Aberrant chromosomal fusion of the Ewing's sarcoma oncogene (EWS) to several different cellular partners produces the Ewing's family of oncoproteins (EWS-fusion-proteins, EFPs) and associated tumors (EFTs). EFPs are potent transcriptional activators, dependent on the N-terminal region of EWS (the EWS-activation-domain, EAD) and this function is thought to be central to EFT oncogenesis and maintenance. Thus EFPs are promising therapeutic targets, but detailed molecular studies will be pivotal for exploring this potential. Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function. Therefore, the lack of manipulatable assays for the EAD presents a significant barrier to progress. Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient, bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.
Animals ; Biological Assay ; Female ; Oncogene Proteins ; genetics ; Oncogenes ; genetics ; Oocytes ; metabolism ; pathology ; RNA-Binding Protein EWS ; genetics ; metabolism ; Sarcoma, Ewing ; genetics ; pathology ; Xenopus

OBJECTIVE: Osteosarcoma(OS) and Ewing's sarcoma (EWS) are the two most common primary malignant bone tumors in children. The aim of the study was to identify key genes in OS and EWS and investigate their potential pathways. METHODS: Expression profiling (GSE16088 and GSE45544) were obtained from GEO DataSets. Differentially expressed genes were identified using GEO2R and key genes involved in the occurrence of both OS and EWS were selected using venn diagram. Gene ontology and pathway enrichment analyses were performed for the ensembl. Protein-protein interaction (PPI) networks were established by STRING. Further, UCSC was used to predict the transcription factors of the cell division cycke 5-like(CDC5L) gene, and GEPIA was used to analyze the correlation between the transcription factors and the CDC5L gene. RESULTS: The results showed that CDC5L gene was the key gene involved in the pathogenesis of OS and EWS. The gene is mainly involved in mitosis, and is related to RNA metabolism, processing of capped intron-containing pre-mRNA, mRNA and pre-mRNA splicing. CONCLUSION CDC5L, as a key gene, plays a role in development of OS and EWS, which may be reliable targets for diagnosis and treatment of these primary malignant tumors.
Bone Neoplasms/pathology* ; Cell Cycle Proteins/genetics* ; Child ; Computational Biology ; Gene Expression Profiling ; Humans ; Osteosarcoma/genetics* ; RNA-Binding Proteins/genetics* ; Sarcoma, Ewing/genetics*

OBJECTIVETo detect tumor specific chromosome translocations and associated fusion transcripts in paraffin-embedded tissue by interphase fluorescence in-situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR), respectively, and to evaluate their diagnostic values for Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET).

METHODSNuclei of the tumor cells and total RNA were extracted from 10 cases of ES/PNET. Interphase FISH was utilized to analyze the EWS gene translocation with a dual color, break apart probe (Vysis company). RT-PCR was used to detect t (11; 22) (q24; q12) and t (21; 22) (q22; q12) fusion transcripts.

RESULTSAmong 10 cases of ES/PNET, the EWS-FLI1 fusion transcript was detected in 8 by RT-PCR. EWS-ERG fusion transcript was not detected in any of the cases. EWS gene translocation was found in 9 of 10 cases by FISH.

CONCLUSIONSInterphase FISH and RT-PCR can be reliably applied to paraffin-embedded tissues for molecular diagnosis of ES/PNET. Between the two approaches, interphase FISH provides a more sensitive and stable result.

Adolescent ; Adult ; Child ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Neuroectodermal Tumors, Primitive, Peripheral ; diagnosis ; genetics ; Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sarcoma, Ewing ; diagnosis ; genetics

Ameloblastoma ; pathology ; Bone Neoplasms ; classification ; genetics ; pathology ; Chondromatosis ; pathology ; Chondrosarcoma ; classification ; pathology ; Enchondromatosis ; pathology ; Humans ; Osteosarcoma ; classification ; pathology ; Sarcoma, Ewing ; pathology ; Societies, Medical ; United States ; World Health Organization

