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 (19)

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 10 March, 2017 using data from PubMed, MeSH and CancerIndex

Mutated Genes and Abnormal Protein Expression (36)

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, EWS-FLI1, 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
323
FLI1 11q24.3 EWSR2, SIC-1 Translocation
Prognostic
-t(11;22)(q24;q12) EWSR1-FLI1 Translocation in Ewing's Sarcoma
-EWSR1-FLI1 Fusion Transcript Structure and Prognosis?
323
ERG 21q22.3 p55, erg-3 Translocation
-t(21;22) EWSR1-ERG Translocations in Ewing's Sarcoma
-ERG and Ewing's Sarcoma
99
TP53 17p13.1 P53, BCC7, LFS1, TRP53 Overexpression
Prognostic
-TP53 Mutations and aberrant expression in a sub-set of Ewing's Sarcoma
36
CDKN2A 9p21.3 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
25
ETV1 7p21.3 ER81 Translocation
- t(7;22)(p22;q12) EWS-ETV1 Translocation in Ewing's Sarcoma
24
FEV 2q36 PET-1, HSRNAFEV Translocation
-t(2;22) EWSR1-FEV Translocation in Ewing's Sarcoma
-FEV and Ewing's Sarcoma
17
NR0B1 Xp21.3 AHC, AHX, DSS, GTD, HHG, AHCH, DAX1, DAX-1, NROB1, SRXY2 -NR0B1 and Ewing's Sarcoma
10
ETV4 17q21.31 E1AF, PEA3, E1A-F, PEAS3 Translocation
-t(17;22)(q12;q12) EWSR1-E1AF Translocation in Ewing's Sarcoma
-ETV4 and Ewing's Sarcoma
8
BCR 22q11.23 ALL, CML, PHL, BCR1, D22S11, D22S662 -BCR and Ewing's Sarcoma
7
CCK 3p22.1 -CCK Expression in Ewing's Sarcoma
7
BCOR Xp11.4 MAA2, ANOP2, MCOPS2 -BCOR and Ewing's Sarcoma
7
GLI1 12q13.2-q13.3 GLI Overexpression
-GLI1 upregulation by EWSR1/FLI1 in Ewing's Sarcoma
7
DUX4L1 4q35 DUX4, DUX10 -DUX4 and 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
PTEN 10q23.31 BZS, DEC, CWS1, GLM2, MHAM, TEP1, MMAC1, PTEN1, 10q23del Amplification
-PTEN Amplification in Ewing's Sarcoma?
6
LIF 22q12.2 CDF, DIA, HILDA, MLPLI -LIF and Ewing's Sarcoma
5
TAF15 17q11.1-q11.2 Npl3, RBP56, TAF2N, TAFII68 -TAF15 and Ewing's Sarcoma
5
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
STAG2 Xq25 SA2, SA-2, SCC3B, bA517O1.1 -STAG2 and Ewing's Sarcoma
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
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
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
SOX6 11p15.2 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
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

He T, Surdez D, Rantala JK, et al.
High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.
Gene. 2017; 596:137-146 [PubMed] Related Publications
A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

Ishiguro M, Yuki M, Fukushige T, et al.
Molecular cytogenetic characterization of two established ESFT cell lines.
Hum Cell. 2017; 30(1):41-48 [PubMed] Related Publications
Ewing's sarcoma/primitive neuroectodermal tumor/Askin's tumor (Ewing`s sarcoma family of tumors: ESFT) is the most common type of malignant tumor of bone and soft tissue in children and young adults, and morphologically is a member of a group of small round cell tumors. We report, here, on the establishment of two human ESFT cell lines, FU-PNET-3 and FU-PNET-4, from the iliac and the chest wall, respectively, the cells of both cell lines were tumorigenic in immunodeficient mice. Histologically, both original and xenograft tumors and cultured cells were composed of small round cells with positive immunoreactivity for CD99 and Nkx2.2. Molecular biological examination demonstrated chimeric transcripts of EWSR1 exon 7 to FLI1 exon 6 in FU-PNET-3 cells, and EWSR1 exon 10 to FLI1 exon 6 in FU-PNET-4 cells. Cytogenetic analysis revealed chromosome translocation t(11;22)(q24;q12) and some secondary changes in both cultured cells. These histological, molecular biological, and cytogenetical findings indicate ESFT in both cell lines. ESFT is well studied, but its recurrent fusion genes are heterogeneous and its biological behaviors are unclear. The FU-PNET-3 and FU-PNET-4 cell lines have been well examined and may become useful tools for studying the genetic and biological behavioral properties of ESFT.

