Comprehensive genomic profiling of EWSR1/FUS::CREBtranslocation-associated tumors uncovers prognostically significant recurrent genetic alterations and methylation-transcriptional correlates

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS::CREBtranslocation-associated tumors, we performed a comprehensive genomic analysis of fusion transcript variants, recurrent genetic alterations (mutations, copy number alterations), gene expression, and methylation profiles across a large cohort of tumor types. The distribution of the EWSR1/FUSfusion partners—ATF1, CREB1, and CREM—and exon involvement was significantly different across different tumor types. Our targeted sequencing showed that secondary genetic events are associated with tumor type rather than fusion type. Of the 39 cases that underwent targeted NGS testing, 18 (46%) had secondary OncoKB mutations or copy number alterations (29 secondary genetic events in total), of which 15 (52%) were recurrent. Secondary recurrent, but mutually exclusive, TERTpromoter and CDKN2Amutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/Bhomozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF, CDH19, PARVB, and PFKPwas found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4and XAF1were differentially upregulated and hypomethylated in AFH but not CCS. Unsupervised clustering of methylation profiles revealed that CREB family translocation-associated tumors form neighboring but tight, distinct clusters. A sarcoma methylation classifier was able to accurately match 100% of CCS cases to the correct methylation class; however, it was suboptimal when applied to other histologies. In conclusion, our comprehensive genomic profiling of EWSR1/FUS::CREBtranslocation-associated tumors uncovered mostly histotype, rather than fusion-type associated correlations in transcript variants, prognostically significant secondary genetic alterations, and gene expression and methylation patterns.