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

Abstract

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS - CREB translocation-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 / FUS fusion 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, TERT promoter and CDKN2A mutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/B homozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF , CDH19 , PARVB , and PFKP was found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4 and XAF1 were differentially upregulated and hypomethylated in AFH but not CCS. 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 - CREB translocation-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.

Inflammatory and Nested Testicular Sex Cord Tumor

“…2,23,24 The structural variants found in the 3 IN-TSCTs that underwent DNA sequencing consisted of in-frame gene fusions that contained exons 1 to 7 or 1 to 6 of EWSR1 and exons 4 to 7 of ATF1. 50 These fusion genes are expected to result in protein products with the characteristics described above. Hypothetically, high-level amplification of ATF1 could have oncogenic effects similar to those of constitutively activated fusion proteins that contain its DNA-binding domain.…”

Primary Clear Cell Sarcoma of Bone

“…Although the immunophenotypic findings are indistinguishable, cytogenetic or molecular diagnostic assays can easily separate CCS from melanoma. While CCS harbors EWSR1 rearrangements, most often EWSR1::ATF1 ,7,8 rarely EWSR1::CREB1 or EWSR1::CREM ,13 melanomas lack such translocations 17. In contrast, most melanomas harbor mitogen-activated protein kinase alterations, such as activating mutations in the BRAF or NRAS genes, or inactivating mutations in NF1 17…”

“…negative CCS harbor either EWSR1::CREB1 or EWSR1:: CREM fusions, 13,14 believed to introduce similar transcriptomic changes. Unlike melanomas, CCS exhibits quiet genomes with a low mutational burden and an absence of mitogenactivated protein kinase pathway alterations, 13,15,16 such as BRAF V600E (common in melanomas 17 and amenable to targeted therapy 18 ). These differences in management make it crucial for surgical pathologists to distinguish CCS from melanoma.…”

“…Around 95% of CCS harbor a t(12;22)(q13;q12) translocation,7 resulting in an EWSR1::ATF1 fusion8; this fusion brings a strong transactivation domain of EWSR1 to the DNA binding domain of ATF1, leading to overexpression of ATF1-regulated transcriptome, including genes of the melanocytic lineage 9–12. Most EWSR1::ATF1 negative CCS harbor either EWSR1::CREB1 or EWSR1::CREM fusions,13,14 believed to introduce similar transcriptomic changes.…”

Modeling sarcoma relevant translocations using CRISPR‐Cas9 in human embryonic stem derived mesenchymal precursors

“…The EWSR1 is ubiquitously expressed and belongs to the FET family of RNA binding proteins and, due to its strong transactivator domain, drives the oncogenic potential of its various transcription factor partners. The so‐called “EWSR1‐family of tumors” encompasses a growing spectrum of tumors, including both benign and highly malignant mesenchymal neoplasms, as well as a small subset of carcinomas, myoepithelial carcinomas, and mesotheliomas 36 . Thus, modeling specific EWSR1 ‐related gene fusions needs to consider the cell lineage and differentiation of the specific tumor being driven.…”