Salet MC, Vogels R, Brons P, et al.
Maturation toward neuronal tissue in a Ewing sarcoma of bone after chemotherapy.
Diagn Pathol. 2016; 11(1):74 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Ewing sarcoma is the second most common bone tumor, occurring mainly in children and young adults. It shows a typical primitive, small round cell morphology and a characteristic fusion oncogene involving EWSR1 and members of the ETS family in most of the cases. Neuronal maturation after chemotherapy is a rare phenomenon and we herein describe such an exceptional case.
CASE PRESENTATION: An 8-year old boy was diagnosed with a Ewing sarcoma in the left femur. On biopsy the morphology was typical and there was an EWSR1-FLI1 gene fusion. He underwent neo-adjuvant chemotherapy and resection of the tumor. On microscopic evaluation, part of the tumor showed ganglioneuroblastoma-like differentiation with expression of neuronal markers. The continued presence of EWSR1 rearrangement in both the blue round cell component and the ganglioneuroblastoma-like component was shown by FISH analysis.
CONCLUSIONS: In conclusion, this case describes the possibility of a Ewing sarcoma to differentiate into a ganglioneuroblastoma-like lesion after neo-adjuvant chemotherapy treatment; the prognostic value of this phenomenon remains questionable.

Machado I, Navarro L, Pellin A, et al.
Defining Ewing and Ewing-like small round cell tumors (SRCT): The need for molecular techniques in their categorization and differential diagnosis. A study of 200 cases.
Ann Diagn Pathol. 2016; 22:25-32 [PubMed] Related Publications
BACKGROUND: Differentiation of Ewing sarcoma family of tumors (ESFT) and Ewing-like tumors remains problematic. Certain ESFT with morphological and immunohistochemical (IHC) profiles lack the EWSR1-ETS transcript. To improve diagnostic accuracy we investigated the presence of several specific transcripts in 200 small round cell tumors (SRCT) displaying ESFT morphology and immunophenotype in which EWSR1 FISH analysis was non-informative or negative.
DESIGN: 200 tumors (formalin-fixed, paraffin-embedded) were analyzed by RT-PCR. All tumors were tested for EWSR1-ETS, EWSR1/WT1, PAX3/7-FOX01 or SYT/SSX transcripts, and the negative tumors were subsequently analyzed for CIC/DUX4, BCOR/CCNB3 and CIC/FOX04 transcripts.
RESULTS: 133 (66.5%) ESFT displayed one of the above EWSR1-ETS translocations. Three cases (1.5%) revealed the SYT-SSX transcript for Synovial sarcoma, and one (0.5%) a EWSR1-WT1 transcript for Desmoplastic Small Round Cell tumor. The CIC-DUX4 translocation was found in six Ewing-like tumors (3%) with CD99 positivity. The BCOR-CCNB3 gene fusion was observed in 5 tumors (2.5%) displaying round or spindle cells with strong CCNB3 IHC expression in 3 tumors. Moreover, RT-PCR failed to detect any gene fusion transcripts in 19 tumors (9.5%) and were considered "undifferentiated small round cell sarcoma" (SRCS). Molecular biology results were non-informative in 33 SRCTs (16.5%) due to RNA degradation through inadequate fixation and/or decalcification.
CONCLUSION: Our analysis of 200 SRCTs confirms the molecular heterogeneity of neoplasms with ESFT morphology and highlight that molecular studies with RT-PCR including new emerging gene fusion transcripts are mandatory for the diagnosis when EWSR1 FISH is negative or non-informative. The incidence of CIC-DUX4, BCOR-CCNB3 and CIC-FOX04 transcripts was relatively low. A small group of Ewing-like sarcomas or undifferentiated SRCS remains unclassified. Adopting appropriate tissue fixation and processing protocols is important to avoid degradation of fixed/embedded tissue when no frozen tumor is available.

