Ewing Sarcoma Breakpoint Research Articles

Desmoplastic small round cell tumor with sphere‐like clusters mimicking adenocarcinoma

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive neoplasm that predominantly affects young men. DSRCT often presents as multiple nodules on the serosal surface and is histologically categorized as a small round cell tumor. However, the cytological spectrum of DSRCT is not fully understood because of its rarity. Here, we report an unusual case of DSRCT that showed spheres of cells without stromal cores in pleural fluid cytology material, a finding that is typically associated with metastatic adenocarcinoma and mesothelioma. The specimen from a simultaneous needle biopsy showed the classic histology of DSRCT, comprising nests of small round cells set in desmoplasia. The diagnosis of DSRCT was further supported by immunohistochemical coexpression of cytokeratin and desmin, as well as Ewing sarcoma breakpoint region 1 gene rearrangement, which was determined by fluorescence in situ hybridization. The unusual cytological finding in this case illustrates a potential pitfall of the cytological diagnosis of pleural fluid or ascites. DSRCT should not be excluded from the differential diagnosis when sphere-like round cell clusters are observed in pleural or abdominal effusion, particularly in young male patients.

EWSR1 and ATF1 rearrangements in clear cell odontogenic carcinoma: presentation of a case

Clear cell odontogenic carcinoma (CCOC) is a rare odontogenic tumor of the jaws that is more common in the mandible than maxilla and has a female preponderance with a peak incidence in the sixth decade. It is characterized by locally aggressive behavior and has the potential to metastasize. This tumor was recently reported to have a rearrangement of the Ewing sarcoma breakpoint region 1 gene (EWS RNA-binding protein 1, EWSR1) in 5 of 8 cases tested and of the activating transcription factor 1 gene (ATF1) in 1 case tested. We report a case of CCOC in the premolar area of the mandible in a 59-year-old woman. This case demonstrated the presence of both EWSR1 and ATF1 gene rearrangements by fluorescence in situ hybridization.

Extraskeletal myxoid chondrosarcoma with non-EWSR1-NR4A3 variant fusions correlate with rhabdoid phenotype and high-grade morphology.

Extraskeletal myxoid chondrosarcomas (EMC) are rare soft tissue sarcomas with distinctive histology and uncertain histogenesis, characterized by Ewing sarcoma breakpoint region 1-nuclear receptor subfamily 4, group A, member 3 (EWSR1-NR4A3) fusion in 75% of the cases. A smaller proportion of cases show NR4A3 fused to other gene partners including TATA binding protein-associated factor 15 (TAF15), transcription factor 12 (TCF12), and TRK-fused gene (TFG). The impact of various gene fusions on morphology and outcome has not been previously evaluated. We investigated 26 consecutive EMCs and correlated the genetic findings with morphology and clinical outcome. There were 5 females and 21 males (median age, 49.5 years). Mean size of the tumors was 11 cm. Fluorescence in situ hybridization analysis showed EWSR1-NR4A3 gene fusion in 16 cases (62%), TAF15-NR4A3 gene fusion in 7 cases (27%), and TCF12-NR4A3 gene fusion in 1 case (4%). Two cases showed only NR4A3 gene rearrangements. Morphologically, most EWSR1-rearranged tumors (10/16) showed low cellularity, minimal cytologic atypia, and low mitotic counts. In contrast, 80% of EMCs with variant (non-EWSR1) NR4A3 gene fusions (TAF15, TCF12) had high-grade morphology with increased cellularity, proliferation, and cytologic atypia, showing a plasmacytoid/rhabdoid morphology in half the cases. Follow-up showed that only 1 of 16 patients with EWSR1-rearranged tumors died of disease, in contrast to 3 (43%) of 7 TAF15-rearranged tumors. In conclusion, EMCs with variant NR4A3 gene fusions show a higher incidence of rhabdoid phenotype, high-grade morphology, and a more aggressive outcome compared with the EWSR1-NR4A3 positive tumors. Furthermore, fluorescence in situ hybridization assay for NR4A3, along with EWSR1, may be an additional ancillary test to confirm diagnosis of EMCs.