Objective: To investigate the clinicopathological characteristics, pathological diagnosis of Ewing's sarcoma of the central nervous system. Methods: Six cases of Ewing's sarcoma of the central nervous system diagnosed at the First Affiliated Hospital of Nanjing Medical University, Nanjing, China from 2015 to 2022 were collected. The clinical manifestations, histological morphology, immunophenotype and molecular genetics of these cases were analyzed. The related literature was reviewed. Results: There were four males and two females, with a male to female ratio of 2∶1. The onset age was 17-40 years, with a median age of 23 years. All 6 tumors were located in the spinal cord (2 cases of cervical vertebra, 1 case of thoracic vertebra, 2 cases of lumbar vertebra, and 1 case of sacral vertebra). The patients' clinical manifestations were mostly lumbago, weakness and numbness of lower limbs/limbs. In 1 case, the tumor recurred and metastasized to the suprasellar region and the third ventricle. Microscopically, the tumor showed diffuse infiltrative growth. In some cases, the tumor was closely related to the spinal meninges. The tumor cells were arranged in sheet, lobular, thin-rope, and nest-like patterns. Homer-Wright rosette was visible. The tumor cells were small to medium in size, and most of them had scant cytoplasm. A few cells had clear cytoplasm. Some areas were rhabdoid. The tumor cell nuclei showed focal mild pleomorphism. The chromatin was uniform and delicate while the nucleoli were not obvious. Mitosis was commonly seen. The tumor was separated by fibrous connective tissue and may be accompanied by mucinous degeneration. Immunohistochemistry showed that all tumors were positive for CD99, NKX2.2, Fli1, ERG. ATRX, H3K27me3, INI1 and BRG1 were all retained. Immunohistochemical stains for EMA, GFAP and Olig2 were negative. The Ki-67 proliferation index was 30%-70%. EWSR1 break-apart FISH test was positive. Conclusions: Ewing's sarcoma is rare in the central nervous system and needs to be distinguished from a variety of neoplasms with primitive undifferentiated small cell morphology. Immunohistochemistry and molecular genetics may be required for a proper diagnosis.
Humans ; Male ; Female ; Young Adult ; Adult ; Adolescent ; Sarcoma, Ewing/pathology* ; Proto-Oncogene Protein c-fli-1 ; Immunohistochemistry ; Biomarkers, Tumor/genetics* ; Central Nervous System/pathology*

Extraskeletal Ewing's sarcoma (EES) is a branch of neuroectodermal tumor (PNET), which is very rare soft tissue sarcoma. We report a case of EES/PNET arising is the lung of a 67-yr-old man. Computed tomography, bone scintigraphy, and positron emission tomography confirmed the mass to have a primary pulmonary origin. The mass showed positive reactivity in the Periodic Acid Schiff (PAS) stain and MIC-2 immunoreactivity in immunohistochemical stain. Fluorescence in situ hybridization (FISH) was performed, which revealed an EWSR1 (Ewing sarcoma breakpoint region 1) 22q12 rearrangement. The diagnosis was confirmed both pathologically and genetically. The mass lesion was resected, and the patient is currently undergoing chemotherapy.
Aged ; Calmodulin-Binding Proteins/genetics ; Chromosome Breakage ; Chromosomes, Human, Pair 22/genetics ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms/*diagnosis/genetics/metabolism/pathology ; Male ; Neuroectodermal Tumors, Primitive, ; RNA-Binding Proteins/genetics ; Sarcoma, Ewing's/*diagnosis/genetics/metabolism/pathology

Base Sequence ; Formaldehyde ; chemistry ; Humans ; Molecular Sequence Data ; Oncogene Proteins, Fusion ; genetics ; Paraffin Embedding ; Proto-Oncogene Protein c-fli-1 ; genetics ; RNA, Neoplasm ; genetics ; metabolism ; RNA-Binding Protein EWS ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rhabdomyosarcoma ; genetics ; Sarcoma, Ewing ; genetics ; Sarcoma, Synovial ; genetics ; Soft Tissue Neoplasms ; genetics ; Tissue Fixation

Extraskeletal Ewing's sarcoma (EES) is a rare soft tissue tumor morphologically indistinguishable from the more common Ewing's sarcoma of bone. We report a case of EES arising in the hard palate of 34-yr-old male patient. Microscopically, the monotonous small round cells without neuronal differentiation showed membranous positive immunoreactivity for MIC2/CD99 and vimentin. Ultrastructurally, the tumor cells showed a few intracytoplasmic organelles without evidence of neurosecretory granules or neurofilaments. The EWS-FLI1 chimeric gene was identified using the nested reverse transcriptase-polymerase chain reaction.
Adult ; Antigens, CD/analysis ; Cell Adhesion Molecules/analysis ; Humans ; Immunohistochemistry ; Male ; Oncogene Proteins, Fusion/genetics ; Palatal Neoplasms/genetics/metabolism/*pathology ; Palate, Hard/metabolism/*pathology ; Proto-Oncogene Protein c-fli-1/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sarcoma, Ewing's/genetics/metabolism/*pathology ; Transcription, Genetic ; Vimentin/analysis

BACKGROUNDEwing's sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is often difficult to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival materials, we evaluated the feasibility of detecting EWS-Ets including EWS-Fli1 and EWS-ERG fusion transcripts in paraffin-embedded tissues and its diagnostic value for detecting ES/pPNET.

