(P 252) THE GENOMIC LANDSCAPE OF CLEAR CELL SARCOMA RESEARCH INITIATIVE

Objective: Background: Clear cell sarcoma of soft tissue (CCS) is a rare cancer occurring predominantly in young adults, with a peak incidence in the third and fourth decades of life. They are deep soft tissue tumours and more than half of them arise in the lower extremities associated with tendons and aponeuroses. CCS are aggressive with local recurrence rates approaching 40%. The genetic hallmarks of CCS are EWSR1 gene fusions with members of the CREB gene family (ATF1, CREB or CREM). The outcome of these patients has not improved significantly over decades with a 10-year disease specific survival rate of only ~33%. These cancers are difficult to treat and show a profound resistance to chemo and radiotherapy. There are very limited new therapeutic interventions available for patients with CCS. A recent multi-national CCS clinical trial targeting MET, a putative downstream transcriptional target of EWSR1-ATF1 with Crizotinib, showed no overall benefit in survival although limited responses were seen using RECIST criteria. There is therefore an unmet need for a clearer understanding of the CCS biology and the identification of new therapeutic targets.
Hypothesis: We hypothesise that selection of well-characterized cases along with comprehensive molecular analysis will reveal novel correlations between molecular markers and clinical features of CCS, and potentially lead to identification of therapeutic targets.

Methods: In 2021, the Edward Showler Foundation (ESF) partnered with University College London (UCL), UK and the Royal National Orthopaedic Hospital NHS Trust, in an effort to 1) undertake a comprehensive molecular characterisation of CCS, 2) create a genomics resource which will be made available to the sarcoma research community and 3) to create a tissue resource comprising fresh frozen and formalin-fixed paraffin embedded CCS samples harmonised with the molecular and clinical information.

Results: Phase 1 of the program is now underway and includes a cohort of 47 frozen primary CCS, 40 with matching blood or adjacent normal tissue together with deep clinical annotations were collected from 4 tissue biobanks across the United Kingdom. 5/47 patients were recruited to the 100,000 Genomes Project in England, UK where whole genome sequencing (WGS) of the primary tumour DNA (100X depth of coverage) and matching germline DNA (30X depth of coverage) was performed. 3/5 cases showed varying levels of chromosomal instability with potentially complex copy number patterns which will be deconvolved using Oxford Nanopore long read sequencing of DNA and RNA. The remaining 42 primary cases will undergo whole genome sequencing of tumour and paired normal specimens as well as bulk RNA sequencing. 10/47 cases will undergo single cell RNAseq and spatial transcriptomic analysis to evaluate the extent of transcriptional heterogeneity in CCS. 21/47 cases have longitudinal samples (5/21 had 2 or more relapses) collected from local recurrences and/or metastases which comprise the Phase 2 cohort where these samples will undergo whole exome, RNAseq and methylation array analysis to investigate tumour heterogeneity and sarcoma evolution. Tissue microarrays of all cases and an additional 20 FFPE samples have been constructed for deep immunophenotyping analysis and will be made accessible to the sarcoma research community. Phase 3 of the project will involve collaborating with other specialist oncology and pathology units to access archival CCS samples in order to expand the tissue resource available for research.

Conclusion: The ESF-UCL-RNOH CCS research effort will provide a comprehensive molecular landscape of CCS, insights into genomic and transcriptional heterogeneity and evolution. These genomics and tissue resources have the potential to advance research for CCS biology and treatment.