3′-UTR Poly(T/U) Tract Deletions and Altered Expression of EWSR1 Are a Hallmark of Mismatch Repair–Deficient Cancers

The genome-wide accumulation of DNA replication errors known as microsatellite instability (MSI) is the hallmark lesion of DNA mismatch repair (MMR)-deficient cancers. Although testing for MSI is widely used to guide clinical management, the contribution of MSI at distinct genic loci to the phenotype remains largely unexplored. Here, we report that a mononucleotide (T/U)16 tract located in the 3' untranslated region (3'-UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene is a novel MSI target locus that shows perfect sensitivity and specificity in detecting mismatch repair-deficient cancers in two independent populations. We further found a striking relocalization of the EWSR1 protein from nucleus to cytoplasm in MMR-deficient cancers and that the nonprotein-coding MSI target locus itself has a modulatory effect on EWSR1 gene expression through alternative 3' end processing of the EWSR1 gene. Our results point to a MSI target gene-specific effect in MMR-deficient cancers.

Congenital Extrarenal Extra-Central Nervous System Malignant Rhabdoid Tumor of the Upper Extremity: A Case Report.

Malignant rhabdoid tumor (MRT) is an extremely rare and highly aggressive tumor that occurs in infancy or childhood1. MRT was first described as a rhabdomyosarcomatoid variant of Wilms tumor because of its histologic resemblance to rhabdomyoblasts and its most frequent presenting location as a renal tumor2,3. Since then, MRT has also been found in the central nervous system (CNS), ileum, lung, heart, liver, skin, and intestines4-7. We present a rare case of congenital extrarenal extra-CNS MRT of the forearm in an infant with an aggressive course and poor outcome. The patient’s parents were informed that data concerning the case would be submitted for publication, and they provided consent. A four-month-old otherwise well baby girl had presented shortly after birth with a 0.5-cm area of bluish discoloration over the volar aspect of the left forearm. This discoloration had remained stable until three months of age, when this area began to rapidly enlarge and become more discolored. Ultrasound revealed a large heterogeneous soft-tissue mass in the forearm with areas of cystic change consistent with an infantile hemangioma versus sarcoma. Computed tomography (CT) of the chest showed left axillary lymphadenopathy. The patient underwent CT-guided needle biopsy of the lesion, which measured 7.8 cm × 3.4 cm. Histologic evaluation and immunohistochemical studies of the biopsy were demonstrative of a high-grade sarcoma, consistent with MRT (Figs. 1-A through 1-D⇓). Fluorescent in situ hybridization (FISH) was performed. Because it was negative for the Ewing sarcoma breakpoint region, the break-apart FISH ruled out Ewing and desmoplastic small round cell tumor; because it was negative for the FKHR gene, the break-apart FISH ruled out alveolar rhabdomyosarcoma. Karyotyping for the chromosomal t(X;18) translocation was negative, thus ruling out synovial sarcoma. Subsequent staging evaluation included bilateral bone marrow aspirate …

Compound clear cell sarcoma misdiagnosed as a Spitz nevus

Clear cell sarcoma (CCS) typically presents as a tumor in the deep soft tissue of extremities, but when centered in the dermis it may be confused with a melanocytic nevus, primary nodular or metastatic melanoma. Compound variants of CCS, i.e. tumor cells present in both the epidermis and underlying soft tissue have not yet been described. Herein we report such a case, which initially presented as a nodule on the left wrist of a young woman at 19 years of age. The lesion was then interpreted as 'Spitz nevus, compound type'. Twelve years later the patient noticed an enlarged lymph node in the right axilla. The excised lymph node was nearly completely replaced by malignant tumor cells, which were immunoreactive for S100 protein. They resembled the tumor cells of the wrist lesion. Cytogenetic analysis of the metastatic tumor revealed a t(12;22) translocation. Fluorescence in situ hybridization confirmed Ewing's sarcoma breakpoint region 1 (EWSR1) rearrangement in 70% of the tumor cells, thereby supporting the diagnosis of metastatic CCS. Our case is of interest because it documents that CCS can involve the epidermis. This observation expands the morphological spectrum associated with this tumor.

