Ewing's Sarcoma Translocation Research Articles

Activation of recombinational repair in Ewing sarcoma cells carrying EWS-FLI1 fusion gene by chromosome translocation

Chromosome translocation (TL) is an important mode of genomic changes underlying human tumorigenesis, the detailed mechanisms of which are, however, still not well understood. The two major modalities of DNA double strand break repair, i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ), have been hypothesized. In a typical TL+ human neoplasm, Ewing sarcoma, which is frequently associated with t(11;22) TL encoding the EWS-FLI1 fusion gene, NHEJ has been regarded as a model to explain the disease-specific TL. Using comprehensive microarray approaches, we observed that expression of the HR genes, particularly of RAD51, is upregulated in TL+ Ewing sarcoma cell lines, WE-68 and SK-N-MC, as in the other TL+ tumor cell lines and one defective in DNA mismatch repair (MMR). The upregulated RAD51 expression indeed lead to frequent focus formation, which may suggest an activation of the HR pathway in these cells. Furthermore, sister chromatid exchange was frequently observed in the TL+ and MMR-defective cells. Intriguingly, ionizing irradiation revealed that the decrease of 53BP1 foci was significantly retarded in the Ewing sarcoma cell lines, suggesting that the NHEJ pathway may be less active in the cells. These observations may support an HR involvement, at least in part, to explain TL in Ewing sarcoma.

PET-CT imaging features that differentiate between positive and negative EWSR1 translocation in Ewing sarcoma.

Ewing sarcoma breakpoint region 1 (EWSR1) translocation-negative tumors represent a minor portion of small round cell tumors consistent with Ewing sarcoma morphology. The purpose of this study was to differentiate EWSR1 translocation-positive tumors from EWSR1 translocation-negative tumors using PET-computed tomography features. In this retrospective study 27, Ewing sarcoma patients (December 2011 to November 2016) were divided into two groups, EWSR1 translocation-positive and EWSR1 translocation-negative based on cytogenetic analysis. Pretreatment standardized uptake value maximum (SUVmax) and Hounsfield Units (HU) were measured in the primary tumor in the axial slice with the largest tumor diameter.The associations between SUVmax, HU and the presence of EWSR1 translocation were evaluated. Receiver operating characteristic curve analysis was used to determine cut-off levels of SUVmax and HU suggestive of EWSR1-negative tumors. Twenty-one patients were classified as EWSR1-positive and six as EWSR1-negative. Eighteen had SUVmax and 21 had HU measurements. EWSR1-negative tumors had significantly higher SUVmax values (P = 0.003) and significantly lower HU values (P = 0.008). Receiver operating characteristic curve analysis showed that SUVmax had diagnostic ability to discriminate between EWSR1-negative and EWSR1-positive tumors (area under the curve = 0.964, P = 0.006). A SUVmax of at least 10 had a sensitivity of 100% and specificity of 85.7% for EWSR1-negative tumors. HU had lower diagnostic ability than SUVmax (area under the curve = 0.881, P = 0.012). A HU up to 57 had a sensitivity of 81.3% and specificity of 80.0% for EWSR1-negative tumors. Higher SUVmax and lower HU may differentiate between EWSR1-positive and EWSR1-negative tumors. This may reflect EWSR1-negative tumor aggressiveness.

The association between let-7, RAS and HIF-1α in Ewing Sarcoma tumor growth.

Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family.We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment.Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.

Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription

Chromosomal translocations involving transcription factor genes have been identified in an increasingly wide range of cancers. Some translocations can create a protein "chimera" that is composed of parts from different proteins. How such chimeras cause cancer, and why they cause cancer in some cell types but not others, is not understood. One such chimera is EWS-FLI, the most frequently occurring translocation in Ewing Sarcoma, a malignant bone and soft tissue tumor of children and young adults. Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately, transcriptional dysregulation.