Zhang N, Liu H, Yue G, et al.
Molecular Heterogeneity of Ewing Sarcoma as Detected by Ion Torrent Sequencing.
PLoS One. 2016; 11(4):e0153546 [PubMed] Free Access to Full Article Related Publications
Ewing sarcoma (ES) is the second most common malignant bone and soft tissue tumor in children and adolescents. Despite advances in comprehensive treatment, patients with ES metastases still suffer poor outcomes, thus, emphasizing the need for detailed genetic profiles of ES patients to identify suitable molecular biomarkers for improved prognosis and development of effective and targeted therapies. In this study, the next generation sequencing Ion AmpliSeq™ Cancer Hotspot Panel v2 was used to identify cancer-related gene mutations in the tissue samples from 20 ES patients. This platform targeted 207 amplicons of 2800 loci in 50 cancer-related genes. Among the 20 tissue specimens, 62 nonsynonymous hotspot mutations were identified in 26 cancer-related genes, revealing the molecular heterogeneity of ES. Among these, five novel mutations in cancer-related genes (KDR, STK11, MLH1, KRAS, and PTPN11) were detected in ES, and these mutations were confirmed with traditional Sanger sequencing. ES patients with KDR, STK11, and MLH1 mutations had higher Ki-67 proliferation indices than the ES patients lacking such mutations. Notably, more than half of the ES patients harbored one or two possible 'druggable' mutations that have been previously linked to a clinical cancer treatment option. Our results provided the foundation to not only elucidate possible mechanisms involved in ES pathogenesis but also indicated the utility of Ion Torrent sequencing as a sensitive and cost-effective tool to screen key oncogenes and tumor suppressors in order to develop personalized therapy for ES patients.

Town J, Pais H, Harrison S, et al.
Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target.
Proc Natl Acad Sci U S A. 2016; 113(13):3603-8 [PubMed] Free Access to Full Article Related Publications
The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.

Zhang Z, Li Y, Huang L, et al.
Let-7a suppresses macrophage infiltrations and malignant phenotype of Ewing sarcoma via STAT3/NF-κB positive regulatory circuit.
Cancer Lett. 2016; 374(2):192-201 [PubMed] Related Publications
The interaction between tumors cells, tumor-derived humoral factors and the bone marrow in the bone niches has been shown to be essential for bone tumor initiation and promotion. Among the tumor stromal cells, tumor-associated macrophages (TAMs) are usually the most abundant immune population. Previously, we reported that let-7a functions as a tumor suppressor in ES. Herein, we found that the suppressive effects are not only limited on the malignant phenotype of tumor cells but also on the regulation of macrophage infiltration. We observed that the let-7a expression is negatively related to macrophage infiltrations in ES. Moreover, overexpression of putative ts-miRNA let-7a significantly suppressed the recruitment of PBMCs in vitro and decreased the macrophage infiltrations in ES-xenografted tumors in vivo. Most importantly, a positive regulatory feedback loop consisting of let-7a, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) (let-7a/STAT3/NF-κB) was involved in let-7a-mediated suppressive effects. These data might provide evidence of a novel intracellular signaling network function in ES pathogenesis, and manipulating this novel feedback loop will have therapeutic potential for ES patients.

