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Author(s): Nathan C Sheffield [1]; Gaelle Pierron [2]; Johanna Klughammer [1]; Paul Datlinger [1]; Andreas Schönegger [1]; Michael Schuster [1]; Johanna Hadler [1]; Didier Surdez [3]; Delphine Guillemot [2]; Eve [...], Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

Author(s): Aparna Gorthi [1, 2]; July Carolina Romero [1, 2]; Eva Loranc [2]; Lin Cao [2]; Liesl A. Lawrence [1, 2]; Elicia Goodale [2]; Amanda Balboni Iniguez [3, 4]; Xavier [...]

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag[2.sup.-/-] [[gamma].sub.c.sup.- /-] mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated sternness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor. epigenetic regulation | Ewing tumor | sternness

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma, However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewing's sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewing's sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChiP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NROB1, a gene required for Ewing's sarcoma oncogenesis. The microsatellite in the NROB1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis. ChiP-chip | transcription | gene regulation | ETS | NROB1

Byline: M. Gary Hadfield (1), Martha M. Quezado (1), Robert L. Williams (2), Vivian Y. Luo (2) Keywords: Key words: central nervous system, Ewing's family of tumors, prognosis and treatment, molecular pathology and genetics Abstract: This review consolidates information gleaned from several case reports and larger series on Ewing's sarcoma family of tumors (EFT) involving structures related to and found in the central nervous system (CNS). These tumors involve the skull, the spinal column, adjacent soft tissues, the meninges, and the brain. We have separated the cases by skull region and spinal column level, and we discuss the attendant differences in prognosis following treatment by neurosurgery, radiation, and chemotherapy. Light and electron microscopic features can be used to differentiate EFT from other small round blue cell tumors that involve the CNS (central primitive neuroectodermal tumor, lymphoma, etc.). Recent molecular and genetic findings in EFT provide new diagnostic methods. We conclude that EFT involving the CNS and adjacent structures is not so rare as previously stated and that the prognosis is more favorable, as a rule, than for the more common examples arising in the long bones and pelvis. Author Affiliation: (1) Division of Neuropathology, Medical College of Virginia Campus/Virginia Commonwealth University, Richmond, VA 23298, USA, US (2) Department of Pathology, Medical College of Virginia Campus/Virginia Commonwealth University, Richmond, VA 23298, USA, US Article note: Received February 3, 1999 accepted November 22, 1999.

Confirmed cases of Ewing's sarcoma or neuroectodermal tumor seem to have identifiable changes in the genetic material of the tumor which can be rapidly detected with the PCR technique. Researchers collected and classified 114 tumor samples from patients who were diagnosed with Ewing's sarcoma or related tumors. RNA was taken from each sample and analyzed with the PCR technique. Investigators found structural rearrangements in the genetic material of 89 tumors. Seventy-eight tumors had an EWS gene joined to the FLI1 gene. Eleven tumors had the EWS gene fused to the ERG gene. Genetic rearrangement was observed in 97% of Ewing's sarcomas and 93% of non-typical Ewing's sarcomas or neuroectodermal tumors. Twelve tumors that did not belong to the Ewing family showed no evidence of genetic rearrangement. Six of 15 tumors that were unclassified contained genetic rearrangements.

EWSR1 and EWSR2, two proteins produced by the chromosome translocation that characterizes Ewing's sarcoma and related neuroectodermal tumors, were used to demonstrate that the translocation changes the open reading frame of an expressed gene on the chromosome 22 gene by inserting a sequence encoding a RNA-binding domain in place of the DNA-binding domain for the human homologue of the mouse gene Fli-1. This finding should stimulate further genetic research on the neuroectodermal tumors.

Cholecystokinin (CCK) is a peptide hormone that has been detected in two tumor cell lines derived from human neuroepitheliomas. An investigation was conducted to determine the expression of CCK in a larger subset of human tumor lines. The results showed that CCK is also expressed in neuroeptihelioma, Ewing sarcoma and human rhabdomyosarcoma cell lines. The expression of CCK was not detected in any of the neuroblastoma cell lines tested. The expression of CCK in these cell lines may serve as a marker to distinguish these types of tumors.

Detection of an abnormal gene fusion in Ewing's sarcoma, atypical Ewing's sarcoma, and primitive neuroectodermal tumors (PNETs) may impact patient treatment and prognosis. Ewing's sarcoma, atypical Ewing's sarcoma, and PNETs belong to the same tumor group and can be difficult to differentiate. Research using the polymerase chain reaction (PCR) revealed that 83 of 87 tumors of this type had an abnormal gene fusion. There seems to be no relationship between the type of gene fusion and the biological appearance of the disease, tumor location, or patient's age. Further research is needed in this area. The most appropriate treatment for patients with these types of tumors must also be determined through clinical studies. Although the presence of gene fusion is common to all groups, the disease processes are different. Gene therapy that targets the abnormal gene fusions may offer a future treatment for children with these tumors.