Putative tumor suppressor miR‐145 inhibits colon cancer cell growth by targeting oncogene friend leukemia virus integration 1 gene

Tumor suppressor microRNA miR-145 is commonly down-regulated in colon carcinoma tissues, but its specific role in tumors remains unknown. In this study, the authors identified the Friend leukemia virus integration 1 gene (FLI1) as a novel target of miR-145. FLI1 is involved in t(11;22)(q24:q12) reciprocal chromosomal translocation in Ewing sarcoma, and its expression appears to be associated with biologically more aggressive tumors. The authors demonstrated that miR-145 targets a putative microRNA regulatory element in the 3'-untranslated region (UTR) of FLI1, and its abundance is reversely associated with FLI1 expression in colon cancer tissues and cell lines. By using a luciferase/FLI1 3'-UTR reporter system, they found that miR-145 down-regulated the reporter activity, and this down-regulation was reversed by anti-miR-145. Mutation of the miR-145 microRNA regulatory element sequence in the FLI1 3'-UTR abolished the activity of miR-145. miR-145 decreased FLI1 protein but not FLI1 mRNA, suggesting a mechanism of translational regulation. Furthermore, the authors demonstrated that miR-145 inhibited cell proliferation and sensitized LS174T cells to 5-fluorouracil-induced apoptosis. Taken together, these results suggest that miR-145 functions as a tumor suppressor by down-regulating oncogenic FLI1 in colon cancer.

Malignant transformation of teratoma to primitive neuroectodermal tumor (PNET): Outcome analysis with retroperitoneal lymph node dissection and PNET specific chemotherapy

5081 Background: Malignant transformation of teratoma to PNET is a rare entity. Surgical resection has been the mainstay of therapy because these tumors are not curable with cisplatin based chemotherapy. We report long-term survival and potential cure with retroperitoneal lymph node dissection (RPLND) and PNET specific chemotherapy. Methods: Retrospective review of 75 patients (pts) with PNET in the testis or at distant metastasis treated from Jan 1988 to Dec 2007. 74 had RPLND as part of initial treatment or at relapse. PNET specific chemotherapy consisted of cyclophosphamide, doxorubicin, vincristine alternating with ifosfamide and etoposide. Available PNET specimens were tested for the Ewing's sarcoma (EWS) translocation using a FISH-based method. Results: The median follow-up was 40 months (range 2 to 235). 27 pts presented with clinical stage I disease. 18 underwent primary RPLND with PNET in the retroperitoneum in 5. 4 are dead of disease (DOD). 9 elected surveillance or adjuvant chemotherapy. 8 relapsed with PNET. 4 are DOD. 48 pts presented with metastatic disease. 20 are DOD, 24 have no evidence of disease (NED) and 4 are alive with disease. 50 of 75 pts had PNET documented metastasis with an estimated 5 years disease specific survival of 47%. 10 of these were treated with PNET specific chemotherapy for unresectable disease. 8 of the 10 achieved objective response with the duration of response ranging from 4 to 73 months. 2 pts are NED. 2 additional pts were treated with PNET specific chemotherapy as adjuvant to RPLND. Both are continuously NED. Specimens from 14 pts were tested for the EWS translocation, 2 were positive. Conclusions: Malignant transformation of teratoma to PNET carries an adverse prognosis. RPLND is an integral part of the therapeutic strategy. PNET specific chemotherapy, adjuvant to RPLND or for treatment of unresectable disease followed by surgery, may result in long-term survival and potential cure. No significant financial relationships to disclose.

Contribution of Cytogenetics to the Management of Poorly Differentiated Sarcomas

Sarcomas constitute a heterogeneous group of rare tumors that in recent years have been shown by cytogenetic analysis to have a remarkably high incidence of specific and primary alterations. These genetic alterations not only have guided molecular studies in establishing the underlying genes involved, thereby yielding important pathogenetic information, but have also provided clinicians with a valuable tool to add to their diagnostic armamentarium. The addition of molecular cytogenetic (fluorescence in situ hybridization [FISH]) and molecular approaches (reverse transcriptase-polymerase chain reaction [RT-PCR]) has further enhanced the sensitivity and accuracy of detecting nonrandom chromosomal imbalances and/or structural rearrangements in sarcomas, including assessment in formalin-fixed, paraffin-embedded tissues. Poorly differentiated sarcomas represent a significant challenge to the pathologist as these neoplasms lack an identifiable hematoxylin and eosin-stained phenotype and often have lost diagnostic immunohistochemical or ultrastructural features as well. In contrast, primary cytogenetic changes and associated molecular events such as the 11;22 translocation in Ewing sarcoma are retained as a given tumor metastasizes or becomes less differentiated, as their presence appears to be vital for sustaining neoplastic transformation. Consequently, demonstration of characteristic, tumor-specific chromosomal aberrations is especially useful in the management of poorly differentiated sarcomas.

