Ewing's Sarcoma - Molecular Biology

Overview

Over 90% of Ewing's sarcomas contain a t(11;22)(q24;q12) translocation which fuses the EWS gene on chromosome 22 with the FLI1 gene on chromosome 11. This genetic feature defines the Ewing's family of tumours (Ewing's sarcomas, peripheral primitive neuroectodermal tumours (pPNET) and Askin's tumours). In a minority of Ewing's tumours the EWS gene has a different fusion partner including either the ERG, ETV1 and the EA1F and genes. The Ewing's family of tumours also strongly express the MIC2 (CD99) antigen.

See also: Ewing's Sarcoma - clinical resources (18)

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 08 August, 2015 using data from PubMed, MeSH and CancerIndex

Mutated Genes and Abnormal Protein Expression (35)

How to use this data tableClicking on the Gene or Topic will take you to a separate more detailed page. Sort this list by clicking on a column heading e.g. 'Gene' or 'Topic'.

GeneLocationAliasesNotesTopicPapers
EWSR1 22q12.2 EWS, bK984G1.4 Translocation
Prognostic
-t(21;22) EWSR1-ERG Translocations in Ewing's Sarcoma
-t(11;22)(q24;q12) EWSR1-FLI1 Translocation in Ewing's Sarcoma
-EWSR1-FLI1 Fusion Transcript Structure and Prognosis?
-t(17;22)(q12;q12) EWSR1-E1AF Translocation in Ewing's Sarcoma
- t(7;22)(p22;q12) EWS-ETV1 Translocation in Ewing's Sarcoma
-t(2;22) EWSR1-FEV Translocation in Ewing's Sarcoma
-t(20;22) EWSR1-NFATC2 in Ewing's Sarcoma
286
FLI1 11q24.1-q24.3 EWSR2, SIC-1 Translocation
Prognostic
-t(11;22)(q24;q12) EWSR1-FLI1 Translocation in Ewing's Sarcoma
-EWSR1-FLI1 Fusion Transcript Structure and Prognosis?
286
ERG 21q22.3 p55, erg-3 Translocation
-t(21;22) EWSR1-ERG Translocations in Ewing's Sarcoma
-ERG and Ewing's Sarcoma
96
TP53 17p13.1 P53, BCC7, LFS1, TRP53 Overexpression
Prognostic
-TP53 Mutations and aberrant expression in a sub-set of Ewing's Sarcoma
31
ETV1 7p21.3 ER81 Translocation
- t(7;22)(p22;q12) EWS-ETV1 Translocation in Ewing's Sarcoma
24
CDKN2A 9p21 ARF, MLM, P14, P16, P19, CMM2, INK4, MTS1, TP16, CDK4I, CDKN2, INK4A, MTS-1, P14ARF, P19ARF, P16INK4, P16INK4A, P16-INK4A Deletion
Prognostic
-CDKN2A Deletion in Ewing's Sarcoma
22
FEV 2q36 PET-1, HSRNAFEV Translocation
-t(2;22) EWSR1-FEV Translocation in Ewing's Sarcoma
-FEV and Ewing's Sarcoma
16
NR0B1 Xp21.3 AHC, AHX, DSS, GTD, HHG, AHCH, DAX1, DAX-1, NROB1, SRXY2 -NR0B1 and Ewing's Sarcoma
10
ETV4 17q21 E1AF, PEA3, E1A-F, PEAS3 Translocation
-t(17;22)(q12;q12) EWSR1-E1AF Translocation in Ewing's Sarcoma
-ETV4 and Ewing's Sarcoma
8
CCK 3p22.1 -CCK Expression in Ewing's Sarcoma
7
TGFBR2 3p22 AAT3, FAA3, LDS2, MFS2, RIIC, LDS1B, LDS2B, TAAD2, TGFR-2, TGFbeta-RII Underexpression
-TGFBR2 Suppression by Ewing's Sarcoma EWS/FLI1 Fusion Genes
6
ID2 2p25 GIG8, ID2A, ID2H, bHLHb26 -Upregulation of ID2 in Ewing's Sarcoma
6
GLI1 12q13.2-q13.3 GLI Overexpression
-GLI1 upregulation by EWSR1/FLI1 in Ewing's Sarcoma
6
TAF15 17q11.1-q11.2 Npl3, RBP56, TAF2N, TAFII68 -TAF15 and Ewing's Sarcoma
5
DUX4L1 4q35 DUX4, DUX10 -DUX4 and Ewing's Sarcoma
5
LIF 22q12.2 CDF, DIA, HILDA, MLPLI -LIF and Ewing's Sarcoma
5
BCOR Xp11.4 MAA2, ANOP2, MCOPS2 -BCOR and Ewing's Sarcoma
4
NFATC2 20q13.2 NFAT1, NFATP Translocation
-t(20;22) EWSR1-NFATC2 in Ewing's Sarcoma
4
SH2D1B 1q23.3 EAT2 Overexpression
-SH2D1B (EAT2) is Upregulated by Ewing's Sarcoma EWS/FLI1 Fusion Genes
4
SKP2 5p13 p45, FBL1, FLB1, FBXL1 -SKP2 and Ewing's Sarcoma
3
MCF2 Xq27 DBL, ARHGEF21 -MCF2 and Ewing's Sarcoma
3
NKX2-2 20p11.22 NKX2B, NKX2.2 -NKX2-2 and Ewing's Sarcoma
3
ERBB3 12q13 HER3, LCCS2, ErbB-3, c-erbB3, erbB3-S, MDA-BF-1, c-erbB-3, p180-ErbB3, p45-sErbB3, p85-sErbB3 -ERBB3 and Ewing's Sarcoma
3
FOXO4 Xq13.1 AFX, AFX1, MLLT7 -FOXO4 and Ewing's Sarcoma
3
STAG2 Xq25 SA2, SA-2, SCC3B, bA517O1.1 -STAG2 and Ewing's Sarcoma
2
FOXM1 12p13 MPP2, TGT3, HFH11, HNF-3, INS-1, MPP-2, PIG29, FKHL16, FOXM1B, HFH-11, TRIDENT, MPHOSPH2 Overexpression
-FOXM1 upregulation by EWSR1/FLI1 in Ewing's Sarcoma
2
PATZ1 22q12.2 ZSG, MAZR, PATZ, RIAZ, ZBTB19, ZNF278, dJ400N23 -PATZ1 and Ewing's Sarcoma
2
SOX6 11p15.3 SOXD, HSSOX6 Amplification
-SOX6 Amplification in Ewing's Sarcoma?
1
EGR2 10q21.1 AT591, CMT1D, CMT4E, KROX20 -EGR2 and Ewing's Sarcoma
1
ZNF384 12p12 NP, CIZ, NMP4, CAGH1, ERDA2, TNRC1, CAGH1A -ZNF384 and Ewing's Sarcoma
1
SERPINC1 1q25.1 AT3, AT3D, ATIII, THPH7 -SERPINC1 and Ewing's Sarcoma
1
PTEN 10q23.3 BZS, DEC, CWS1, GLM2, MHAM, TEP1, MMAC1, PTEN1, 10q23del Amplification
-PTEN Amplification in Ewing's Sarcoma?
1
DGCR8 22q11.2 Gy1, pasha, DGCRK6, C22orf12 -DGCR8 and Ewing's Sarcoma
1
DROSHA 5p13.3 RN3, ETOHI2, RNASEN, RANSE3L, RNASE3L, HSA242976 -DROSHA and Ewing's Sarcoma
1
CD99 Xp22.32 and Yp11.3 MIC2, HBA71, MIC2X, MIC2Y, MSK5X Overexpression
-MIC2 expression in the Differential Diagnosis of Ewing's Sarcoma

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Research Publications

Svoboda LK, Harris A, Bailey NJ, et al.
Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epigenetic regulation of developmental transcription programs.
Epigenetics. 2014; 9(12):1613-25 [PubMed] Related Publications
The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between HOXD13 and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1.

