stricter calling criterion (Tumor logarithm of odds [TLOD] scores >6.3). Gene CNVs were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio >0.60 were called as gained, regions with a log ratio <−0.60 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage of the pretreatment and post-treatment samples are noted in Supplemental Table 3.

RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to the GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Non-tumor gene expression data was not provided, thus region-specific unmatched skeletal muscle tissue gene expression data was accessed from the GTEx project to serve as a population normal and to identify underexpressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion. Sequencing coverage of the pretreatment and post-treatment samples are noted in Supplemental Table 3.

Western Blot Analysis
Cells were extracted in RIPA lysis buffer (ThermoFisher), supplemented with complete protease inhibitor and phosphatase inhibitor cocktail (ThermoFisher). After incubation on ice for 1 h, samples were centrifuged at 13,000g for 20 min at 4°C. Protein samples were separated in SDS-PAGE gel and then transferred onto a 0.2-mm polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane using wet transfer method at 90 V for 90 min. Primary antibodies anti-EF1α (Millipore #05-235) and anti-GAPDH (Cell Signaling Cat #2118S) were used with 1:1000 and 1:10,000 dilutions, respectively. Secondary antibodies HRP goat anti-rabbit (Vector Cat# PI-1000) and HRP horse anti-mouse (Vector Cat# PI-2000) were both diluted at 10,000. Proteins were detected using IVIS Lumina Imaging System.

ADDITIONAL INFORMATION Data Deposition and Access
Raw data was deposited to our CuReFAST database, Foundation Medicine, and Human Longevity Inc. and can be accessed upon request. Raw sequencing data from our biopsy samples and cell lines were deposited to Gene Expression Omnibus (GEO) and European Genome-phenome Archive (EGA) under accession numbers GSE138269 and EGAS00001003981. The RNA and DNA sequencing data from this patient's normal and tumor samples will be submitted separately to EGA by the laboratory of Dr. E. Alejandro Sweet-Cordero (UCSF). Variants found from this case have been submitted to ClinVar (https://www .nlm.ncbi.nih.gov/clinvar/) under accession numbers SCV001146981-SCV001146999.

Ethics Statement
Genetic studies and publication of clinical details were Institutional Review Board (IRB)approved, and full written informed consent was obtained from all participants. The parents provided consent for the proband.

Acknowledgments
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/). Translation factor eEF1A1 overexpression in a CIC-rearranged sarcoma case N.E.B. was supported by research funding in memory of Ava Sentell. We greatly appreciate the participation and kind tissue donation of the patient and we thank the family for facilitating helpful discussion and participation in the study. We are also tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative. We acknowledge those that have contributed to our sample collection: Dr. Marielle E. Yohe from the National Cancer Institute for providing the embryonal rhabdomyosarcoma cell line, CCA; Dr. Jonathon A. Fletcher from Brigham and Women's Hospital for providing the embryonal rhabdomyosarcoma cell line, RMS559; the Jackson Laboratory for providing WES and RNA sequencing data from PDX models; and the Gene Expression Omnibus (GEO) for sharing WES and RNA sequencing data form cell lines. We thank Dr. Atiya Mansoor, Dr. Amit Sabnis, and Dr. E. Alejandro Sweet-Cordero for providing their time and expertise.