title: Molecular diagnosis of Ewing tumors: improved detection of EWS-FLI-1 and EWS-ERG chimeric transcripts and rapid determination of exon combinations.
abstract: Most Ewing tumors (ET), including Ewing sarcomas, peripheral primitive neuroectodermal tumors (PNET), and Askin's tumors, can be defined according to the specific chromosomal translocations t(11;22)(q24;q12) (EWS-FLI-1) or t(21;22)(q21;q12) (EWS-ERG). Detection of the chimeric RNA transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) has greatly facilitated the diagnosis of ET. Because of variable chromosomal breakpoint locations, however, the EWS gene fusions with FLI-1 and ERG genes are highly heterogenous, resulting in different sizes of the amplification products. To improve the diagnostic usefulness of the RT-PCR assay, we have developed an assay to detect chimeric mRNA transcripts by nested RT-PCR, followed by digestion of the PCR fragments with three different restriction endonucleases. This allows confirmation of the specificity of the PCR product and provides a rapid method to determine the combination of exons present in a transcript. In the 12 Ewing tumors tested, five different exon combinations were detected. In nine repeat biopsies of four patients, the case-specific translocation remained unchanged. One additional central PNET had no ET-specific translocation. In conclusion, the suggested combination of RT-PCR and restriction analysis of the PCR products allows a rapid and specific determination of ET-specific translocations.
authors.affiliation: Institute for Pathology, University of Basel, Switzerland.
authors.forename: V S
authors.initials: VS
authors.lastname: Meier
authors.forename: T
authors.initials: T
authors.lastname: Kühne
authors.forename: G
authors.initials: G
authors.lastname: Jundt
authors.forename: F
authors.initials: F
authors.lastname: Gudat
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002880:Chromosomes, Human, Pair 11; D002891:Chromosomes, Human, Pair 21; D002892:Chromosomes, Human, Pair 22; D004268:DNA-Binding Proteins; D005091:Exons / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D015513:Oncogene Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016133:Polymerase Chain Reaction; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D012194:RNA-Directed DNA Polymerase; D012512:Sarcoma, Ewing / Q000175:diagnosis* / Q000235:genetics*; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D000071230:Transcriptional Regulator ERG; D014178:Translocation, Genetic
chemical_list: D004268:DNA-Binding Proteins; C072312:ERG protein, human; C106031:EWS-FLI fusion protein; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D015534:Trans-Activators; D014157:Transcription Factors; D000071230:Transcriptional Regulator ERG; D012194:RNA-Directed DNA Polymerase