Chen S, Deniz K, Sung YS, et al.
Ewing sarcoma with ERG gene rearrangements: A molecular study focusing on the prevalence of FUS-ERG and common pitfalls in detecting EWSR1-ERG fusions by FISH.
Genes Chromosomes Cancer. 2016; 55(4):340-9 [PubMed] Free Access to Full Article Related Publications
The genetics of Ewing sarcoma (ES) are characterized by a canonical fusion involving EWSR1 gene and a member of the ETS family of transcription factors, such as FLI1 and ERG. In fact, ERG gene rearrangements represent the second most common molecular alteration, with EWSR1-ERG being identified in 5-10% of cases, while only a handful of reports document a FUS-ERG fusion. In this study, we focus on ES with ERG gene abnormalities, specifically to investigate the prevalence and clinicopathologic features of FUS-ERG fusions in a large cohort of small blue round cell tumors (SBRCTs) and compare to the eight reported FUS-positive ES. Among the 85 SBRCTs tested, seven (8.2%) cases harbored FUS gene rearrangements; six fused to ERG and one with FEV. During this investigation we came across a number of ERG-rearranged ES lacking both EWSR1 and FUS abnormalities by FISH. In one case, RNA sequencing identified an EWSR1-ERG transcript despite the negative EWSR1 rearrangements by FISH. Additional 3-color FISH fusion assay demonstrated the fusion of EWSR1 and ERG signals in all four cases negative for break-apart EWSR1 FISH. These results emphasize a potential pitfall of relying on EWSR1 FISH assay alone for diagnosis of ES. In cases with classic morphology and/or strong CD99 and ERG immunoreactivity, additional molecular testing should be applied, such as ERG FISH or RT-PCR/next generation sequencing, for a more definitive diagnosis. Although our study group is small, there were no differences noted between the clinical, morphologic features and immunoprofile of the different subsets of ERG-rearranged SBRCTs.

Yoshida A, Goto K, Kodaira M, et al.
CIC-rearranged Sarcomas: A Study of 20 Cases and Comparisons With Ewing Sarcomas.
Am J Surg Pathol. 2016; 40(3):313-23 [PubMed] Related Publications
The CIC gene rearrangement exists in a subset of small round cell sarcomas. As the nosologic relationship of these sarcomas to Ewing sarcomas remains undetermined, we examined 20 CIC-rearranged sarcomas to compare their clinicopathologic features with those of Ewing sarcomas. The CIC-rearranged sarcomas were from a group of 14 men and 6 women with a median age of 24.5 years. The primary tumor sites included the limbs, trunk wall, internal trunk, lung, cerebrum, and pharynx. A comparison of the demographic and clinical characteristics of the 20 patients with CIC-rearranged sarcomas with those of the 53 near-consecutive patients with EWSR1-rarranged Ewing sarcomas showed that there were no differences with respect to their ages and sexes. Although none of the CIC-rearranged sarcomas arose in the bone, 40% of the Ewing sarcomas primarily affected the skeleton. The overall survival of patients with Ewing sarcomas was significantly better than that for patients with CIC-rearranged sarcomas. A histologic comparison of the CIC-rearranged sarcomas with 20 EWSR1-rearranged Ewing sarcomas showed significantly higher degrees of lobulation, nuclear pleomorphism, the prominence of the nucleoli, spindle cell elements, and myxoid changes in the CIC-rearranged sarcomas. Distinguishing immunohistochemical features included heterogenous CD99 reactivity, nuclear WT1 expression, and calretinin expression in the CIC-rearranged sarcomas and NKX2.2 expression in the Ewing sarcomas. CIC-rearranged sarcomas are distinct from Ewing sarcomas clinically, morphologically, and immunohistochemically, and they should be considered a separate entity rather than being grouped within the same family of tumors.