Li Z, Yu X, Shen J, et al.
MicroRNA expression and its clinical implications in Ewing's sarcoma.
Cell Prolif. 2015; 48(1):1-6 [PubMed] Related Publications
Ewing's sarcoma (EWS) is the second most common primary bone cancer, and is a predominant childhood malignant disease. Due to limited understanding of its pathogenesis and frequent occurrence of resistance to conventional types of treatment, its management remains difficult, and mortality is frequent. Development of EWS is a multistep process involving genetic and epigenetic alterations of protein-coding proto-oncogenes and tumour-suppressor genes. MicroRNAs (miRNAs) have recently been discovered as a new category of non-protein coding; small RNA molecules that regulate gene expression at the post-transcriptional level. Substantial numbers of deregulated miRNAs have been documented in EWS and their biological significance has been confirmed in multiple functional experiments. Several studies have confirmed involvement of miRNAs in various steps of EWS pathogenesis, from occurrence to metastasis. Functionally, miRNA dysregulation may promote cell-cycle progression, confer resistance to apoptosis, and enhance invasiveness and metastasis. These miRNAs have opened a novel field in cancer research with potential clinical utilization for screening, diagnosis, prognostics and prediction of response to treatment. Elucidating biological aspects of miRNA dysregulation may help better understand pathogenesis of EWS and promote development of miRNA directed-therapeutics against it.

Denny CT
ChIP-ping away at EWS/ETS transcription networks.
Cancer Cell. 2014; 26(5):595-6 [PubMed] Related Publications
In this issue of Cancer Cell, Riggi and colleagues use a genomic approach to define two distinct molecular mechanisms through which the chimeric EWS/FLI1 oncoprotein regulates target genes in Ewing sarcoma, expanding a framework upon which to model the target gene network and test strategies for antagonizing growth of this tumor.

Lerman DM, Monument MJ, McIlvaine E, et al.
Tumoral TP53 and/or CDKN2A alterations are not reliable prognostic biomarkers in patients with localized Ewing sarcoma: a report from the Children's Oncology Group.
Pediatr Blood Cancer. 2015; 62(5):759-65 [PubMed] Article available free on PMC after 01/05/2016 Related Publications
BACKGROUND: A growing collection of retrospective studies have suggested that TP53 mutations and/or CDKN2A deletions have prognostic significance in Ewing sarcoma. We sought to evaluate these variables in patients with localized disease treated prospectively on a single Children's Oncology Group protocol.
PROCEDURE: Of the 568 patients enrolled on Children's Oncology Group protocol AEWS0031 (NCT00006734), 112 had tumor specimens of sufficient quality and quantity to allow for analysis of TP53 mutations status by DNA sequencing, and CDKN2A deletion by dual color fluorescent in situ hybridization.
RESULTS: Eight of 93 cases (8.6%) were found to have TP53 point mutations and 12 of 107 cases (11.2%) demonstrated homozygous CDKN2A deletion. Two cases were found to have an alteration in both genes. There was no significant difference in event-free survival of patients with TP53 mutations and/or CDKN2A deletions compared to patients with normal TP53/CDKN2A gene status, as demonstrated by log rank test (p = 0.58).
CONCLUSIONS: Although previous retrospective studies suggest their significance, TP53 mutation and/or CDKN2A deletion are not reliable prognostic biomarkers in localized Ewing sarcoma.

Riggi N, Knoechel B, Gillespie SM, et al.
EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma.
Cancer Cell. 2014; 26(5):668-81 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation.

Doyle LA, Wong KK, Bueno R, et al.
Ewing sarcoma mimicking atypical carcinoid tumor: detection of unexpected genomic alterations demonstrates the use of next generation sequencing as a diagnostic tool.
Cancer Genet. 2014 Jul-Aug; 207(7-8):335-9 [PubMed] Related Publications
Increasingly, tumors are being analyzed for a variety of mutations and other genomic changes, with the goals of guiding personalized therapy and directing patients to appropriate clinical trials based on genotype, as well as identifying previously unknown genomic changes in different tumor types and thereby providing new insights into the pathogenesis of human cancers. Next generation sequencing is a powerful research tool now gaining traction in the clinic. In this report, we demonstrate the utility of next generation sequencing assays in providing diagnostic information when evaluating tumor specimens. This is illustrated by a case previously thought to represent an atypical carcinoid tumor, in which an EWSR1-ERG translocation was detected during next generation sequencing using a hybrid capture approach, leading to a revised diagnosis of Ewing sarcoma. The role of translocation detection in these assays is also discussed.

Tirode F, Surdez D, Ma X, et al.
Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations.
Cancer Discov. 2014; 4(11):1342-53 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
UNLABELLED: Ewing sarcoma is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples.
SIGNIFICANCE: Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers.

Crompton BD, Stewart C, Taylor-Weiner A, et al.
The genomic landscape of pediatric Ewing sarcoma.
Cancer Discov. 2014; 4(11):1326-41 [PubMed] Related Publications
UNLABELLED: Pediatric Ewing sarcoma is characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, representing a paradigm for studying cancers driven by transcription factor rearrangements. In this study, we describe the somatic landscape of pediatric Ewing sarcoma. These tumors are among the most genetically normal cancers characterized to date, with only EWS-ETS rearrangements identified in the majority of tumors. STAG2 loss, however, is present in more than 15% of Ewing sarcoma tumors; occurs by point mutation, rearrangement, and likely nongenetic mechanisms; and is associated with disease dissemination. Perhaps the most striking finding is the paucity of mutations in immediately targetable signal transduction pathways, highlighting the need for new therapeutic approaches to target EWS-ETS fusions in this disease.
SIGNIFICANCE: We performed next-generation sequencing of Ewing sarcoma, a pediatric cancer involving bone, characterized by expression of EWS-ETS fusions. We found remarkably few mutations. However, we discovered that loss of STAG2 expression occurs in 15% of tumors and is associated with metastatic disease, suggesting a potential genetic vulnerability in Ewing sarcoma.

