Ewing sarcoma with novel translocation t(2;16) producing an in-frame fusion of FUS and FEV

Abstract

Ewing family tumors are molecularly characterized by expression of chimeric transcripts generated by specific chromosomal translocations, most commonly involving fusion of the EWS gene to a member of the ETS family of transcription factors (including FLI1, ERG, ETV1, E1AF, and FEV). Approximately 85% of reported cases of Ewing sarcoma bear an EWS-FLI1 fusion. In rare cases, FUS can substitute for EWS, with translocation t(16;21)(p11;q24) producing a FUS-ERG fusion with no EWS rearrangement. We report a case of Ewing sarcoma, presenting as a pathological fracture of the distal clavicle in a 33-year-old male, in which cytogenetic analysis revealed a single t(2;16)(q35;p11) balanced translocation. Fluorescence in situ hybridization using a commercially available diagnostic probe was negative for an EWS gene rearrangement; instead, break-apart fluorescence in situ hybridization probes for FUS and FEV were positive for a translocation involving these genes. Cloning and sequencing of the breakpoint region demonstrated an in-frame fusion of FUS to FEV. In conclusion, this represents the first reported case of Ewing family tumors demonstrating a variant translocation involving FUS and FEV and highlights the need to consider alternative permutations of fusion partners for molecular diagnosis of sarcomas.

Figure 1

A: Representative histology of the tumor specimen showing an aggressive small round blue cell tumor infiltrating through bone (H&E; ×40 magnification). B: Immunohistochemical staining of the tumor cells for CD99 showing diffusely strong membranous positivity (×400 magnification).

Figure 2

Representative metaphase from the tumor sideline showing the t(2;16)(q35;p11) (arrows) and extra copies of chromosomes 4, 16, and 21.

Figure 3

Representative FISH images. A: A sideline metaphase with +16 hybridized with the break-apart probe for FUS showing two red-green fused signals on the two intact copies of FUS on two #16 chromosomes, a green signal on the der(2) and a red signal on the der(16). B: A stemline metaphase hybridized with a 2p12 control probe (RP11-270E5-green) and the FEV BAC probe (RP11-207M4-red) showing splitting of the FEV probe with signals on the der(16) and the der(2).

Figure 4

Sequence analysis of the FUS-FEV fusion transcript. A: RT-PCR amplification product of the predicted 1.4-kb size. B: Sequence chromatogram of the fusion transcript at the breakpoint region (shown as the reverse complement sequence), illustrating the fusion of FUS (ending at the 3′-end of exon 10) to FEV (beginning at the 5′-end of exon 2). The fusion sequence is in-frame, as shown in (C).

Figure 5

Schematic representation of the structure of the TET family proteins EWS and FUS and the ETS family protein FEV (adapted from Janknechkt17), as well as the novel FUS-FEV fusion transcript described in this article. Exons are demarcated by dotted lines, with numeric annotations above denoting the exon number. Domains important in the function of the fusion transcript include the SYQG-rich transcriptional activating domain of the TET proteins and the DNA-binding domain of the ETS protein. Other domains include the RNA-recognition motifs, arginine-glycine-glycine-rich (RGG-rich) regions involved in nuclear import signaling, the zinc finger (Zn) nucleic acid-binding domain, and an alanine-rich (Ala-rich) region involved in transcriptional repression. Small arrowheads represent the most common breakpoint sites in EFT. Large arrowheads denote the breakpoints seen in the novel FUS-FEV transcript.