Detection of EWSR1 translocation with nuclear extraction-based fluorescence in situ hybridization for diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor.

Objective: To investigate nuclear extraction-based fluorescence in situ hybridization (NE-FISH) in EWSR1 translocation of Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) in formalin-fixed, paraffin-embedded (FFPE) tissue, compared to thin-section FISH (TS-FISH) and nested reverse-transcription polymerase chain reaction (RT-PCR).

Methods: Nuclei of the tumor cells and total ribonucleic acid (RNA) were extracted from 10 cases of ES/PNET. NE-FISH and TS-FISH were used to analyze the EWS gene translocation. RT-PCR was used to detect t(11;22)(q24;q12) and t(21;22)(q22;q12) fusion transcripts.

Results: Of 10 cases, 9 (90%) were abnormal by NE-FISH and TS-FISH. The average rate of abnormal split signals detected by NE-FISH was significantly higher than by TS-FISH. We analyzed 10 ES/PNET cases and 8 various normal human tissues by NE-FISH and nested RT-PCR. Fusion signals were found in 9 (90%) ES/PNET cases by NE-FISH and 8 (80%) by nested RT-PCR. Of 8 normal tissues, none was abnormal by either FISH or RT-PCR.

Conclusions: NE-FISH is more reliable than TS-FISH in detecting EWSR1 translocation of ES/PNET in FFPE tissue, although the FISH techniques were concordant. Nested RT-PCR could be more sensitive than conventional RT-PCR. A combination of NE-FISH and nested RT-PCR would improve accuracy of molecular diagnosis of ES/PNET.