Wei S, Siegal GP
Round cell tumors of bone: an update on recent molecular genetic advances.
Adv Anat Pathol. 2014; 21(5):359-72 [PubMed] Related Publications
Round cell tumors of bone are a divergent group of neoplasms that largely constitute Ewing sarcoma/primitive neuroectodermal tumor, small cell osteosarcoma, Langerhans cell histiocytosis, mensenchymal chondrosarcoma, and hematopoietic malignancies including lymphoma and plasmacytoma/myeloma, along with metastatic round cell tumors including neuroblastoma, rhabdomyosarcoma, and small cell carcinoma. These lesions share many histomorphologic similarities and often demonstrate overlapping clinical and radiologic characteristics, but typically have a diverse clinical outcome, thus warranting differing therapeutic modalities/regimens. Recent advances in molecular and cytogenetic techniques have identified a number of additional novel entities, including round cell sarcomas harboring CIC-DUX4 and BCOR-CCNB3 fusions, respectively. These novel findings have not only enhanced our understanding of the pathogenesis of round cell tumors, but also allowed us to reclassify some entities with potential therapeutic and prognostic significance. This article provides an overview focusing on recent molecular genetic advances in primary, nonhematologic round cell tumors of bone.

Marino MT, Grilli A, Baricordi C, et al.
Prognostic significance of miR-34a in Ewing sarcoma is associated with cyclin D1 and ki-67 expression.
Ann Oncol. 2014; 25(10):2080-6 [PubMed] Related Publications
BACKGROUND: At diagnosis, identification of reliable biological indicators of prognosis to allow stratification of patients according to different risks is an important but still unresolved aspect in the treatment of Ewing sarcoma (EWS) patients. This study aimed to explore the role of miR-34A expression on prognosis of EWS patients.
PATIENTS AND METHODS: Specimens from 109 patients with non-metastatic EWS treated at the Rizzoli Institute with neoadjuvant chemotherapy (protocols ISG/SSGIII, EW-1, EW-2, EW-REN2, EW-REN3, EW-PILOT) and 17 metastases were studied. Sixty-eight patients (62%) remained disease-free and 41 (38%) relapsed (median follow-up: 67 months, range 9-241 months). Expression of miR-34a and of some of its targets (cyclin D1, bcl-2, SIRT1 and YY1) was evaluated by qRT-PCR using TaqMan MicroRNA Assays and/or by immunohistochemistry on tissue microarrays from the same patients.
RESULTS: High expression of miR-34a in localized tumors was significantly related to better event-free and overall survival (P = 0.004). Relevance of miR-34a was confirmed by using different calibrators (normal mesenchymal stem cells and different normal tissues). By multivariate Cox regression analysis, low miR-34a expression as well as nontotal necrosis and high levels of lactate dehydrogenase were all confirmed as independent risk factors associated with poor outcome. Expression of miR-34a was lower in metastases than in primary tumors. It inversely correlated with expression of cyclin D1 and Ki-67.
CONCLUSIONS: By demonstrating its relationship with clinical outcome, we propose evaluation of miR-34a at diagnosis of EWS patients to allow early risk stratification. Validation of these results would nonetheless ultimately need a prospective assessment.

Brohl AS, Solomon DA, Chang W, et al.
The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.
PLoS Genet. 2014; 10(7):e1004475 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Przybyl J, Kozak K, Kosela H, et al.
Gene expression profiling of peripheral blood cells: new insights into Ewing sarcoma biology and clinical applications.
Med Oncol. 2014; 31(8):109 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
Ewing sarcoma (ES) is a group of highly aggressive small round cell tumors of bone or soft tissue with high metastatic potential and low cure rate. ES tumors are associated with a rapid osteolysis and necrosis. The currently accepted clinical prognostic parameters do not accurately predict survival of high-risk patients. Moreover, neither the subtype of EWS-FLI1/ERG in the tumor, nor the detection of fusion transcripts in the peripheral blood (PB) samples, has prognostic value in ES patients. We evaluated the prevalence of circulating tumor cells (CTCs) in 34 adult ES patients. Since CTCs were confirmed in only small subset of patients, we further explored the expression profiles of PB leukocytes using a panel of genes associated with immune system status and increased tumor invasiveness. Moreover, we analyzed the alterations of the routine blood tests in the examined cohort of patients and correlated our findings with the clinical outcome. A uniform decrease in ZAP70 expression in PB cells among all ES patients, as compared to healthy individuals, was observed. Monocytosis and the abnormal expression of CDH2 and CDT2 genes in the PB cells significantly correlated with poor prognosis in ES patients. Our study supports the previously proposed hypothesis of systemic nature of ES. Based on the PB cell expression profiles, we propose a mechanism by which immune system may be involved in intensification of osteoclastogenesis and disease progression in ES patients. Moreover, we demonstrate the prognostic value of molecular PB testing at the time of routine histopathological diagnosis.

Sugita S, Arai Y, Tonooka A, et al.
A novel CIC-FOXO4 gene fusion in undifferentiated small round cell sarcoma: a genetically distinct variant of Ewing-like sarcoma.
Am J Surg Pathol. 2014; 38(11):1571-6 [PubMed] Related Publications
Differential diagnosis of small round cell sarcomas (SRCSs) grouped under the Ewing sarcoma family of tumors (ESFT) can be a challenging situation for pathologists. Recent studies have revealed that some groups of Ewing-like sarcoma show typical ESFT morphology but lack any EWSR1-ETS gene fusions. Here we identified a novel gene fusion, CIC-FOXO4, in a case of Ewing-like sarcoma with a t(X;19)(q13;q13.3) translocation. The patient was a 63-year-old man who had an asymptomatic, 30-mm, well-demarcated, intramuscular mass in his right posterior neck, and imaging findings suggested a diagnosis of high-grade sarcoma. He was treated with complete resection and subsequent radiotherapy and chemotherapy. He was alive without local recurrence or distant metastasis 6 months after the operation. Histologic examination revealed SRCS with abundant desmoplastic fibrous stroma suggesting a desmoplastic small round cell tumor. Immunohistochemical analysis showed weak to moderate and partial staining for MIC2 (CD99) and WT1, respectively. High-throughput transcriptome sequencing revealed a gene fusion, and the genomic rearrangement between the CIC and FOXO4 genes was identified by fluorescence in situ hybridization. Aside from the desmoplastic stroma, the CIC-FOXO4 fusion sarcoma showed morphologic and immunohistochemical similarity to ESFT and Ewing-like sarcomas, including the recently described CIC-DUX4 fusion sarcoma. Although clinicopathologic analysis with additional cases is necessary, we conclude that CIC-FOXO4 fusion sarcoma is a new type of Ewing-like sarcoma that has a specific genetic signature. These findings have important implications for the differential diagnosis of SRCS.

Sadri N, Barroeta J, Pack SD, et al.
Malignant round cell tumor of bone with EWSR1-NFATC2 gene fusion.
Virchows Arch. 2014; 465(2):233-9 [PubMed] Related Publications
Gene rearrangements involving the Ewing sarcoma breakpoint region 1 (EWSR1) gene are seen in a broad range of sarcomas and some nonmesenchymal neoplasms. Ewing sarcoma is molecularly defined by a fusion of the EWSR1 gene (or rarely the related FUS gene) to a member of the E26 transformation-specific (ETS) family of transcription factors, frequently the EWSR1-FLI1 fusion. More recently, EWSR1 gene fusion to non-ETS family members, including the nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) gene, has been reported in a histological variant of Ewing sarcoma. Here, we report a malignant round cell tumor of bone with an EWSR1-NFATC2 fusion gene. This report builds upon the unusual morphological and clinical presentation of bone neoplasms containing an EWSR1-NFATC2 fusion gene.

