MOLECULAR DIAGNOSIS OF EWING TUMORS - IMPROVED DETECTION OF EWS-FLI-1AND 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 acc ording to the specific chromosomal translocations t(11;22)(q24;q12) (E WS-FLI-1) or t(21,22)(q21; q12) (EWS-ERG). Detection of the chimeric R NA 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 F LI-1 and ERG genes are highly heterogenous, resulting in different siz es of the amplification products. To improve the diagnostic usefulness of the RT-PCR assay, we have developed an assay to detect chimeric mR NA transcripts by nested RT-PCR, followed by digestion of the PCR frag ments with three different restriction endonucleases. This allows conf irmation of the specificity of the PCR product and provides a rapid me thod to determine the combination of exons present in a transcript. In the 12 Ewing tumors tested, five different exon combinations were det ected. In nine repeat biopsies of four patients, the case-specific tra nslocation remained unchanged. One additional central PNET had no ET-s pecific translocation. In conclusion, the suggested combination of RT- PCR and restriction analysis of the PCR products allows a rapid and sp ecific determination of ET-specific translocations.