METHODSThirteen paraffin-embedded samples of ES/pPNETs were retrieved from archives. Thirteen cases of other tumors with small round cell features (including rhabdomyosarcoma, neuroblastoma, lymphoma, small cell carcinoma, and desmoplastic small round cell tumor) were used as negative controls. Beta-actin and beta2-microglobulin were used as internal controls. A nested reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay was performed to detect the EWS-Fli1 and EWS-ERG fusion transcripts.

RESULTSBeta-actin and beta2-microglobulin were detected in 10/13 and 13/13 ES/pPNETs, respectively. EWS-Fli1 fusion transcripts were detected in 11 of 13 (85%) ES/pPNETs. Three chimeric transcripts, all EWS-Fli1, were detected in ES/pPNET samples. Among 11 EWS-Fli1-positive cases, 7 cases had a type I fusion transcript involving fusion of EWS exon 7 with Fli1 exon 6, 2 cases had a type II fusion transcript involving EWS exon 7 with Fli1 exon 5, and 2 cases expressed fusion transcripts involving EWS exon 7 and Fli1 exon 8. Type I EWS-Fli1 fusion predominated over other types. Fusion types could not be distinguished in the remaining 2 cases. Thirteen negative controls did not show detectable chimeric messages. There was a significant relationship between EWS-Fli1 fusion transcripts and CD99 expression.

CONCLUSIONSMolecular detection of EWS-Fli1 fusion transcripts in formalin-fixed paraffin-embedded material by nested RT-PCR is feasible and is useful for the diagnosis and differential diagnosis of ES/pPNETs.

Adolescent ; Adult ; Child ; Female ; Humans ; Male ; Middle Aged ; Neuroectodermal Tumors, Primitive, Peripheral ; genetics ; pathology ; Oncogene Proteins, Fusion ; genetics ; Polymerase Chain Reaction ; Proto-Oncogene Protein c-fli-1 ; RNA, Messenger ; analysis ; RNA-Binding Protein EWS ; Sarcoma, Ewing ; genetics ; pathology ; Transcription Factors ; genetics

OBJECTIVETo find out a possible approach to improve the effectiveness of radiotherapy and chemotherapy for Ewing's sarcoma by constructing a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia and to evaluate the effects of this HRE regulated HSV-TK system on killing effect of gancyclovir (GCV) on Ewing's sarcoma cell line SK-ES under hypoxic condition.

METHODSThe HRE was synthesized according to the literature and cloned into the enhancer site of pIRES(2)-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the multiple clone site of pIRES(2)-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing's sarcoma cell line SK-ES was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxic (21% oxygen) or hypoxic (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing's sarcoma cell line SK-ES transfected with pTK or pHRE-TK recombinant plasmid to the anti-tumour drug GCV was determined with the method of tetrazolium (MTT) after treating with GCV for five days.

RESULTS(1) The result of sequencing showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction. (2) Comparison of fluorescent optical density (FOD) showed that (1) the EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia (P < 0.01); (2) when the cells were exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group (P < 0.01); (3) there was no significant difference among the four groups of cells when they were exposed to normoxia (P > 0.05). (3) Comparison of the sensitivity of four groups of cells to GCV showed that (1) the cells in pHRE-TK and pTK groups were much more sensitive to GCV than the cells in pHRE group under hypoxia condition (P < 0.01), the higher the GCV concentration, the greater the difference; (2) the cells of pHRE-TK group were more sensitive to GCV than those in pTK group under hypoxic condition (P < 0.01), but was almost equally sensitive under normoxic condition (P > 0.05); (3) the pHRE-TK group cells had higher sensitivity to GCV under hypoxia than normoxia (P < 0.01) while the pTK group cells had almost the same sensitivity to GCV under hypoxia and normoxia (P > 0.05).

CONCLUSION(1) The eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed successfully. (2) HRE could up-regulate expression of EGFP by SK-ES cells under hypoxia condition. (3) HRE could enhance the killing effect of HSV-TK/GCV system on human Ewing's sarcoma cell line SK-ES under hypoxic condition.

Antiviral Agents ; pharmacology ; Cell Hypoxia ; drug effects ; genetics ; Cell Line, Tumor ; Ganciclovir ; pharmacology ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Humans ; Microscopy, Fluorescence ; Plasmids ; Polymerase Chain Reaction ; Response Elements ; genetics ; Sarcoma, Ewing ; drug therapy ; metabolism ; Simplexvirus ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Transfection