Sankar S, Theisen ER, Bearss J, et al.
Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth.
Clin Cancer Res. 2014; 20(17):4584-97 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
PURPOSE: Ewing sarcoma is a pediatric bone tumor that absolutely relies on the transcriptional activity of the EWS/ETS family of fusion oncoproteins. While the most common fusion, EWS/FLI, utilizes lysine-specific demethylase 1 (LSD1) to repress critical tumor suppressors, small-molecule blockade of LSD1 has not yet been thoroughly explored as a therapeutic approach for Ewing sarcoma. We therefore evaluated the translational potential of potent and specific LSD1 inhibition with HCI2509 on the transcriptional program of both EWS/FLI and EWS/ERG as well as the downstream oncogenic phenotypes driven by EWS/ETS fusions in both in vitro and in vivo models of Ewing sarcoma.
EXPERIMENTAL DESIGN: RNA-seq was used to compare the transcriptional profiles of EWS/FLI, EWS/ERG, and treatment with HCI2509 in both EWS/FLI- and EWS/ERG-containing cell lines. We then evaluated morphologic phenotypes of treated cells with immunofluorescence. The induction of apoptosis was evaluated using caspase-3/7 activation and TUNEL staining. Colony forming assays were used to test oncogenic transformation and xenograft studies with patient-derived cell lines were used to evaluate the effects of HCI2509 on tumorigenesis.
RESULTS: HCI2509 caused a dramatic reversal of both the up- and downregulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of morphologic and oncogenic phenotypes modulated by EWS/FLI. Importantly, HCI2509 displayed single-agent efficacy in multiple xenograft models.
CONCLUSIONS: These data support epigenetic modulation with HCI2509 as a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins.

Li Y, Luo H, Liu T, et al.
The ets transcription factor Fli-1 in development, cancer and disease.
Oncogene. 2015; 34(16):2022-31 [PubMed] Related Publications
Friend leukemia virus-induced erythroleukemia-1 (Fli-1), an E26 transformation specific (ETS) transcription factor, was isolated a quarter century ago through a retrovirus mutagenesis screen. Fli-1 has since been recognized to play critical roles in normal development and homeostasis. For example, it transcriptionally regulates genes that drive normal hematopoiesis and vasculogenesis. Indeed, Fli-1 is one of 10 key regulators of hematopoietic stem/progenitor cell maintenance and differentiation. Aberrant expression of Fli-1 also underlies a number of virally induced leukemias, including Friend virus-induced erythroleukemia and various types of human cancers, and it is the target of chromosomal translocations in childhood Ewing's sarcoma. Abnormal expression of Fli-1 is important in the etiology of autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. These studies establish Fli-1 as a strong candidate for drug development. Despite difficulties in targeting transcription factors, recent studies identified small-molecule inhibitors for Fli-1. Here we review past and ongoing research on Fli-1 with emphasis on its mechanistic function in autoimmune disease and malignant transformation. The significance of identifying Fli-1 inhibitors and their clinical applications for treatment of disease and cancer with deregulated Fli-1 expression are discussed.

Paronetto MP, Bernardis I, Volpe E, et al.
Regulation of FAS exon definition and apoptosis by the Ewing sarcoma protein.
Cell Rep. 2014; 7(4):1211-26 [PubMed] Related Publications
The Ewing sarcoma protein EWS is an RNA and DNA binding protein implicated in transcription, pre-mRNA splicing, and DNA damage response. Using CLIP-seq, we identified EWS RNA binding sites in exonic regions near 5' splice sites. A prominent target was exon 6 of the FAS/CD95 receptor, which is alternatively spliced to generate isoforms with opposing activities in programmed cell death. Depletion and overexpression experiments showed that EWS promotes exon 6 inclusion and consequently the synthesis of the proapoptotic FAS/CD95 isoform, whereas an EWS-FLI1 fusion protein characteristic of Ewing sarcomas shows decreased activity. Biochemical analyses revealed that EWS binding promotes the recruitment of U1snRNP and U2AF65 to the splice sites flanking exon 6 and therefore exon definition. Consistent with a role for EWS in the regulation of programmed cell death, cells depleted of EWS show decreased sensitivity to FAS-induced apoptosis, and elevated EWS expression enhances apoptosis in EWS-haploinsufficient Ewing sarcoma cells.

Bledsoe KL, McGee-Lawrence ME, Camilleri ET, et al.
RUNX3 facilitates growth of Ewing sarcoma cells.
J Cell Physiol. 2014; 229(12):2049-56 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

Mariño-Enríquez A, Fletcher CD
Round cell sarcomas - biologically important refinements in subclassification.
Int J Biochem Cell Biol. 2014; 53:493-504 [PubMed] Related Publications
Round cell sarcomas are a heterogeneous group of tumors that often affect children and young adults and, if untreated, often pursue a very aggressive clinical course. Specific subtypes of round cell sarcoma, like Ewing sarcoma or rhabdomyosarcoma, respond to well-defined therapeutic regimens so that proper classification is crucial for appropriate patient management. A subset of round cell sarcomas, however, lack specific clinical, morphologic, and immunophenotypic features and cannot be unequivocally classified based on such features. Systematic application of cytogenetics and molecular genetic techniques has allowed for the identification of an increasing number of genetically defined subgroups within this category of undifferentiated tumors. Although the clinical relevance of these molecular categories is yet to be proven, the systematic identification of lesions that share reproducible biologic, and often morphologic and immunophenotypic features, has great impact in terms of biologic understanding and coherent classification schemes, and will help to guide the potential development of rational new therapies. In this review we discuss the main categories of undifferentiated round cell sarcoma, in relation to Ewing sarcoma and its molecular variants, with particular emphasis on the genetic and biologic features of recently described entities including desmoplastic small round cell tumor and CIC-DUX4 as well as BCOR-CCNB3-associated round cell sarcomas. This article is part of a Directed Issue entitled: Rare Cancers.

Vanhapiha N, Knuutila S, Vettenranta K, Lohi O
Burkitt lymphoma and Ewing sarcoma in a child with Williams syndrome.
Pediatr Blood Cancer. 2014; 61(10):1877-9 [PubMed] Related Publications
Williams syndrome (WS) is a relatively rare multisystem neurodevelopmental disorder caused by a hemizygous deletion of contiguous genes on chromosome 7q11.23. Although WS does not predispose carriers to cancers, alterations of chromosome 7 are common in several human neoplasms. We report here a patient with WS and two different cancers, Burkitt lymphoma and Ewing sarcoma. Array-CGH analysis of the patient blood revealed a constitutive 1.4 million base pair deletion at 7q11.23, compatible with WS diagnosis.