Ewing's Sarcoma/Primitive Neuroectodermal Tumor Arising from the Adrenal Gland: A Case Report and Literature Review

We report a rare case of Ewing's sarcoma (ES)/primitive neuroectodermal tumor (PNET) arising from the adrenal gland. A 17-year-old Japanese woman presented with left upper abdominal pain and high fever. Computed tomography and magnetic resonance imaging revealed a 15 × 10 cm tumor replacing the adrenal gland. Preoperative diagnosis was an adrenocortical carcinoma. Resection of the tumor was performed. We obtained the final diagnosis of ES/PNET by immunohistochemical molecular study with positive staining for the MIC2 gene product (CD99) and a Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangement. Local recurrence was observed one month after the surgery. The patient was then treated with systemic chemotherapy and localized radiotherapy.

Abstract 1910: 3′UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers.

Abstract Background: Microsatellite instability (MSI), the genome-wide accumulation of DNA replication errors, is the hallmark lesion of DNA mismatch repair (MMR) deficient cancers, present in Lynch syndrome (LS)-related and 10-20% of sporadic colorectal (CRC), gastric and endometrial cancers. MSI testing is widely used to guide clinical management but the functional significance of MSI at distinct genic loci remains largely elusive. Here, we characterize a novel MSI target locus consisting of a mononucleotide (T/U)16 tract located in the 3’untranslated region (UTR) of the Ewing sarcoma break point region 1 (EWSR1) gene (EWS16T). Methods: The diagnostic accuracy of EWS16T to identify MMR-deficient cancers was determined by analyzing 319 cancers (240 colorectal, 72 gastric, 7 endometrial) from two different populations. The functional consequences of EWS16T contractions were assessed in vitro (siRNA-mediated poly(A) site selection and pull-down assays) and in vivo (mRNA and protein expression by qPCR and tissue microarray analysis). Results: The EWS16T locus discriminates MMR-proficient (n=128) from deficient (n=191) cancers with perfect diagnostic sensitivity (100%) and specificity (100%). Biochemical analyses indicate that EWS16T contractions alter poly(A) site selection by promoting SFPQ-mediated distal poly(A) site usage in EWSR1 pre-mRNAs and result in decreased mRNA as well as EWS protein expression. In contrast to their MMR-proficient counterparts (n=64), MMR-deficient, LS-related CRC (n=94) display altered subcellular localization of EWS (p<0.001). Conclusions: The EWS16T locus represents a novel, quasi-monomorphic MSI target locus to accurately identify both hereditary and sporadic MMR-deficient cancers. Contractions therein affect multiple regulatory mechanisms implicating the RNA-/DNA-binding Ewing sarcoma protein in MSI-associated colorectal tumorigenesis. Citation Format: Salvatore Piscuoglio, Shivendra Kishore, Michal Kovac, Annette Gylling, Friedel Wenzel, Francesca Trapani, Hans Joerg Altermatt, Valentina Mele, Giancarlo Marra, Päivi Peltomaki, Luigi Terracciano, Mihaela Zavolan, Karl Heinimann. 3′UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2013-1910

EWSR1 Genetic Rearrangements in Salivary Gland Tumors

The Ewing sarcoma breakpoint region 1 (EWSR1) is translocated in many sarcomas. Recently, its rearrangement has been described in salivary gland hyalinizing clear cell carcinomas (HCCCs) and in a subset of soft tissue myoepitheliomas. This study examines the presence of the EWSR1 rearrangement in a variety of salivary gland lesions including classic myoepitheliomas and HCCCs. Using a tissue microarray and whole-mount sections, fluorescence in situ hybridization (FISH) was performed on a variety of salivary gland lesions including HCCCs. The EWSR1 rearrangement was detected in 87% of HCCCs (13 of 15); all other salivary gland lesions including classic myoepitheliomas had intact EWSR1. Patients with HCCCs with rearranged EWSR1 included 1 man, 10 women, and 2 of unknown sex. Ages ranged from 35 to 83 years; the tumor size ranged from 0.8 to 5.5 cm, and the involved locations included: palate (2), base of the tongue (2), mandible (2), submandibular gland (2), lip (1), floor of the mouth (1), sublingual gland (1), inner cheek (1), and nasopharynx (1). All HCCCs were composed of sheets and nests of monotonous cells with clear cytoplasm within a hyalinized stroma. All tested cases were immunoreactive with antibodies to p63 and were nonreactive with antibodies to more conventional myoepithelial antigens (e.g., smooth muscle actin and S100 protein). These findings show that the EWSR1 rearrangement is almost a defining feature of HCCCs and also confirm that classic salivary gland myoepitheliomas are distinct from these tumors and do not share a pathogenetic relationship with their soft tissue counterparts.

