Figure 1.
Circos plots of representative Ewing sarcoma family tumors.
Circos plot tracks represent somatic mutations, from outside circle: mutated genes including missense (Black), indel (Red) and nonsense (Orange); genomic location; genome copy number alterations (Grey); lesser allele frequency (Green); LOH (dotted track); density of heterozygous SNPs (Orange); density of homozygous SNPs (Blue); Intrachromasomal (Grey) and interchromasomal (Red) rearrangements. Tumor EWS2006 (A) contains only 2 somatic coding mutations. Tumor EWS2017 (B) has 4 somatic coding mutations including a frameshift mutation in STAG2. Both tumors shown have the characteristic EWSR1-FLI1 fusion and a modest degree of aneuploidy.
Figure 2.
Molecular profiling of Ewing sarcoma family tumors using RNA sequencing data.
EFT clinical samples that lack an EWSR1-fusion have a distinct profile. A) Hierarchical clustering based on RNA expressional profile shows the fusion negative (blue) and alternate fusion (red) samples to separate from the majority of EWSR1-fusion positive EFTs. B) Expression profile of Ewing sarcoma signature genes (top) and genes correlating with EWSR1-FLI1 target NROB1 (bottom) in normal tissues and EFT cohort demonstrating the lack of typical expressional profile in EWSR1-fusion negative samples (Alt).
Figure 3.
Mutational spectrum in STAG2 (A) and TP53 (B) on linear protein models.
Exonic point mutations and small indels are shown in relation to the functional domains of these genes. Larger structural mutations and non-exonic mutations in STAG2 are not pictured and include multi-exon intragenic deletions (3), intronic splice site mutations (3), intragenic duplication events (2), 5′ (1) and 3′ UTR (1) mutations.
Figure 4.
Examples of immunohistochemistry showing STAG2 expression in Ewing sarcoma tumor samples.
A) STAG2 is robustly expressed in EFT harboring wild-type STAG2 alleles (top), but is completely lost in the subset of EFT harboring truncating mutations of the STAG2 gene (bottom). Expression is retained within the non-neoplastic stromal and endothelial cells, demonstrating the somatic nature of STAG2 loss in these tumors. B) Sequence trace demonstrating the E984X STAG2 nonsense mutation present in EFT sample NCI-0047 that is shown in A.
Figure 5.
Western blots analysis of STAG2, TP53, p21WAF1/CIP1, and p16INK4a on a panel of 36 unique EFT cell lines.
13 of 36 cell lines have complete absence of STAG2 protein, an additional two cell lines (6647 and TC-215) have STAG2 isoforms with altered molecular weight due to large intragenic in-frame insertions or deletions, one additional cell line (ES-7) has intact STAG2 expression despite a frameshift mutation occurring at amino acid residue 1212 that is C-terminal to the epitope recognized by the antibody, and two additional cell lines have intact STAG2 expression but harbor a small in-frame insertion (CHLA-9) and a missense mutation (ES-6). Absence of p16INK4a protein is seen in 25/36 cell lines including all 16 with identified CDKN2A deletion.
Table 1.
Mutational frequency of recurrently altered genes in Ewing sarcoma family tumors and cell lines.
Figure 6.
Summary of sequencing findings in EFT tumors (red) and cell lines (dark blue) highlighting recurrent alterations.
There are frequent alterations in STAG2, TP53, and CDKN2A in EFT tumors and cell lines. 57/97 (58.7%) of samples containing an EWSR1-ETS fusion have a secondary mutation in one of these three tumor suppressor genes. Notable variants in BRAF, PI3KCA, RAD51 and BRCA2 are also shown.