Villasante A, Marturano-Kruik A, Vunjak-Novakovic G
Bioengineered human tumor within a bone niche.
Biomaterials. 2014; 35(22):5785-94 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Monolayer cultures of tumor cells and animal studies have tremendously advanced our understanding of cancer biology. However, we often lack animal models for human tumors, and cultured lines of human cells quickly lose their cancer signatures. In recent years, simple 3D models for cancer research have emerged, including cell culture in spheroids and on biomaterial scaffolds. Here we describe a bioengineered model of human Ewing's sarcoma that mimics the native bone tumor niche with high biological fidelity. In this model, cancer cells that have lost their transcriptional profiles after monolayer culture re-express genes related to focal adhesion and cancer pathways. The bioengineered model recovers the original hypoxic and glycolytic tumor phenotype, and enables re-expression of angiogenic and vasculogenic mimicry features that favor tumor adaptation. We propose that differentially expressed genes between the monolayer cell culture and native tumor environment are potential therapeutic targets that can be explored using the bioengineered tumor model.

Arnold MA, Ballester LY, Pack SD, et al.
Primary subcutaneous spindle cell Ewing sarcoma with strong S100 expression and EWSR1-FLI1 fusion: a case report.
Pediatr Dev Pathol. 2014 Jul-Aug; 17(4):302-7 [PubMed] Related Publications
Ewing sarcoma is described classically as a small, round cell tumor of bone and soft tissue in children and young adults. Ewing sarcoma most often is characterized by a fusion of the Ewing sarcoma breakpoint region 1 (EWSR1) and the Friend leukemia virus integration 1 (FLI1) genes, forming an EWSR1-FLI1 fusion transcript. We report an exceptional case of primary subcutaneous Ewing sarcoma in a 16-year-old female composed entirely of spindle cells with focal fascicular growth and exhibiting strong, diffuse immunohistochemical reactivity for S100, unlike classic Ewing sarcoma. However, reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed the presence of a rare variant of the EWSR1-FLI1 fusion transcript, featuring fusion of EWSR1 exon 10 to FLI1 exon 6. To our knowledge, the combined histologic, molecular, and clinical features have not been reported previously in Ewing sarcoma, and raise a broad differential diagnosis emphasizing the importance of molecular techniques in the diagnosis of this tumor.

Specht K, Sung YS, Zhang L, et al.
Distinct transcriptional signature and immunoprofile of CIC-DUX4 fusion-positive round cell tumors compared to EWSR1-rearranged Ewing sarcomas: further evidence toward distinct pathologic entities.
Genes Chromosomes Cancer. 2014; 53(7):622-33 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Round cell sarcomas harboring CIC-DUX4 fusions have recently been described as highly aggressive soft tissue tumors of children and young adults. Due to partial morphologic and immunohistochemical overlap with Ewing sarcoma (ES), CIC-DUX4-positive tumors have generally been classified as ES-like and managed similarly; however, a systematic comparison at the molecular and immunohistochemical levels between these two groups has not yet been conducted. Based on an initial observation that CIC-DUX4-positive tumors show nuclear immunoreactivity for WT1 and ETS transcription factors, FLI1 and ERG, we performed a detailed immunohistochemical and molecular analysis including these markers, to further investigate the relationship between CIC-DUX4 tumors and ES. The study group included 21 CIC-DUX4-positive sarcomas and 20 EWSR1-rearranged ES. Immunohistochemically, CIC-DUX4 sarcomas showed membranous CD99 positivity in 18 (86%) cases, but only 5 (24%) with a diffuse pattern, while WT1 and FLI1 were strongly positive in all cases. ERG was positive in 18% of cases. All ES expressed CD99 and FLI1, while ERG positivity was only seen in EWSR1-ERG fusion positive ES. WT1 was negative in all ES. Expression profiling validated by q-PCR revealed a distinct gene signature associated with CIC-DUX4 fusion, with upregulation of ETS transcription factors (ETV4, ETV1, and ETV5) and WT1, among top overexpressed genes compared to ES, other sarcomas and normal tissue. In conclusion, the distinct gene signature and immunoprofile of CIC-DUX4 sarcomas suggest a distinct pathogenesis from ES. The consistent WT1 expression may provide a useful clue in the diagnosis in the context of round cell sarcomas negative for EWSR1 rearrangement. © 2014 Wiley Periodicals, Inc.

Choi EY, Gardner JM, Lucas DR, et al.
Ewing sarcoma.
Semin Diagn Pathol. 2014; 31(1):39-47 [PubMed] Related Publications
Classification of small round cell tumors of bone is often challenging due to overlapping clinicopathologic features. The purpose of this article is to review the clinical, radiological, histologic, and molecular features of Ewing sarcoma and to provide a discussion of the differential diagnosis of small round cell tumors of bone.

Karnuth B, Dedy N, Spieker T, et al.
Differentially expressed miRNAs in Ewing sarcoma compared to mesenchymal stem cells: low miR-31 expression with effects on proliferation and invasion.
PLoS One. 2014; 9(3):e93067 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
Ewing sarcoma, the second most common bone tumor in children and young adults, is an aggressive malignancy with a strong potential to metastasize. Ewing sarcoma is characterised by translocations encoding fusion transcription factors with an EWSR1 transactivation domain fused to an ETS family DNA binding domain. microRNAs are post-transcriptional regulators of gene expression and aberrantly expressed microRNAs have been identified as tumor suppressors or oncogenes in most cancer types. To identify potential oncogenic and tumor suppressor microRNAs in Ewing sarcoma, we determined and compared the expression of 377 microRNAs in 40 Ewing sarcoma biopsies, 6 Ewing sarcoma cell lines and mesenchymal stem cells, the putative cellular origin of Ewing sarcoma, from 6 healthy donors. Of the 35 differentially expressed microRNAs identified (fold change >4 and q<0.05), 19 were higher and 16 lower expressed in Ewing sarcoma. In comparisons between Ewing sarcoma samples with EWS-FLI or EWS-ERG translocations, with differing dissemination characteristics and of primary samples and metastases no significantly differential expressed microRNAs were detected using various stringency criteria. For miR-31, the microRNA with lowest expression in comparison to mesenchymal stem cells, functional analyses were performed to determine its potential as a tumor suppressor in Ewing sarcoma. Two of four miR-31 transfected Ewing sarcoma cell lines showed a significantly reduced proliferation (19% and 33% reduction) due to increased apoptosis in one and increased length of G1-phase in the other cell line. All three tested miR-31 transfected Ewing sarcoma cell lines showed significantly reduced invasiveness (56% to 71% reduction). In summary, we identified 35 microRNAs differentially expressed in Ewing sarcoma and demonstrate that miR-31 affects proliferation and invasion of Ewing sarcoma cell lines in ex vivo assays.

Burdach S
Molecular precision chemotherapy: overcoming resistance to targeted therapies?
Clin Cancer Res. 2014; 20(5):1064-6 [PubMed] Related Publications
Cytotoxic drugs may have specific effects on oncogenes and their downstream targets. Increase of cancer cell sensitivity due to repression of an oncogene downstream target can be specifically addressed by combined precision chemotherapy, increasing the therapeutic index of chemotherapy and overcoming resistance to highly selective targeted therapies.