Molecular detection and targeting of EWSR1 fusion transcripts in soft tissue tumors

Soft tissue tumors are a heterogeneous group of tumors, traditionally classified according to morphology and histogenesis. Molecular classification divides sarcomas into two main categories: (a) sarcomas with specific genetic alterations and (b) sarcomas showing multiple complex karyotypic abnormalities without any specific pattern. Most chromosomal alterations are represented by translocations which are increasingly detected. The identification of fusion transcripts, in fact, not only support the diagnosis but also provides the basis for the development of new therapeutic strategies aimed at blocking aberrant activity of the chimeric proteins. One of the genes most susceptible to breakage/translocation in soft tissue tumors is represented by Ewing sarcoma breakpoint region 1 (EWSR1). This gene has a large number of fusion partners, mainly associated with the pathogenesis of Ewing’s sarcoma but with other soft tissue tumors too. In this review, we illustrate the characteristics of this gene/protein, both in normal cellular physiology and in carcinogenesis. We describe the different fusion partners of EWSR1, the molecular pathways in which is involved and the main molecular biology techniques for the identification of fusion transcripts and for their inhibition.

Characterization of Ewing sarcoma associated cancer/testis antigens

The prognosis of patients suffering from tumors of the Ewing family (EFT) is still poor. Immunotherapy strategies are pursued and EFT-specific antigens have to be identified as targets for cytotoxic T-lymphocytes (CTL). Due to the lack of expression of cancer/testis antigens (CTA) in normal tissues, these antigens are partially able to induce immune responses in cancer patients. Therefore, they are promising targets for immunotherapy. EFT are characterized by chromosomal rearrangements involving members of the TET (translocated in liposarcoma, Ewing sarcoma breakpoint region 1, TATA box binding protein-associated factor 15) family of RNA binding proteins and members of the E-26 (ETS) family of transcription factors. The resulting onco-fusion proteins are highly specific for EFT and downstream targets of TET-ETS represent candidate tumor specific antigens. In order to identify new EFT-associated CTA, we analyzed microarray-data sets from EFT and normal tissues from the Gene Expression Omnibus (GEO) database. The impact of TET-ETS on expression of CTA was analyzed using GEO data sets from transgenic mesenchymal stem cells. One CTA with high specificity for EFT is lipase I (LIPI, membrane-associated phospholipase A1-β). CTL specific for LIPI-derived peptides LDYTDAKFV and NLLKHGASL were able to lyse HLA-A2 positive EFT cells in vitro which confirms the possible role of LIPI and other CTA for EFT-immunotherapy.

Peripheral Primitive Neuroectodermal Tumor with Osseous Component of the Small Bowel Mesentery: A Case Study

A case of peripheral primitive neuroectodermal tumor of the small bowel mesentery with osseous component is reported. A 23-year-old man was admitted to our hospital because of acute severe abdominal pain. Abdominal computed tomography revealed a large solid and cystic, oval shaped mass, measuring 11.0×6.0 cm in the pelvic cavity. Histologically the resected lesion consisted of sheets of undifferentiated small round cells forming Homer-Wright rosettes and perivascular pseudorosettes, and showed areas of osteoid and bone formation. Immunohistochemical studies revealed that tumor cells expressed positivity against CD99 (MIC2), CD57, neuron-specific enolase, and vimentin. Fluorescence in situ hybridization study revealed Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangement on chromosome 22q12. To the authors' knowledge this is the first documentation of a peripheral neuroectodermal tumor with osteoid and bone formation of the small bowel mesentery.

Small cell osteosarcoma with Ewing sarcoma breakpoint region 1 gene rearrangement detected by interphase fluorescence in situ hybridization

Because of its characteristic morphologic appearance, small cell osteosarcoma (SCO) can be confused with other small round cell malignancies of the bone, most importantly with Ewing sarcoma, making this distinction difficult. A specific tool used in separating SCO from Ewing sarcoma has been the detection of Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangements in Ewing sarcoma and their absence in SCO. However, there are rare case reports that have documented the existence of EWSR1 gene rearrangement in SCO. In this report, we describe another case of SCO with an EWSR1 gene rearrangement detected by interphase fluorescence in situ hybridization. Our finding adds support to the existing evidence that SCO is a tumor that can be characterized by EWSR1 gene arrangements. Therefore, we caution the pathology community not to rely solely on molecular studies in distinguishing SCO from Ewing sarcoma.

