Our experiences with molecular diagnosis of Ewing´s sarcoma in paraffin-embedded tissue

Introduction: Ewing´s sarcoma is relatively uncommon tumor representing 6-8 percent of malignant bone tumors with variable morphology. No specific immunohistochemical marker of this tumor exists to date. Cytogenetically, Ewing´s sarcomas are characterized by a specific reciprocal chromosomal translocation t (11 ;22) (q24;q12). The presence of this chromosomal translocation has been detected in approximately 85 percent of cases. The translocation results in the fusion of EWS gene (Ewing sarcoma breakpoint region 1 gene) from chromosome 22 to FLI1 gene (Friend leukemia virus integration 1 gene) at 11q24 which is a member of ETS (v-ets erythroblastosis virus E26 oncogene homolog) family of transcription factors. Moreover, another chromosomal translocation t (11 ;22) (q22;q12) has been found in 10-15 percent of cases, which results in the expression of EWS-ERG fusion transcript. In 1 % or less cases t (7;22), t (17;22), and t (2;22) translocations and inv (22) have been described. The above mentioned secondary chromosomal aberration resulted in fusion between EWS gene and one of the ETS su-perfamily: Ets variant gene 1 (ETV1), Ets variant gene 4 (E1AF), fifth Ewing variant gene (FEV), and zinc finger sarcoma gene (ZSG), respectively.

Aim: In this study, we performed a comparison of two molecular diagnostic strategies, namely RT-PCR and FISH, in fresh, frozen and formalin-fixed paraffin-embedded tissues.

Results and discussion: We found that all PCR-negative Ewing´s sarcoma cases which we tested gave positive FISH results. We found that the storage time of paraffin block, even for as long as 10 years, did not affect the quality of the FISH results. We detected chromosomal aberrations in paraffin-embedded tissues stored as long as 31 years. However, the sensitivity of RT-PCR analysis in paraffin-embedded tissue could be increased if a smaller-sized amplicon (approximately 100 bp) was used for PCR because of degradation of RNAs in paraffin-embedded tissue.

Conclusion: We conclude that FISH is a more sensitive technique than RT-PCR for the diagnosis of Ewing´s sarcoma family in formalin-fixed paraffin-embedded tissue, although RT-PCR analysis provides additional information regarding fusion transcript subtype, which may play a role in prediction of prognosis. In conclusion, molecular pathology techniques, using reverse transcription-polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) are valuable diagnostic tools for the evaluation of undifferentiated small round-cell tumors like Ewing´s sarcoma. Finally, we assume that the diagnosis and prognosis of the Ewing´s sarcoma family can benefit from molecular testing with both techniques.