Molecular diagnosis of Ewing tumors: improved detection of EWS-FLI-1 and EWS-ERG chimeric transcripts and rapid determination of exon combinations.

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.