br Conclusions br Acknowledgement Grant support Ministry of
Acknowledgement Grant support: Ministry of Health (n. of grant: RBAP10447J_004 to N. Baldini), Italian Association for Cancer Research (n. of grant: 11426 to N. Baldini).
Introduction Ewing sarcomas (ES) and primitive neuroectodermal tumors (PNET) are related childhood tumors. They have in common a t(11;22)(q24;q12), which occurs in 85% of cases 1, 2. Variants of this translocation have been reported with the most common being t(21;22)(q22;q12) occurring in >10% of ES cases  and the least frequent being t(7;22)(p22;q12) , t(17;22)(q12;q12) , and t(2;22)(q33;q12). In all these rearrangements, the EWS gene localized to 22q12 fuses to genes of the ETS family: FLI1 at 11q24, ERG at 21q22, ETVI at 7p22, EIAF at 17q12, and FEV at 2q33 (for review see ref. ). In addition to the primary abnormality t(11;22), nonrandom secondary changes have been observed. Trisomy 8 occurs in 44% and der(16)t(1;16) in 18% of ES cases . Chromosomal translocations are the most common mechanism that lead to EWS gene fusion to ETS-family genes. Insertion of chromosomal material is another mechanism, which may result in gene fusion. This was observed in one case of ES in which the EWS gene was fused to the ERG gene on chromosome 21 . We report a cryptic fusion of EWS and FLI1, which was identified by FISH using locus-specific probes containing the FLI1 and EWS gene sequences. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies revealed the presence of two EWS/FLI1 fusion gene products. The cryptic rearrangement, which was identified in BWX 46 having trisomy 8 as the sole cytogenetic anomaly, appears to have occurred as an insertion of EWS into the FLI-1 gene on chromosome 11.
Case report A 6-year-old boy presented with a posterior mediastinal mass underwent left thoracotomy. Sections of the tumor showed large circumscribed lobules with sharp borders separating the malignant cells from connective tissue septae. Individual tumor cells were small with a high nuclear to cytoplasmic ratio. PAS staining showed positivity in neoplastic. Immunoperoxidase staining of the tumor for MIC2 with O13 monoclonal antibody revealed immunoreactivity in most of the viable tumor cells. The tumor cells were weakly immunoreactive for other immunostains to include neuron specific enolase, BCL-1, and HER-2 NEU. Electron microscopic examination revealed primitive cells that were separated with a moderate amount of intercellular edema. Within the cytoplasm, large sheets of electron-dense granular glycogen were observed. Nuclei were large with occasional prominent nucleoli.
Materials and methods
Results Cytogenetic analysis of 20 metaphase cells revealed an abnormal clone in all cells examined at 450 band resolution (Fig. 1a). Trisomy 8 was found in every cell as the sole cytogenetic abnormality (47,XY,+8). Fluorescence in situ hybridization analysis of 5 metaphase and 40 interphase cells hybridized with specific probes directed onto 11q24, 22q12, and the centromere of chromosome 8 showed three large aqua signals of CEP 8, two green signals of EWS probe on both homologues 22, one orange signals of FLI-1 probe on one chromosome 11 homologue and one fusion signal (green and orange signals) on the other copy of chromosome 11 [der(11)] (Figs. 1b & 1c) in all cells. These cells were subsequently washed and rehybridized with whole chromosome painting probes of chromosomes 11 and 22. Results showed complete painted chromosomes 11 and 22 with no obvious translocation between them (Fig. 1d). Therefore, FISH results are suggestive of an interstitial insertion of EWS into FLI-1 on 11q and absence of the typical reciprocal translocation (11;22). Reverse transcriptase-polymerase chain reaction analysis revealed two amplification products, one the size of 394 bp consistent with a type II EWS-FLI-1 fusion transcript , and the other, a 700 bp EWS-FLI-1 fusion transcript variant (Fig. 2). Sequencing studies revealed an in-frame junction between exon 7 of EWS and exon 5 of FLI-1 for the 394 bp product and an in-frame junction between exon 10 of EWS and exon 5 of FLI-1 for the 700 bp product.