Genomic EWS-FLI1 fusion sequences in Ewing sarcoma resemble breakpoint characteristics of immature lymphoid malignancies

Abstract

Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85-90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5' region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma.

Figure 1. Genomic fusion site sequencing.

(A) Genomic organization of the EWS and FLI1 genes and corresponding breakpoint cluster regions (BCR). Nested primer sets for der22 are shown as double headed arrows. (B) Representative breakpoint sequencing workflow. Left: Gel electrophoresis of MLR-PCR products from two tumor samples in lane 1 and 2 (lane 3 negative control DNA; lane 4 ddH₂O; lane 5 positive control DNA; M = DNA ladder). Center: Gel electrophoresis of single long-range PCR products from 1^st round MLR-PCR product of sample 1 (lane 1–11; lane 12 positive control) to identify FLI1 and EWS primers next to the fusion sites and to reduce amplification product size for direct sequencing. Right: Sequencing of the shortest amplification product and alignment to EWS and FLI1 reference sequences.

Figure 2. Breakpoint distribution in the BCR of EWS and FLl1.

(A) Vertical bars above or below the breakpoint regions indicate individual breakpoint positions of Ewing sarcoma patients. Black boxes represent exons, and gray boxes correspond to repeat elements. (B) Results of Kernel density analysis (dashed line = breakpoint density; gray line = lower limit of 95% confidence band determined by bootstrapping procedure; black line = 95% confidence interval of a density function resulting from simulations at randomly distributed pseudo-breakpoints). X-axes indicate the BCR nucleotide positions within the respective reference gene. (C) Scatterblot of gender-specific EWS-FLI1 breakpoints. Circles represent female, and squares represent male subjects. (D) Number of microhomologies and filler nucleotides at EWS-FLI1 (der22; black bar) and FLI1-EWS (der11; gray bar) fusion sites. Each bar on the x-axis represents one individual.