(P 45) MONITORING EWSR1 FUSIONS IN SARCOMA PATIENTS USING PERSONALIZED LIQUID BIOPSY ASSAYS

Objective: Sarcomas and sarcoma-like tumors harbor gene fusions involving the EWSR1 gene. Using liquid biopsies to analyze cell-free DNA (cfDNA) to detect these gene fusions could allow improved disease management without ionizing radiation. However, the structural complexity and individual intronic breakpoints poses challenges for the detection in patient plasma. We present a novel approach that involves mapping each patient's unique EWSR1 fusion breakpoint, designing personalized primers, developing a customized TaqMan assay, and using it to longitudinally track tumor burden in plasma cfDNA.

Methods: Patients with EWSR1 fusion positive sarcomas are identified through clinical RNA-based sequencing. Tumor DNA is sequenced to map the intronic DNA breakpoint and customized primers spanning the breakpoint are designed and validated for each patient, and subsequently Taqman assays are designed. Plasma timepoints are collected at regular intervals, and the tumor burden is quantified and correlated with clinical imaging studies.

Results: We consented 16 patients to participate in this study, with a total of 48 collected plasma timepoints. TaqMan assays were designed for 8 patients, and the remaining 8 patients are currently under analysis. One patient was followed over the period of three years with a total of 12 plasma timepoints. The patient started with a low number of EWSR1-FLI1 copies of < 1ng/ml plasma for 18 months while on vincristine, irinotecan, and temozolomide combination therapy. Due to complications, the patient was switched to a temozolomide monotherapy. The next plasma timepoint after 4 months still showed a low number of fusion copies < 1ng/ml plasma. Ten months after starting monotherapy, disease progression correlated with an increase of EWSR1-FLI1 fusion copies to 3ng/ml plasma. Despite different treatment approaches, the patient continued to progress, with an increase in fusion copies to 21ng/ml plasma.

Conclusion: Our pilot assay was able to detect ESWR1 fusions in cfDNA in plasma of sarcoma patients. In two patients who had serial testing, the level of ESWR1 fusion in cfDNA correlated with disease status. This exciting finding will need to be confirmed in future work with a larger number of homogenous patients to confirm if this biomarker could be an adjunct assay to imaging. Nevertheless, our approach using an individually tailored cfDNA TaqMan assay for each patient could provide a valuable monitoring tool to inform clinicians about tumor progression.