Reverse transcriptase polymerase chain reaction
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.
Discussion We are reporting a cryptic insertion of chromosome 22 material (EWS) to the long arm of chromosome 11 (11q24, FLI-1) recognizable by FISH in BADGE exhibiting trisomy 8 as the sole cytogenetic anomaly. In ES and PNET tumors, trisomy 8 is the most common secondary abnormality . Additional copies of chromosome 8 are often reported as a secondary aberration and considered to be related to tumor progression. The frequent identification of trisomy 8 as secondary to other major hematological disorders  has lead investigators to consider that trisomy 8 may be a marker of cryptic rearrangements . In fact, the presence of trisomy 8 in our case, which was clinicopathologically suggestive of ES, prompted us to search for a possible cryptic t(11;22)(q24;q12). Similar coexistence of masked fusion and trisomy 8 was previously reported in a variant case of ES in which EWS was inserted into another gene of the ETS family (ERG gene on chromosome 21) . With the introduction of molecular techniques, masked fusions have been uncovered in several hematological disorders. It has been shown that there are mechanisms in addition to a cytogenetic translocation, which may lead to the formation of chimeric genes. For example, PML/RARA, AMLI/ETO, and CBFB/MYHI fusion genes were demonstrated to exist in patients where conventional cytogenetic revealed no evidence of t(15;17) , t(8;21) , and inv(16) , respectively. In these reported cases, the fusion was the result of an insertion as demonstrated by FISH and PCR. A similar mechanism appears to be responsible for the abnormalities in the current case. The frequency of chromosomal insertion, which creates functional fusion gene in hematological malignancies, is low. However, this may change as more molecular cytogenetic studies are simultaneously performed. Notably, trisomy 8 was detected in 90% of these cryptic fusion cases raising again the question whether an available diagnostic molecular test should be performed in specific cases in which trisomy 8 is found as a sole anomaly. The orientation of EWS and FLI on chromosomes 22 and 11, respectively, resides in the same orientation  5′ centromeric and 3′ telomeric. A typical balanced translocation [t(11;22)(q24;q12)] usually leads to the fusion of the 5′ portion of the EWS gene and the 3′ portion of the FLI-1 gene. Two-color hybridization of the BAC clone 222M10 for EWS and 428J4 for FLI-1 usually shows a yellow fusion signal on the derivative 22 . With the same set of probes, our case showed a fusion signal in an unexpected location on 11q instead of 22q. In addition, painting of chromosomes 11 and 22 showed no translocation of chromosome 22q12 to 11q24. Therefore, the presence of fusion on derivative 11 indicates that two breaks must have occurred on the chromosome 22 segment containing the 5′ portion of the EWS gene and one break within the FLI-1 gene. For the orientation to be 5′ centromeric and 3′ telomeric, this segment of EWS must have been inverted and inserted within the FLI-1 gene. The molecular data indicate that the common type II EWS/FLI-1 fusion transcript product is present as well as an EWS-FLI-1 variant product with fusion of different exons. The two EWS-FLI-1 fusion RNA can be produced by alternative splicing of transcripts from a single type “10-5” EWS-FLI1 fusion gene. The presence of two EWS/FLI-1 fusion transcript products in the same case has been described in other reports .