Li J, You T, Jing J
MiR-125b inhibits cell biological progression of Ewing's sarcoma by suppressing the PI3K/Akt signalling pathway.
Cell Prolif. 2014; 47(2):152-60 [PubMed] Related Publications
OBJECTIVES: Increasing evidence has suggested the close relationship between microRNAs (miRNAs) dysregulation and the carcinogenesis of Ewing's sarcoma (ES), among of which miR-125b has been reported to be decreased in ES tissues recently. Strikingly, ectopic expression of miR-125b could suppress cell proliferation of ES cell line A673, suggesting the tumor suppressor role of miR-125b in ES. However, the other accurate mechanistic functions and relative molecule mechanisms are largely unknown.
MATERIALS AND METHODS: Herein, we completed a series of experiments to investigate the role of miR-125b in Ewing's sarcoma. We restored the expression of miR-125b in ES cell line A673 through transfection with miR-125b mimics. To further understand the role of miR-125b in ES, we detected the effects of miR-125b on the cell proliferation, migration and invasion, cell cycle as well as cell apoptosis.
RESULTS: We found that restored expression of miR-125b in ES cell line A673 inhibited cell proliferation, migration and invasion, arrested cell cycle progression, and induced cell apoptosis. Moreover, bioinformatic prediction suggested the oncogene, phosphoinositide-3-kinase catalytic subunit delta (PIK3CD), was a target gene of miR-125b in ES cells. Further quantitative RT-PCR and western blot assays identified over-expression of miR-125b suppressed the expression of PIK3CD mRNA and protein. PIK3CD participates in regulating the PI3K signaling pathway, which has been reported to play an important role in the development of ES. Suppression of PIK3CD down-regulated the expression of phospho-AKT and phospho-mTOR proteins and inhibited the biologic progression of A673 cells.
CONCLUSIONS: Collectively, these data suggest that miR-125b functions as a tumor suppressor by targeting the PI3K/Akt/mTOR signaling pathway, and may provide potential therapy strategy for ES patients by targeting miRNA expression.

Amaral AT, Manara MC, Berghuis D, et al.
Characterization of human mesenchymal stem cells from ewing sarcoma patients. Pathogenetic implications.
PLoS One. 2014; 9(2):e85814 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
BACKGROUND: Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin.
MATERIALS AND METHODS: In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples.
RESULTS: We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99.
CONCLUSIONS: In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis.

Zhou Z, Yu L, Kleinerman ES
EWS-FLI-1 regulates the neuronal repressor gene REST, which controls Ewing sarcoma growth and vascular morphology.
Cancer. 2014; 120(4):579-88 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
BACKGROUND: RE1-silencing transcription factor (REST), a neuronal repressor gene, regulates neuronal stem cell differentiation. Ewing sarcoma may originate from neural crest cells. In the current study, the authors investigated whether REST plays a role in the growth of this tumor.
METHODS: REST expression was determined by Western blot analysis and reverse transcription-polymerase chain reaction in 3 human Ewing sarcoma cell lines and 7 patient tumor samples. The role of REST in tumor growth and tumor vascular morphology was determined using a Ewing sarcoma xenograft model. Immunofluorescence staining, Hypoxyprobe, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to investigate the impact of REST on pericyte marker expression, hypoxia, and apoptosis in vivo.
RESULTS: High levels of REST were expressed in all 3 human Ewing sarcoma cell lines and in 6 of the 7 patient tumor samples. Overexpression of EWS-FLI-1 in human mesenchymal stem cells and human neural progenitor cells was found to increase REST expression. Inhibition of EWS-FLI-1 using small interfering RNA decreased REST expression in human Ewing sarcoma cells. Inhibition of REST did not affect EWS-FLI-1, but significantly suppressed tumor growth in vivo, reduced the tumor vessel pericyte markers α- smooth muscle actin (SMA) and desmin, increased hypoxia and apoptosis in tumor tissues, and decreased the expression of delta-like ligand 4 (DLL4) and Hes1.
CONCLUSIONS: Inhibition of REST suppressed tumor growth, inhibited pericyte marker expression, and increased tumor hypoxia and apoptosis. Because tumor vessel function has been linked to tumor growth and metastases, REST may be a new therapeutic target in patients with Ewing sarcoma.

Zhang Z, Huang L, Yu Z, et al.
Let-7a functions as a tumor suppressor in Ewing's sarcoma cell lines partly by targeting cyclin-dependent kinase 6.
DNA Cell Biol. 2014; 33(3):136-47 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
MicroRNAs play an important role in the development and progression of Ewing's sarcoma (ES). Especially, the expression of let-7a has been reported to be significantly downregulated in various cancers, and can affect the initiation and maintenance of tumor progression. However, the relative effects of let-7a on ES cells and relative mechanisms are largely unknown. In this study, we identified the underexpression of let-7a in human ES cells comparing with the human mesenchymal stem cells. Then, we sought to compensate for its loss through exogenous transfection with let-7a mimic into ES cell lines A673 and SK-ES-1. Restored let-7a expression inhibited cell proliferation, migration, as well as invasion; arrested cell cycle progression; and induced cell apoptosis of both cell lines. Moreover, bioinformatic prediction suggested that cyclin-dependent kinase 6 (CDK6), which is overexpressed and functions as an oncoprotein in ES cells, is a putative target gene of let-7a. Using mRNA and protein expression analysis and luciferase assays, we further identified the target role of CDK6. Finally, we found that restored CDK6 expression in ES cells that had been treated with let-7a mimic before could partly dampen let-7a-mediated tumor suppression. Taken together, our results showed that let-7a acted as a tumor suppressor in ES by targeting CDK6, and it may provide novel diagnostic and therapeutic options for human Ewing sarcoma clinical operation in future.

Recurrent Chromosome Abnormalities

Selected list of common recurrent structural abnormalities

This is a highly selective list aiming to capture structural abnormalies which are frequesnt and/or significant in relation to diagnosis, prognosis, and/or characterising specific cancers. For a much more extensive list see the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer.

Trisomy 8 in Ewing's Sarcoma

Approximately half of all cases of Ewing's sarcoma have gain of chromosome 8 (e.g. 52% in a series of 134 patients reported by Hattinger et al, 2002). There are mixed reports whether it is of prognostic significance.

Maurici D, Perez-Atayde A, Grier HE, et al.
Frequency and implications of chromosome 8 and 12 gains in Ewing sarcoma.
Cancer Genet Cytogenet. 1998; 100(2):106-10 [PubMed] Related Publications
Ewing sarcoma (ES) is the second most common primary malignant tumor of bone in children and young adolescents. Most ES contain a pathognomonic translocation t(11;22)(q24;q12) that is likely a pivotal event in the tumorigenesis of these neoplasms. Many ES also contain nonrandom, numerical chromosomal aberrations, the most common of which are trisomies 8 and 12. In this study we evaluated the hypothesis that these trisomies might occur during neoplastic progression and might be associated with differences in biologic behavior. We tested this hypothesis using a combined cytogenetic and dual color fluorescence in situ hybridization approach to determine chromosome 8 and 12 copy number in 52 ES. Relative gains, primarily trisomies, of chromosomes 8 and 12 were found in 24 (46%) and 17 (33%) cases, respectively. Trisomy 8 and trisomy 12 were independent events acquired in a flexible order during ES genetic progression. Our preliminary findings also suggest a higher frequency of trisomies 8 and 12 in relapses than in primary tumors. Prospective studies will be required to determine whether either trisomy is prognostic in newly-diagnosed ES.

