47MO - Activating EZH2 mutations define a new subset of aggressive Ewing sarcomas

Background

While the primary genetic alteration of Ewing Sarcoma (ES) is fusion of EWSR1 and ETS family transcription factors, genomic profiling studies have uncovered recurrent secondary alterations in STAG2, TP53, and ERF, usually associated with a more aggressive phenotype. Mutations in EZH2, have also been sporadically reported in ES. Here, we systematically evaluated the frequency of EZH2 mutations, their relationship to other secondary alterations, and examined their role in ES biology.

Methods

Patients with ES harboring activating EZH2 mutations were identified from a cohort of MSKCC patients prospectively tested with the MSK-IMPACT large panel NGS assay and a cohort of ES patients from MSCNRIO in Warsaw tested with the Oncomine Comprehensive assay. The functional effects of EZH2 mutations were assessed in engineered isogenic ES cell lines (TC71, CHP100) expressing EZH2^wt, EZH2^Y646F or EZH2^A692V. We assessed proliferation, migration/invasion, and histone methylation, and performed transcriptome and epigenetic profiling.

Results

Among 222 cases, we identified 8 patients (3.6%) with 9 EZH2 mutations, of which 8 were located in the enzymatic SET domain (Y646F/H/N, A692G, A692V) and one in the CXC domain (K515R). Over a median follow-up of 55 months, 5 patients developed distant metastases, four of whom died of disease. EZH2 mutant samples showed strong and diffuse H3K27me3 nuclear expression. TC71 and CHP100 cells with EZH2^Y646F or EZH2^A692V had enhanced H3K27 trimethylation by Western blot and epigenetic profiling. No difference in growth were observed between EZH2 mutant and wild-type cells in vitro. RNA sequencing of cell lines with EZH2^Y646F revealed a highly repressive transcriptional landscape with significant downregulation of 623 genes and increased expression of 257 genes compared to EZH2^wt cells. Cancer testis antigens (CTAs) MAGE-C1, MAGE-C2, MAGE-C3 and TEX-19 were among the most upregulated genes.

Conclusions

Recurrent activating EZH2 mutations lead to broad transcriptional changes that may define a distinct clinical and biological subset of ES. While this aggressive subset is small, it is of particular therapeutic interest given the availability of EZH2 inhibitors and because overexpressed CTAs may also make it a candidate for immunotherapy.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Pawel Sobczuk has received funding from the Polish National Agency for Academic Exchange and the Kosciuszko Foundation.

Disclosure

P. Sobczuk: Financial Interests, Personal, Other, Travel grant: Novartis; Financial Interests, Personal, Other, Travel Grant: MSD, Pierre Fabre, BMS; Financial Interests, Personal, Invited Speaker: Swixx BioPharma, BMS, Gilead; Financial Interests, Personal, Stocks/Shares: CelonPharma; Non-Financial Interests, Personal, Leadership Role, Board Member, Chair of Young Oncologists Section: Polish Society of Clinical Oncology. P. Rutkowski: Financial Interests, Personal, Invited Speaker, honoraria for lectures: MSD, BMS, Pierre Fabre; Financial Interests, Personal, Advisory Board: MSD, BMS, Pierre Fabre, Merck, Sanofi, Blueprint Medicines, Philogen; Financial Interests, Personal, Invited Speaker: Merck, Sanofi, Novartis; Financial Interests, Institutional, Research Grant, research grant for ISS: Pfizer; Financial Interests, Institutional, Funding, research grant for institution: BMS; Non-Financial Interests, Personal, Invited Speaker: Polish Society of Surgical Oncology; Non-Financial Interests, Personal, Officer: ASCO; Non-Financial Interests, Personal, Invited Speaker, President Elect: Polish Oncological Society. All other authors have declared no conflicts of interest.