Nugent M
microRNA and Bone Cancer.
Adv Exp Med Biol. 2015; 889:201-30 [PubMed] Related Publications
MicroRNA molecules have a variety of roles in cellular development and proliferation processes, including normal osteogenesis. These effects are exerted through post-translational inhibition of target genes. Altered miRNA expression has been demonstrated in several cancers, both in the tumor tissue and in the peripheral circulation. This may influence carcinogenesis if the specific miRNA targets are encoded by tumor suppressor genes or oncogenes. To date, most research investigating the role of microRNAs and primary bone tumors has focused on osteosarcoma and Ewing sarcoma. Several microRNAs including the miR-34 family have been implicated in osteosarcoma tumorigenesis via effects on the Notch signaling pathway. Progression, invasion, and metastasis of osteosarcoma tumor cells is also influenced by microRNA expression. In addition, microRNA expression may affect the response to chemotherapy in osteosarcoma and thus hold potential for future use as either a prognostic indicator or a therapeutic target. The EWS-FLI1 fusion protein produced in Ewing sarcoma has been shown to induce changes in miRNA expression. MicroRNA expression profiling may have some potential for prediction of disease progression and survival in Ewing sarcoma. There is limited evidence to support a role for microRNAs in other primary bone tumors, either malignant or benign; however, early work is suggestive of involvement in chondrosarcoma, multiple osteochondromatosis, and giant cell tumors of bone.

Ryland KE, Hawkins AG, Weisenberger DJ, et al.
Promoter Methylation Analysis Reveals That KCNA5 Ion Channel Silencing Supports Ewing Sarcoma Cell Proliferation.
Mol Cancer Res. 2016; 14(1):26-34 [PubMed] Free Access to Full Article Related Publications
UNLABELLED: Polycomb proteins are essential regulators of gene expression in stem cells and development. They function to reversibly repress gene transcription via posttranslational modification of histones and chromatin compaction. In many human cancers, genes that are repressed by polycomb in stem cells are subject to more stable silencing via DNA methylation of promoter CpG islands. Ewing sarcoma is an aggressive bone and soft-tissue tumor that is characterized by overexpression of polycomb proteins. This study investigates the DNA methylation status of polycomb target gene promoters in Ewing sarcoma tumors and cell lines and observes that the promoters of differentiation genes are frequent targets of CpG-island DNA methylation. In addition, the promoters of ion channel genes are highly differentially methylated in Ewing sarcoma compared with nonmalignant adult tissues. Ion channels regulate a variety of biologic processes, including proliferation, and dysfunction of these channels contributes to tumor pathogenesis. In particular, reduced expression of the voltage-gated Kv1.5 channel has been implicated in tumor progression. These data show that DNA methylation of the KCNA5 promoter contributes to stable epigenetic silencing of the Kv1.5 channel. This epigenetic repression is reversed by exposure to the DNA methylation inhibitor decitabine, which inhibits Ewing sarcoma cell proliferation through mechanisms that include restoration of the Kv1.5 channel function.
IMPLICATIONS: This study demonstrates that promoters of ion channels are aberrantly methylated in Ewing sarcoma and that epigenetic silencing of KCNA5 contributes to tumor cell proliferation, thus providing further evidence of the importance of ion channel dysregulation to tumorigenesis.

Gill SJ, Travers J, Pshenichnaya I, et al.
Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma.
PLoS One. 2015; 10(10):e0140988 [PubMed] Free Access to Full Article Related Publications
Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.

Sand LG, Scotlandi K, Berghuis D, et al.
CXCL14, CXCR7 expression and CXCR4 splice variant ratio associate with survival and metastases in Ewing sarcoma patients.
Eur J Cancer. 2015; 51(17):2624-33 [PubMed] Related Publications
PURPOSE: Ewing sarcoma (EWS) is the second most common sarcoma of bone in children and young adults. Patients with disseminated disease at diagnosis or early relapse have a poor prognosis. Our goal was to identify novel predictive biomarkers for these patients, focusing on chemokines, specifically genes involved in the CXCR4-pathway because of their established role in metastasis and tumour growth.
METHODS: Total RNA isolated from therapy-naïve tumour samples (n=18; panel I) and cell lines (n=21) was used to study expression of CXCR4-pathway related genes and CXCR4 splice variants (CXCR4-2: Small and CXCR4-1: Large) by RT-Q-PCR. Expression levels were correlated to overall survival (OS) and event free survival (EFS). Study results were validated in an independent series of 26 tumour samples (panel II) from therapy-naïve tumour samples.
RESULTS: CXCL12, CXCR4, CXCR7 and CXCL14 were expressed and high CXCR7 and CXCL14 expression showed a positive correlation with EFS and OS and a negative correlation with metastasis development. Both splice variants CXCR4 were expressed in cell lines and tumour samples and CXCR4-1/CXCR4-2 ratio was significantly higher in tumour samples compared to cell lines and correlated with an improved EFS and OS. The results from the test panel were validated in an independent sample panel.
CONCLUSIONS: We identified a set of genes involved in CXCR4 signalling that may be used as a marker to predict survival and metastasis development in Ewing sarcoma.

