Nested set information derived from fusion genes in Ewing sarcoma and other cancers.

Abstract

e23521 Background: Ewing family tumors including Ewing sarcoma (ES) and desmoplastic small round cell tumor (DSRCT) are characterized by EWSR1 and ETS fusion partners including EWS-FLI1, EWS-ERG, EWS-WT1, and others. Since 2019 our Next Generation Sequencing (NGS) sarcoma panel (N = 338) identified both fusion partners and exons containing the EWS breakpoint in ES and DSRCT. Hence, it should be possible to learn more about the exact breakpoints of EWS fusion genes. The molecular diversity and functionality of these fusion transcripts, especially the exact sequence, which could be identical, similar, or unique, may have significant biological implications for diagnostics and treatment options. We have used in-frame analysis of EWS gene fusion breakpoints to identify corresponding polypeptides spanning the breakpoints of the most common EWS gene fusions to provide useful insights about ES and DSRCT. Methods: Pathology reports and EWS gene fusions were analyzed using the Cleveland Clinic NGS panel which is based on anchored multiplex polymerase chain reaction (PCR) enriched for 34 gene targets. The amplicons were subjected to massively parallel sequencing with 151x2 cycle pair-end reads. An informatics pipeline was used for read alignment (GRCh37 as reference genome), fusion identification, and annotation. The DNA sequence across fusion junction and translational amino acid sequences were extracted for comparison in a preliminary teaching set (37/338 EWS fusions). Results: EWS-FLI1 fusions at exon 7-7 (chr22:29683123-chr11:128675261) and exon 7-6 (chr22:29683123-chr11:128651853) were the most common fusion genes in ES (N = 24/32;75%). Other ES gene fusions included exons 10-6, 7-10, 9-8, 7-2 EWS-FEV, 7-8 EWS-ERG, 7-9 EWS-ERG, and 10-9 EWS-ERG. EWS-WT1 gene fusions (chr22:29683123-chr11:32414301) in DSRCT exon 7-7 occurred in 4 of 5 cases (80%). In frame analysis of the common gene fusions in 75% of ES and DSRCT (total n = 37) yielded identical corresponding polypeptides that span the breakpoint (table). Conclusions: Analysis of ES and DSRCT fusion genes provides evidence that cancer-specific fusion genes are associated with common identical breakpoints and corresponding in-frame polypeptides. Our results show that the identification of in-frame polypeptides from the fusion gene sequencing can identify potential nested sets of cancer-specific mRNAs and polypeptides. This information may become relevant for diagnostic and therapeutic targets future vaccines and diagnostics against ES and DSRCT as well as other cancers characterized by fusion genes or frameshift mutations. [Table: see text]