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Pediatric and AYA Sarcomas

(P 256) THE SEAN KARL COHORT: A NATIONAL COLLABORATIVE SINGLE-CELL RNASEQ STUDY OF PAIRED PATIENT EWING SARCOMA SPECIMENS

Objective: Ewing sarcoma (EwS) is a fusion oncoprotein-driven primary bone cancer that demonstrates vast intra- and inter-tumoral heterogeneity. Tumor cell subpopulations, tumor progression, and therapeutic vulnerabilities of cell subpopulations are poorly understood. Paired patient samples (primary and metastatic disease or relapse) are rare at any single institution and collaborative efforts are needed to address these pressing biologic questions. A national collaborative effort (the Sean Karl Cohort) has been established to conduct single-cell RNAseq analyses of retrospective paired tumor samples from patients with EwS and serial samples due to metastasis or relapse. Four analytic teams will utilize these data to understand therapeutic vulnerabilities of EwS cell subsets and discern immunobiologic dysfunction.

Methods: A national collaborative effort was established to acquire retrospective, paired patient EwS tumor samples. Individual cells were isolated from FFPE scrolls or slides and prepared for single-cell RNAseq using the GEM-X Flex Gene Expression reagents (10x Genomics). Alex’s Lemonade Stand Foundation (ALSF) Data Lab established a common data processing and cell annotation workflow to create a harmonized dataset for downstream analysis.

Results: We have demonstrated the feasibility of generating quality, single-cell RNAseq data from retrospective FFPE tumor material at scale across institutions. We have generated data from 73 specimens, yielding more than 340,000 cells sequenced to date. Analytic teams are analyzing the processed data to assess tumor cell subpopulations, therapeutic vulnerabilities, immune cell subsets, and the tumor microenvironment as a whole. Processing and sequencing of additional samples is ongoing at the time of abstract submission.

Conclusion: To make progress toward improving outcomes for patients with aggressive EwS, a detailed understanding of heterogenous tumor cell subpopulations and therapeutic vulnerabilities is needed. We have established a multi-institution, collaborative, and multi-analytic approach to facilitate discovery as a first step towards improving outcomes for aggressive EwS.