Kawano M, Tanaka K, Itonaga I, et al.
c-Myc Represses Tumor-Suppressive microRNAs, let-7a, miR-16 and miR-29b, and Induces Cyclin D2-Mediated Cell Proliferation in Ewing's Sarcoma Cell Line.
PLoS One. 2015; 10(9):e0138560 [PubMed] Free Access to Full Article Related Publications
Myc oncogenic transcription factor is known to inhibit tumor suppressive microRNAs (miRNAs), resulting in greater expression of their target protein related to cell cycle, invasion or anti-apoptotic factors in human cancer cells. To explore possible oncogenic factors in Ewing's sarcoma (ES), we conducted microarray-based approach to profile the changes in the expression of miRNAs and its downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). Three miRNAs, let-7a, miR-16 and miR-29b were significantly down-regulated, whereas c-Myc and cyclin D2 (CCND2) were significantly up-regulated in all tested ES cells compared with hMSCs. To verify that let-7a, miR-16 and miR-29b were the targets of c-Myc in ES cell lines, we transfected siRNA against c-Myc and confirmed the coordinate up-regulation of let-7a, miR-16 and miR-29b through the repression of c-Myc. The ES cells transfected with c-Myc-siRNA and let-7a, miR-16 and miR-29b exhibited the inhibition of the cell cycle progression. The increased expression of let-7a, miR-16 and miR-29b resulted in the reduction of CCND2 protein expression. We also demonstrated that c-Myc-siRNA treatment of ES cells was associated with the decreased expression of CCND2 as a down-stream of three miRNAs. Furthermore, the introduction of let-7a, miR-16 and miR-29b in ES cells could inhibit the c-Myc-mediated up-regulation of CCND2 resulted in the prevention of cell cycle progression. In addition, the transfection of let-7a, miR-16 and miR-29b in ES cells suppressed tumor growth ex vivo treatment. These findings suggests that the up-regulation of c-Myc inhibited the expression of let-7a, miR-16 and miR-29b subsequently induced CCND2 expression in ES cells. The present study might identify a novel oncogenic axis that c-Myc regulates the expression of CCND2 via let-7a, miR-16 and miR-29b, leading to the development new therapeutic targets for ES.

Sand LG, Jochemsen AG, Beletkaia E, et al.
Novel splice variants of CXCR4 identified by transcriptome sequencing.
Biochem Biophys Res Commun. 2015; 466(1):89-94 [PubMed] Related Publications
Chemokine receptor CXCR4 is involved in tumor growth, angiogenesis and metastasis. Its function is regulated in many ways and one of them is alternative splicing. We identified two novel coding splice variants (CXCR4-3 and CXCR4-4) of CXCR4 in Ewing sarcoma (EWS) cell lines by whole transcriptome sequencing and validated these with reverse transcriptase- PCR and Sanger sequencing. The novel splice variants were expressed at RNA level in Ewing sarcoma samples and in other tumor cell lines and placenta, but not in lung. Due to inclusion of an additional exon the new isoforms have a 70 and 33 amino acid elongation of the N-terminal end of CXCR4. For validation at protein and functional level, the identified isoforms and normal CXCR4 were cloned into an EYFP tagged vector and ectopically expressed in HEK293T cell line and EWS cell line A673. Of the novel isoforms CXCR4-3 showed cell membrane localization and a functional response after addition of CXCR4 ligand CXCL12a. CXCR4-4 showed strong cytoplasmic accumulation and no response to ligand treatment. The role of the newly discovered isoforms in CXCR4 signaling is likely to be limited. Our data stresses the importance of functional validation of newly identified isoforms.