Molecular characterization of the genomic breakpoint junction in a t(11;22) translocation in Ewing sarcoma

Molecular characterization of the genomic breakpoint junction in a t(11;22) translocation in Ewing sarcoma

Molecular characterization of the genomic breakpoint junction in a t(11;22) translocation in Ewing sarcoma

Polymerase chain reaction (PCR)-based nucleotide sequence analysis was performed in 12 cases of Ewing sarcoma on the cDNA and/or genomic DNA breakpoint regions of a t(11;22)(q24;q12), which joins the EWS gene located on chromosome 22 with the FLI1 gene located on chromosome 11, in order to understand the molecular mechanism of this translocation. Reverse transcriptase-PCR on total tumor cell RNA from the examined cases showed five types of EWS-FLI1 chimeric product, resulting from various junctions between EWS exon 7 or 10 with FLI1 exon 5, 6, or 8. Sequencing of the genomic fusion junctions of EWS-FLI1 in seven cases showing three types of the chimeric cDNA products revealed that most of the breakpoint junctions shared common nucleotide(s) from both genes, and that the breakpoints in EWS introns 7 and 10 clustered within 100 bp and 300 bp, respectively. All the junctions were found to be flanked by various oligomers, among which a consensus sequence, 5'-AGAAAARDRR-3', was found near the breakpoints of both genes in four cases, suggesting that these oligomers may have a functional significance in the genesis of t(11;22). In addition to these oligomers, sequences highly homologous to Alu repeats and/or eukaryotic topoisomerase II cleavage sites were located near, or flanked, or even encompassed, the breakpoints in most of the cases examined. Thus, these sequences may also mediate DNA double-strand breakage and rejoining to generate the t(11;22). Genomic sequence analysis of both EWS-FLI1 and FLI1-EWS chimeric genes in three of the seven cases demonstrated a deletion and duplication of both EWS and FLI1 sequences in two cases and no gain or loss in one case. The present findings suggest that multiple mechanisms may be operative for the break and rejoining of the fragments of chromosomes 11 and 22 in the genesis of t(11;22), and that some of these translocations are asymmetric at the molecular level.

Translocation of chromosomes 11 and 22 in choroidal metastatic ewing sarcoma detected by fluorescent in situ hybridization

PURPOSE: To describe a patient with metastasis of Ewing sarcoma to the choroid and the molecular genetics of the tumor.METHODS: A 26-year-old woman with metastatic Ewing sarcoma developed large choroidal masses in the left eye and died 2 months later. Autopsy of the eyes was performed. Dual-color fluorescent in situ hybridization was used to detect genetic alteration in the ocular tumor with EWS and FLI-1 probes.RESULTS: Histopathology confirmed choroidal metastatic Ewing sarcoma. Molecular analysis showed chromosomal translocation t(11;22)(q24;q12) or EWS/FLI-1 rearrangement in the malignant cells of the eye.CONCLUSIONS: Ewing sarcoma can rarely metastasize to the uvea. Molecular detection of the t(11;22)(q24;q12) translocation in Ewing sarcoma is valuable in the differential diagnosis of small round cell tumors.

Case 6 Intra-Abdominal Polyphenotypic Tumor

The presence of t(11;22)(q24;q12) is often considered diagnostic of Ewing sarcoma and peripheral primitive neuroectodermal tumor (pPNET). We report a case of a polyphenotypic tumor that possessed this translocation as detected by reverse transcriptase polymerase chain reaction (RT-PCR). This tumor was positive for vimentin, desmin, low-molecular-weight keratin, neuron-specific enolase, S-100 protein, and CD57 by immunohistochemistry. Of note, the tumor was negative for MIC2. The tumor had double-minute chromosomes with > 100 copies of the MDM2 gene. Thus, the presence of the t(11;22)(q24;q12) translocation should not be considered diagnostic of Ewing sarcoma and pPNET in the absence of supporting histologic evidence such as positive staining for MIC2. The presence of this translocation in Ewing sarcoma and pPNET has been taken as evidence that these two tumors are related. Rather than extending this relationship to include some polyphenotypic tumors, other tumors may acquire this genetic change during tumor progression. Treatment regimens for tumors may be better based on phenotype rather than genotype when these two profiles are seemingly in conflict.

