Abstract 3657: Customized liquid biopsy assay for monitoring EWSR1 fusions in sarcomas
Authors: Li Annie , Thierauf Julia C. , Gonda Bianca , Purohit Rashi , Kaluziak Stefan T. , Codd Elizabeth , Dybel Stacey N. , Choy Edwin , Cote Gregory M. , Lennerz Jochen K. , Iafrate A John 
Published: March 2024

Background: Gene fusions involving EWSR1 are involved in certain rare sarcomas and sarcoma-like tumors such as, for example, Ewing sarcoma, clear cell sarcoma, hyalinizing clear cell carcinoma, and clear cell odontogenic carcinoma. EWSR1-FLI1 fusions are found in Ewing sarcoma, while EWSR1-ATF1 and EWSR1-CREB are characteristic of clear cell sarcoma among others. Liquid biopsies involve obtaining tumor-derived materials through minimally invasive methods such as blood or other body fluid collection. Analysis of cell-free DNA (cfDNA) is an attractive method to identify gene fusions and monitor tumor recurrence and minimal residual disease (MRD). However, the structural complexity of oncogenic rearrangements poses challenges for the detection using plasma assays. We present a novel assay to monitor fusion positive tumor DNA in a busy oncology clinic.

Methods and Patients: Patients with EWSR1 fusion positive sarcomas are identified through clinical RNA-based sequencing. Tumor DNA isolated from biopsy or surgical samples is sequenced to map the intronic breakpoint and customized primers spanning the breakpoint are designed for each patient. The breakpoint is subsequently confirmed through PCR. The product length of ~100bp accounts for the small shedding size of tumor cfDNA. A quantitative TaqMan assay is designed to confirm EWSR1 fusions. The limit of detection is determined by a dilution series of tumor DNA. cfDNA is extracted from blood samples of the patients at multiple timepoints, the tumor burden is quantified by TaqMan Assay and subsequently correlated with clinical findings from imaging studies such as tumor progression or remission.

Results: We consented 10 patients with EWSR1 fusion positive tumors to participate in this study. Plasma samples were collected for 8, resulting in 26 total timepoints. Breakpoints were identified in 9 patients and confirmed with PCR on original tumor DNA. TaqMan assays were designed for 5 patients and showed correlation between the tumor burden detected in EWSR1-ATF1 copies in cfDNA extracted from patient blood and the therapeutic response to treatment found through imaging studies. One patient was followed over the period of 9 months, and a total of 5 plasma timepoints were collected. The patient started with a low number of EWSR1-ATF1 copies of &lt;1ng/ul, while on a Iurbinectedin/irinotecan therapy and showed a mixed response in the CT scan. After withdrawing from that therapy and starting other therapy regimens, the EWSR1-ATF1 copies increase to 8ng/ul, correlating with tumor progression observed in CT scans.

Conclusion: Our results suggest that ESWR1 fusions in sarcomas can serve as biomarkers to measure tumor burden. Our approach using an individually tailored cfDNA TaqMan assay could provide a valuable monitoring tool to inform clinicians about tumor progression or MRD. With these proof-of-principle findings we can now determine clinical validity and utility.

Citation Format: Annie Li, Julia C. Thierauf, Bianca Gonda, Rashi Purohit, Stefan T. Kaluziak, Elizabeth Codd, Stacey N. Dybel, Edwin Choy, Gregory M. Cote, Jochen K. Lennerz, A John Iafrate. Customized liquid biopsy assay for monitoring EWSR1 fusions in sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3657.