Gomez NC, Davis IJ
Linking germline and somatic variation in Ewing sarcoma.
Nat Genet. 2015; 47(9):964-5 [PubMed] Related Publications
The identification of gene-regulatory polymorphisms that influence cancer susceptibility can identify key oncogenic pathways. A new study links a germline variant to Ewing sarcoma disease susceptibility and EWSR1-FLI1-mediated gene activation.

Grünewald TG, Bernard V, Gilardi-Hebenstreit P, et al.
Chimeric EWSR1-FLI1 regulates the Ewing sarcoma susceptibility gene EGR2 via a GGAA microsatellite.
Nat Genet. 2015; 47(9):1073-8 [PubMed] Free Access to Full Article Related Publications
Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1-dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.

Sand LG, Szuhai K, Hogendoorn PC
Sequencing Overview of Ewing Sarcoma: A Journey across Genomic, Epigenomic and Transcriptomic Landscapes.
Int J Mol Sci. 2015; 16(7):16176-215 [PubMed] Free Access to Full Article Related Publications
Ewing sarcoma is an aggressive neoplasm occurring predominantly in adolescent Caucasians. At the genome level, a pathognomonic EWSR1-ETS translocation is present. The resulting fusion protein acts as a molecular driver in the tumor development and interferes, amongst others, with endogenous transcription and splicing. The Ewing sarcoma cell shows a poorly differentiated, stem-cell like phenotype. Consequently, the cellular origin of Ewing sarcoma is still a hot discussed topic. To further characterize Ewing sarcoma and to further elucidate the role of EWSR1-ETS fusion protein multiple genome, epigenome and transcriptome level studies were performed. In this review, the data from these studies were combined into a comprehensive overview. Presently, classical morphological predictive markers are used in the clinic and the therapy is dominantly based on systemic chemotherapy in combination with surgical interventions. Using sequencing, novel predictive markers and candidates for immuno- and targeted therapy were identified which were summarized in this review.

Agelopoulos K, Richter GH, Schmidt E, et al.
Deep Sequencing in Conjunction with Expression and Functional Analyses Reveals Activation of FGFR1 in Ewing Sarcoma.
Clin Cancer Res. 2015; 21(21):4935-46 [PubMed] Related Publications
PURPOSE: A low mutation rate seems to be a general feature of pediatric cancers, in particular in oncofusion gene-driven tumors. Genetically, Ewing sarcoma is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency.
EXPERIMENTAL DESIGN: Tumor samples of 116 Ewing sarcoma patients were analyzed here. Whole-genome sequencing was performed on two patients with normal, primary, and relapsed tissue. Whole-exome sequencing was performed on 50 Ewing sarcoma and 22 matched normal tissues. A discovery dataset of 14 of these tumor/normal pairs identified 232 somatic mutations. Recurrent nonsynonymous mutations were validated in the 36 remaining exomes. Transcriptome analysis was performed in a subset of 14 of 50 Ewing sarcomas and DNA copy number gain and expression of FGFR1 in 63 of 116 Ewing sarcomas.
RESULTS: Relapsed tumors consistently showed a 2- to 3-fold increased number of mutations. We identified several recurrently mutated genes at low frequency (ANKRD30A, CCDC19, KIAA0319, KIAA1522, LAMB4, SLFN11, STAG2, TP53, UNC80, ZNF98). An oncogenic fibroblast growth factor receptor 1 (FGFR1) mutation (N546K) was detected, and the FGFR1 locus frequently showed copy number gain (31.7%) in primary tumors. Furthermore, high-level FGFR1 expression was noted as a characteristic feature of Ewing sarcoma. RNA interference of FGFR1 expression in Ewing sarcoma lines blocked proliferation and completely suppressed xenograft tumor growth. FGFR1 tyrosine kinase inhibitor (TKI) therapy in a patient with Ewing sarcoma relapse significantly reduced 18-FDG-PET activity.
CONCLUSIONS: FGFR1 may constitute a promising target for novel therapeutic approaches in Ewing sarcoma.

