Complex/cryptic EWSR1::FLI1/ERG Gene Fusions and 1q Jumping Translocation in Pediatric Ewing Sarcomas

Figure 1. Histology of a humerus lesion in patient 3. The histologic evaluation shows monotonous small round blue cells with fine chromatin and scant amphophilic cytoplasm.

Figure 2. Cytogenetic analysis of patient 3 (a) A karyogram of 48,XY, del(9)(q34), t(9;11;22) (q22;q24;q12),add(12)(q24.1),+16,+18,der(18)t(1;18)(q12;p11.3)x2,der(21)t(1;21)(q12;p11.2). Red arrows point to the t(9;11;22) three-way translocation, and other arrows point to other abnormalities (b) Post-G-banded metaphase FISH using EWSR1 break-apart probe shows EWSR1 rearrangements. Green signals correspond to the 3′ portion of the EWSR1 gene on the derivative chromosome 9, and red signals correspond to the 5′ portion of EWSR1 remaining on the derivative 22. (c) EWSR1 FISH signals in the G-banded metaphase cell.

Figure 3. EWSR1::FLI1 FISH analysis in patient 3 (a,b) Metaphase FISH using dual-color dual-fusion EWSR1-FLI1 probe set shows EWSR1::FLI1 fusion on the derivative chromosome 22 involved in the t(9;11;22)(q22;q24;q12) three-way translocation. Green signals are for the FLI1 gene, and red signals are for the EWSR1 gene. (b) FISH signals in the G-banded metaphase cell. (c) Diagrammatic representation of the complex three-way chromosomal rearrangement.

Figure 4. Visualization of the EWSR1::FLI1 fusion identified by the NanoString gene fusion assay in patient 1. The genomic translocation breakpoints were mapped to chr22:29,683, 123 (exon 7) for EWSR1 and chr11:128,675,261 (exon 6) for FLI1.

Figure 5. SNP microarray of patient 3. (a) Gain of 1q (arr[GRCh37] 1q12q44(142,632,577-249,218,992x1, shown in red box) by copy number plot based on LogR signal intensities (red line) and B-allele frequency plots (blue dots). (b) Whole-genome view of SNP microarray data.

Figure 6. Cytogenetic analysis of patient 4 (a) Partial karyogram shows chromosomes 4, 11, and 22. Arrows point to the t(4;22)(q35;q12) reciprocal translocation by chromosome analysis. (b) FISH using EWSR1 break-apart probe shows EWSR1 rearrangements in interphase cells. (c,d) FISH using EWSR1 break-apart probe shows EWSR1 rearrangements in the G-banded metaphase cell. Green signals correspond to the 3′ portion of the EWSR1 gene on the derivative chromosome 4, and red signals correspond to the 5′ portion of EWSR1 remaining on the derivative chromosome 22. (d) EWSR1 FISH signals in the G-banded metaphase cell.

Figure 7. EWSR1::FLI1 FISH and SNP microarray analysis in patient 4 (a,b) Metaphase FISH using dual-color dual-fusion EWSR1-FLI1 probe set shows EWSR1::FLI1 fusion on the derivative chromosome 22 involved in a cryptic t(4;11;22) three-way translocation. Green signals are for the FLI1 gene, and red signals are for the EWSR1 gene. (b) FISH signals in the G-banded metaphase cell. (c) Whole-genome view of SNP microarray data.

Figure 8. Diagrammatic representation of the cryptic three-way translocation by sub-telomere FISH for chromosomes 4qter, 11qter, and 22qter.

Figure 9. Visualization of the EWSR1::FLI1 fusion identified by the NanoString gene fusion assay. The genomic translocation breakpoints were mapped to chr22:29,688,158 (exon 10) for EWSR1 and chr11:128,651,853 (exon 5) for FLI1.

Figure 10. Cytogenetic analysis of patient 8 (a) A karyogram of 47,XY,+8,der(22)add(22) (p11.2)del(22)(q12). Red arrows point to trisomy 8 and the derivative chromosome 22. (b) Interphase FISH using EWSR1 break-apart probe shows EWSR1 rearrangements. Red arrows point to the cells with EWSR1 gene rearrangements. (c) FISH using an EWSR1/FLI1 probe-set shows EWSR1 and FLI1 FISH signals in an inverted 4′,6-diamidino-2-phenylindole (DAPI)-banded metaphase cell.