Armengol G, Tarkkanen M, Virolainen M, et al.
Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization.
Br J Cancer. 1997; 75(10):1403-9 [PubMed] Free Access to Full Article Related Publications
Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.

Trisomy 12 in Ewing's Sarcoma

Mugneret F, Lizard S, Aurias A, Turc-Carel C
Chromosomes in Ewing's sarcoma. II. Nonrandom additional changes, trisomy 8 and der(16)t(1;16).
Cancer Genet Cytogenet. 1988; 32(2):239-45 [PubMed] Related Publications
Chromosomal data from 82 informative, unrelated Ewing's sarcoma (ES) specimens (including 20 personal specimens) were reviewed for secondary changes additional to the t(11;22)(q24;q12). Additional numerical and/or structural changes were found in 75 specimens. Trisomy 8 was observed consistently in half of the 43 cases selected for analysis of numerical changes. A nonrandom der(16) was observed as a result of an unbalanced t(1;16) in 18% of the 82 analyzed for structural changes. Consistent involvement of chromosome #16 in rearrangements with chromosome #1 may be an additional chromosome change specifically associated with ES.

Armengol G, Tarkkanen M, Virolainen M, et al.
Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization.
Br J Cancer. 1997; 75(10):1403-9 [PubMed] Free Access to Full Article Related Publications
Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.

Maurici D, Perez-Atayde A, Grier HE, et al.
Frequency and implications of chromosome 8 and 12 gains in Ewing sarcoma.
Cancer Genet Cytogenet. 1998; 100(2):106-10 [PubMed] Related Publications
Ewing sarcoma (ES) is the second most common primary malignant tumor of bone in children and young adolescents. Most ES contain a pathognomonic translocation t(11;22)(q24;q12) that is likely a pivotal event in the tumorigenesis of these neoplasms. Many ES also contain nonrandom, numerical chromosomal aberrations, the most common of which are trisomies 8 and 12. In this study we evaluated the hypothesis that these trisomies might occur during neoplastic progression and might be associated with differences in biologic behavior. We tested this hypothesis using a combined cytogenetic and dual color fluorescence in situ hybridization approach to determine chromosome 8 and 12 copy number in 52 ES. Relative gains, primarily trisomies, of chromosomes 8 and 12 were found in 24 (46%) and 17 (33%) cases, respectively. Trisomy 8 and trisomy 12 were independent events acquired in a flexible order during ES genetic progression. Our preliminary findings also suggest a higher frequency of trisomies 8 and 12 in relapses than in primary tumors. Prospective studies will be required to determine whether either trisomy is prognostic in newly-diagnosed ES.

der(16)t(1;16) in Ewing's Sarcoma

Mugneret F, Lizard S, Aurias A, Turc-Carel C
Chromosomes in Ewing's sarcoma. II. Nonrandom additional changes, trisomy 8 and der(16)t(1;16).
Cancer Genet Cytogenet. 1988; 32(2):239-45 [PubMed] Related Publications
Chromosomal data from 82 informative, unrelated Ewing's sarcoma (ES) specimens (including 20 personal specimens) were reviewed for secondary changes additional to the t(11;22)(q24;q12). Additional numerical and/or structural changes were found in 75 specimens. Trisomy 8 was observed consistently in half of the 43 cases selected for analysis of numerical changes. A nonrandom der(16) was observed as a result of an unbalanced t(1;16) in 18% of the 82 analyzed for structural changes. Consistent involvement of chromosome #16 in rearrangements with chromosome #1 may be an additional chromosome change specifically associated with ES.

Hattinger CM, Rumpler S, Ambros IM, et al.
Demonstration of the translocation der(16)t(1;16)(q12;q11.2) in interphase nuclei of Ewing tumors.
Genes Chromosomes Cancer. 1996; 17(3):141-50 [PubMed] Related Publications
The der(16)t(1;16) has been detected cytogenetically in a number of malignancies including Ewing tumors (ETs). To enable fast and reliable analysis of der(16) chromosomes, we established an interphase cytogenetic approach. By using two DNA probes hybridizing to the heterochromatic portions on the long arms of chromosomes 1 and 16, this technique allows the detection of this chromosomal aberration in nonproliferating cells. Formation of the der(16) leads to partial excess of 1q material and partial loss of the long arm of chromosome 16. Double-target fluorescence in situ hybridization (FISH) experiments were performed on cytospin slides of 13 ETs, near-triploid tumor cells and normal cells to assess whether the FISH technique used permits the discrimination of nuclei harboring this aberration from nuclei without a der(16) chromosome. In five ETs, we found evidence for the presence of one or two der(16)t(1;16) chromosomes both by FISH and by conventional cytogenetics. Tumor cells displayed two signals for intact chromosomes 1, one or two additional fused signals for the der(16) chromosomes, and one signal for the intact chromosome 16. In one case without fused signals, the presence of a der(16) was demonstrated by hybridizing a painting probe for chromosome 16 simultaneously with the paracentromeric probe for chromosome 1. Our results suggest that double-target FISH on interphase nuclei offers an ideal tool for analyzing tumors prospectively and retrospectively to assess the biological role and the possible prognostic impact of the der(16) in ETs and in other solid tumors.

Stark B, Mor C, Jeison M, et al.
Additional chromosome 1q aberrations and der(16)t(1;16), correlation to the phenotypic expression and clinical behavior of the Ewing family of tumors.
J Neurooncol. 1997; 31(1-2):3-8 [PubMed] Related Publications
The cytogenetic hallmark of the Ewing family of tumors is t(11,22)(q24;q12) in its simple, complex or variant forms and/or its molecular equivalent EWS/FLI, EWS/ERG rearrangement. Additional secondary consistent chromosomal aberrations include the der(16)t(1;16) and frequently, other chromosome 1q abnormalities leading to 1q overdosage. We studied whether these secondary cytogenetic changes are correlated to clinical features and phenotypic expression which may have a prognostic impact. Successful cytogenetic evaluation was performed in eight patients with a Ewing family tumor. In four of these, in addition to the primary aberration, chromosome 1q overdosage (including two with der (16)t(1;16)) was noted. Out of these four patients, two had metastatic disease at the time of evaluation, while in the other four, disease was localized. Morphologically, the tumors with the additional 1q aberration, revealed the pPNET subtype more frequently than the typical Ewing. They also expressed a higher degree of neural differentiation by neural marker immunocytochemistry, in comparison to tumors without the 1q aberration. Determination of the prognostic significance of this finding requires a longer follow-up with a larger group of patients.