Li W, Li Y, Guo J, et al.
Overexpression of miR‑199b‑5p inhibits Ewing's sarcoma cell lines by targeting CCNL1.
Mol Med Rep. 2015; 12(3):3359-64 [PubMed] Free Access to Full Article Related Publications
MicroRNAs (miRNAs) are known to regulate the expression of a variety of genes, which are important in the development of several types of tumor, including Ewing's sarcoma (ES), at the post‑transcriptional level. Although previous studies have identified that the expression of miRNA‑199b‑5p was downregulated in various types of tumor, the expression levels of miR‑199b‑5p in ES cells remain to be elucidated. The mechanism underlying ES via the miRNA pathway remains to be elucidated. The present study demonstrated that miR‑199b‑5p was an important regulator in ES cells and its expression was downregulated in ES originated A673/TC252 cells. The ES cell lines, A673 and TC252, were transfected with an miR‑199b‑5p mimic to overexpress the levels of this miRNA. This forced expression of miR‑199b‑5p suppressed the cell proliferation and invasion, arrested cell cycle progression, and promoted cell apoptosis. Furthermore, CCNL1 was identified by bioinformatic software as a potential target gene of miR‑199b‑5p. Following this, the present study identified CCNL1 as a direct target of miR‑199b‑5p in ES cells. Taken together, the present study established a functional link between ES, miR‑199b‑5p and CCNL1, and suggested that miR‑199b‑5p acts as a tumor suppressor and may be of diagnostic and therapeutic importance for human ES.

Kitagawa T, Okita H, Baron B, et al.
Mutant screening for oncogenes of Ewing's sarcoma using yeast.
Appl Microbiol Biotechnol. 2015; 99(16):6737-44 [PubMed] Related Publications
Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.

Duggimpudi S, Larsson E, Nabhani S, et al.
The cell cycle regulator CCDC6 is a key target of RNA-binding protein EWS.
PLoS One. 2015; 10(3):e0119066 [PubMed] Free Access to Full Article Related Publications
Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma.

Kang MH, Wang J, Makena MR, et al.
Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewing's family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression.
Clin Cancer Res. 2015; 21(5):1139-50 [PubMed] Related Publications
PURPOSE: To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI).
EXPERIMENTAL DESIGN: Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting.
RESULTS: Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines.
CONCLUSIONS: In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted.

Todorova R
Structure-function based molecular relationships in Ewing's sarcoma.
Biomed Res Int. 2015; 2015:798426 [PubMed] Free Access to Full Article Related Publications
Ewing's Sarcoma Oncogene (ews) on chromosome 22q12 is encoding a ubiquitously expressed RNA-binding protein (EWS) with unknown function that is target of tumor-specific chromosomal translocations in Ewing's sarcoma family of tumors. A model of transcription complex was proposed in which the heterodimer Rpb4/7 binds to EAD, connecting it to Core RNA Pol II. The DNA-binding domain, provided by EFP, is bound to the promoter. Rpb4/7 binds RNA, stabilizing the transcription complex. The complex Rpb4/7 can stabilize the preinitiation complexes by converting the conformation of RNA Pol II. EWS may change its conformation, so that NTD becomes accessible. Two different mechanisms of interaction between EWS and RNA Pol II are proposed: (I) an intermolecular EWS-EWS interaction between two molecules, pushing conformation from "closed" to "open" state, or (II) an intramolecular interaction inside the molecule of EWS, pushing conformation of the molecule from "closed" to "open" state. The modified forms of EWS may interact with Pol II subunits hsRpb5 and hsRpb7. The EWS and EFPs binding partners are described schematically in a model, an attempt to link the transcription with the splicing. The proposed model helps to understand the functional molecular interactions in cancer, to find new partners and ways to treat cancer.

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] Free Access to Full Article 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] Free Access to Full Article 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] Free Access to Full Article 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.

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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