High-resolution physical mapping of a 250-kb region of human chromosome 11q24 by genomic sequence sampling (GSS)

A physical map of the region of human chromosome 11q24 containing the FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma and primitive neuroectodermal tumors, was analyzed by genomic sequence sampling. Using a 4- to 5-fold coverage chromosome 11-specific library, 22 region-specific cosmid clones were identified by phenol emulsion reassociation hybridization, with a 245-kb yeast artificial chromosome clone containing the FLI1 gene, and by directed “walking” techniques. Cosmid contigs were constructed by individual clone fingerprinting using restriction enzyme digestion and assembly with the Genome Reconstruction and AsseMbly (GRAM) computer algorithm. The relative orientation and spacing of cosmid contigs with respect to the chromosome was determined by the structural analysis of cosmid clones and by direct visual in situ hybridization mapping. Each cosmid clone in the contig was subjected to “onepass” end sequencing, and the resulting ordered sequence fragments represent ∼5% of the complete DNA sequence, making the entire region accessible by PCR amplification. The sequence samples were analyzed for putative exons, repetitive DNAs, and simple sequence repeats using a variety of computer algorithms. Based upon the computer predictions, Southern and Northern blot experiments led to the independent identification and localization of the FLI1 gene as well as a previously unknown gene located in this region of chromosome 11q24. This approach to high-resolution physical analysis of human chromosomes allows the assembly of detailed sequence-based maps and provides a tool for further structural and functional analysis of the genome.

Diagnostic value of the molecular genetic detection of the t(11;22) translocation in Ewing's tumours.

One consistent feature of the Ewing's tumour family is the presence of a balanced translocation involving band q12 and band q24 of chromosome 22 and chromosome 11. Recent cloning of the chromosome breakpoint regions of t(11;22)(q24;q12) Ewing's sarcoma translocation has revealed that the breakpoints were localized within the Ewing's sarcoma gene (EWS gene) on chromosome 22 and the Fli-1 gene on chromosome 11. Molecular genetic techniques can thus be applied to the detection of the t(11;22) translocation in Ewing's tumours. By reverse transcription and polymerase chain reaction technique (RT-PCR) 11 Ewing's tumour derived cell lines, 12 primary Ewing's tumours, and 11 tumours after treatment were analysed for the occurence of the t(11;22) translocation. Furthermore, blood and bone marrow samples from 5 patients were available for RT-PCR. In 78% of the cell lines and 91% of the primary Ewing's tumours the t(11;22) translocation was detectable by RT-PCR. In bone marrow samples from a Ewing's sarcoma patient presenting in relapse tumour cells were detected by molecular genetic analysis. Our results indicate that molecular genetic detection of the t(11;22) translocation is valuable in the differential diagnosis of small round cell tumours and will provide important information for the staging and prognosis of Ewing's tumour.

A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS–family transcription factor, ERG

The t(11;22)(q24;q12), present in 85% of Ewing's sarcoma and related tumours, fuses the EWS gene from chromosome 22q12 and the ETS family member, FLI-1. This results in the expression of a chimaeric protein containing the amino-terminal portion of EWS fused to the ETS DNA-binding domain of FLI-1. We have identified a second Ewing's sarcoma translocation, t(21;22)(q22;q12), that fuses EWS to a different ETS family member, the ERG gene located on band 21q22. Identical EWS nucleotide sequences found in the EWS/FLI-1 fusion transcripts are fused to portions of ERG encoding an ETS DNA-binding domain resulting in expression of a hybrid EWS/ERG protein. These findings suggest that fusion of EWS to different members of the ETS family of transcription factor genes may result in the expression of similar disease phenotypes.

A novel and rapid method for isolating sequences adjacent to rare cutting sites and their use in physical mapping

We describe a simple PCR based technique which can be used to isolate sequences adjacent to rare cutter sites and can subsequently be employed for the construction of long range physical maps. The method involves the ligation of an adaptor to rare cutter sequences and its use as a target for forward priming in PCR. Primers to Alu repeat elements initiate synthesis of the reverse strand. Using this technique any rare cutter site which has a repeat element within amplification range can be cloned. We have isolated six unique sequences around NotI sites from an irradiation reduced hybrid containing a fragment of human chromosome 22 and are using these for physical mapping around the Ewing's sarcoma translocation breakpoint on chromosome 22.