Cryptic EWSR1-FLI1 fusions in Ewing sarcoma: potential pitfalls in the diagnostic use of fluorescence in situ hybridization probes

Detection of EWSR1 translocations — particularly t(11;22)(q24;q12) — is of great value in the differential diagnosis of the Ewing family of tumors. We report two cases that highlight the problems and pitfalls of identifying Ewing tumors using conventional chromosome analysis and a commercial EWSR1 fluorescence in situ hybridization (FISH) probe. In both cases, the tumor karyotype was abnormal, but a visible t(11;22)(q24;q12) was not present. The commercial EWSR1 “break-apart” probe was not split in either case. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, however, identified EWSR1-FLI1 fusion transcripts in both tumors, and the gene fusions were corroborated by FISH analysis with “in house” probes and confirmed by sequencing RT-PCR products. The occurrence of cryptic EWSR1-FLI1 fusions mandates that RT-PCR should be performed, particularly in those cases in which the genetic findings are not in agreement with the histologic picture.