Mitochondrial DNA mutations in Ewing's sarcoma

Yu et al (2009) reported a high frequency of mitochondrial DNA D-loop mutations in Ewing's sarcoma: 12 of 17 tumor specimens carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletion. The same authors (Yu et al 2013) sequenced the whole mitochondrial genome from 20 cases of EWS specimens and found 70% of cases had 25 somatic mtDNA mutations: 19 (76%) were located in the D-loop control region, 1 (4%) was in the sequence of the tRNA(Val) gene, 1 (4%) was in the mitochondrial ATP synthase subunit 6 gene, and 4 (16%) occurred in genes encoding components of the mitochondrial respiratory complexes.

Yu M, Wan Y, Zou Q, Xi Y
High frequency of mitochondrial DNA D-loop mutations in Ewing's sarcoma.
Biochem Biophys Res Commun. 2009; 390(3):447-50 [PubMed] Related Publications
Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing's sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations' possible involvement in the initiation and/or progression of this rare malignancy.

Yu M, Wan Y, Zou Q
Somatic mutations of the mitochondrial genome in Chinese patients with Ewing sarcoma.
Hum Pathol. 2013; 44(7):1350-6 [PubMed] Related Publications
Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive initiation and progression of human malignancies. Our previous study revealed a high prevalence of somatic mutations in the D-loop region of mtDNA in Ewing sarcoma (EWS). However, it is unclear whether somatic mutations also occur in the coding regions of mtDNA in EWS. To test this possibility, in the present study, we sequenced the whole mitochondrial genome from 20 cases of EWS specimens and their corresponding peripheral blood samples. We identified a total of 6 somatic mutations in the mtDNA coding regions in our EWS series, and 5 of them were missense or frame-shift mutations that have the potential to directly influence proper mitochondrial function. In combination with our earlier observations on the D-loop fragment, 70% (14/20) of EWS tissues appeared to harbor somatic mtDNA mutations. Among the identified 25 somatic mutations, 19 (76%) were located in the D-loop control region, 1 (4%) was in the sequence of the tRNA(Val) gene, 1 (4%) was in the mitochondrial ATP synthase subunit 6 gene, and 4 (16%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, patients carrying somatic mtDNA mutations did not show significant association with their clinicopathologic characteristics. Together, these findings suggest that somatic mtDNA mutations occur in both protein coding and noncoding regions of mtDNA, which may play critical roles in the pathogenesis of EWS and should be further explored for its possible use as a novel marker for monitoring EWS occurrence and advancement.

del(1p36) in Ewing's Sarcoma

Hattinger CM, Rumpler S, Strehl S, et al.
Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors.
Genes Chromosomes Cancer. 1999; 24(3):243-54 [PubMed] Related Publications
Ewing's sarcoma, peripheral primitive neuroectodermal tumors, and Askin tumors are referred to as Ewing tumors (ETs), and are characterized by high MIC2 expression and a t(11;22)(q24;q12) or other rearrangements involving 22q12. In addition to these constant aberrations, facultative numerical and structural aberrations have been reported: gains of chromosomes 8 and 12, the unbalanced translocation t(1;16), and deletions at the short arm of chromosome 1. To evaluate the frequency and to study the biological impact of these facultative aberrations, we analyzed tumor specimens from 58 ET patients by classical cytogenetics and/or in situ hybridization techniques and compared these data with clinical parameters. Gains of chromosomes 8 and 12 were detected in 55% (32/58) and 24% (14/58) of the cases, respectively. Loss of chromosome 16 or der (16)t(1;16) chromosomes were found in 20% (10/51); deletions at 1p36 were observed in 18% (9/51) of the cases evaluated. The presence of these aberrations did not correlate with age and sex of the patients, with the location of the primary tumor or with the extent of disease at diagnosis by chi-square analysis and Fisher's exact test. Patients with tumors harboring gains of chromosome 8 showed a slightly better clinical outcome (n = 14/30, P = 0.17), whereas gains of chromosome 12 did not influence the clinical outcome (n = 7/30, P = 0.63). However, Kaplan and Meier analysis revealed that deletions at the short arm of chromosome 1 were associated with an unfavorable outcome in patients with localized disease (n = 6/22; P = 0.004).

1q Gain in Ewing's Sarcoma

Kullendorff CM, Mertens F, Donnér M, et al.
Cytogenetic aberrations in Ewing sarcoma: are secondary changes associated with clinical outcome?
Med Pediatr Oncol. 1999; 32(2):79-83 [PubMed] Related Publications
BACKGROUND: Ewing sarcoma is associated with a nonrandom pattern of primary and secondary chromosomal aberrations. Whereas the finding of rearrangements of chromosome 22, usually in the form of a balanced translocation t(11;22)(q24;q12), is important diagnostically, nothing is known about the potential prognostic impact of the secondary chromosomal aberrations.
PROCEDURE: During a 1 3-year-period, short-term cultured tumor samples from 21 children and young adults with Ewing sarcoma were cytogenetically analyzed successfully.
RESULTS: Clonal chromosome aberrations were detected in 18 patients, 17 of whom had the characteristic t(11;22)(q24;q12) or variants thereof. The most frequent secondary change was +8, followed by +12, +2, +5, +9, +15, and gain of material from the long and short arms of chromosome 1. The only recurrent secondary change that was restricted to tumors from the ten patients that were dead at latest follow-up was gain of 1q material. Furthermore, all three patients with tumors with chromosome numbers over 50 had died, and the only patient with a tumor karyotype lacking chromosome 22 rearrangement was alive without evidence of disease.
CONCLUSIONS: These data and previously published results indicate that the karyotypic pattern not only may be of diagnostic significance but also may be important prognostically.

Stark B, Mor C, Jeison M, et al.
Additional chromosome 1q aberrations and der(16)t(1;16), correlation to the phenotypic expression and clinical behavior of the Ewing family of tumors.
J Neurooncol. 1997; 31(1-2):3-8 [PubMed] Related Publications
The cytogenetic hallmark of the Ewing family of tumors is t(11,22)(q24;q12) in its simple, complex or variant forms and/or its molecular equivalent EWS/FLI, EWS/ERG rearrangement. Additional secondary consistent chromosomal aberrations include the der(16)t(1;16) and frequently, other chromosome 1q abnormalities leading to 1q overdosage. We studied whether these secondary cytogenetic changes are correlated to clinical features and phenotypic expression which may have a prognostic impact. Successful cytogenetic evaluation was performed in eight patients with a Ewing family tumor. In four of these, in addition to the primary aberration, chromosome 1q overdosage (including two with der (16)t(1;16)) was noted. Out of these four patients, two had metastatic disease at the time of evaluation, while in the other four, disease was localized. Morphologically, the tumors with the additional 1q aberration, revealed the pPNET subtype more frequently than the typical Ewing. They also expressed a higher degree of neural differentiation by neural marker immunocytochemistry, in comparison to tumors without the 1q aberration. Determination of the prognostic significance of this finding requires a longer follow-up with a larger group of patients.

Armengol G, Tarkkanen M, Virolainen M, et al.
Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization.
Br J Cancer. 1997; 75(10):1403-9 [PubMed] Free Access to Full Article Related Publications
Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.

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