Primitiver neuroektodermaler Tumor im Hoden

We describe a case of a testicular primitive neuroectodermal tumor (PNET) with intratubular germ cell neoplasia of the adjacent testicular parenchyma. The occurrence of testicular PNET is rare because malignant transformation of testicular germ cell tumors into somatic malignancy is uncommon. Based on morphological, immunohistochemical and molecular pathological findings these tumors resemble central PNETs as they otherwise typically occur in children without rearrangement of the Ewing sarcoma breakpoint region (EWSR) gene on chromosome 22. This case also showed no evidence for a translocation.

Extraosseous Ewing sarcoma with foci of neuroblastoma-like differentiation associated with EWSR1(Ewing sarcoma breakpoint region 1)/FLI1 translocation without prior chemotherapy

Peripheral primitive neuroectodermal tumor/Ewing sarcoma and neuroblastoma are distinct malignant tumors belonging to the group of undifferentiated solid pediatric tumors. We report a case of a 14-year-old adolescent girl who presented with a right lower quadrant mass. At surgery, a mobile retroperitoneal mass was entirely removed. Histologic evaluation revealed 2 distinct components; the first, consisting of sheets of undifferentiated cells, was CD99+ and CD56-, whereas the second, consisting of multiple foci of neuropil and maturing neuroblasts, was CD99- and CD56+. Fluorescence in situ hybridization analysis revealed the presence of EWSR1/FLI1 translocation in both histologic distinct components. MYCN (myelocytomatosis viral related oncogene, neuroblastoma derived) was not amplified. The tests for t(11;22) and t(21;22) performed by reverse transcription-polymerase chain reaction were negative. The final diagnosis corresponds to an extraosseous Ewing sarcoma with foci of neuroblastoma-like differentiation. This is the first case, documented by molecular studies, in which neuroblastoma-like differentiation has been noted in primitive neuroectodermal tumor/Ewing sarcoma without prior chemotherapy.

Molecular investigation of Ewing sarcoma: about detecting translocations.

Molecular investigation of Ewing sarcoma: about detecting translocations.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

EWSR1 (Ewing sarcoma breakpoint region 1)

Review on EWSR1 (Ewing sarcoma breakpoint region 1), with data on DNA, on the protein encoded, and where the gene is implicated.

A novel EWSR1‐CREB3L1 fusion transcript in a case of small cell osteosarcoma

Cellular morphology of small cell osteosarcoma, an aggressive variant of osteosarcoma, is similar to Ewing sarcoma, but its molecular pathogenesis is largely unknown. We report the case of a 12-year-old girl with multifocal small cell osteosarcoma positive for the Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangement by interphase fluorescent in situ hybridization and negative for EWSR1-FLI1, EWSR1-ERG, and EWSR1-WT1 fusion transcripts by reverse transcriptase PCR. Rapid amplification of cDNA ends revealed exon 6 of the cAMP-responsive element binding protein 3-like 1 gene (CREB3L1, also known as "OASIS," NM_52854.2) fused in-frame to the EWSR1 exon 11, consistent with the EWSR1-CREB3L1 fusion transcript expressed in tumor tissue. The corresponding chimeric gene was confirmed by amplification and subsequent sequencing of the genomic breakpoint between introns 11 and 5 of EWSR1 and CREB3L1, respectively. An ∼70 kDa product in the tumor tissue lysate reacted with the CREB3L1 carboxyterminal antibody, consistent with a 656-amino acid predicted chimeric protein. Immunohistochemistry with the same antibody showed signal translocation from the physiologic perinuclear compartment observed in glia and unrelated osteoblasts to nuclei of tumor cells, consistent with the likely function of EWSR1-CREB3L1 as a transcriptional regulator predicted by its structure. This is the first report of a fusion transcript in osteogenic sarcoma; it demonstrates a relation between molecular mechanisms of small cell osteogenic and Ewing sarcomas. The 3'-end partner and the inferred structure of EWSR1-CREB3L1, however, are different from those of Ewing sarcoma, suggesting different targets of the new oncogene.

Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening

Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable." We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided. Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P < .001). Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.