(P 251) EVALUATION OF POTENTIAL TARGETS FOR FLUORESCENCE-GUIDED SURGERY IN PEDIATRIC EWING SARCOMA: A PRECLINICAL PROOF-OF-CONCEPT STUDY

Objective: Ewing sarcoma (ES) is an aggressive bone and soft‐tissue tumor. Treatment generally consists of chemotherapy followed by surgery with- or without radiotherapy. Intraoperative distinction between healthy and tumorous tissue is challenging, especially after chemotherapy and at complex anatomical locations. Targeted fluorescence-guided surgery (FGS) can facilitate complete and less mutilating tumor resections by visualizing tumor tissue during surgery. This can be done with a tumor-specific target, a targeting moiety conjugated to a fluorophore (tracer), and a dedicated camera system. A previous systematic review selected CD99, CXCR4, NPY-R-Y1, LINGO-1, IGF-1R, and CD117 as promising targets for FGS of ES. Additionally, GD2 could be a promising target due to its known expression of 40-90% in ES biopsy samples and the clinical availability of a FGS tracer for neuroblastoma. As a crucial step towards developing targeted FGS for ES, this study aimed to evaluate the selected targets through an immunohistochemical analysis conducted on biopsies and resection specimens, including adjacent healthy tissue. Subsequently, three tracers with the highest potential for FGS were identified. Binding specificity of these tracers was assessed using cell line-based flow cytometry and immunofluorescence microscopy experiments. Finally, as proof-of-concept, a topical application experiment with the three selected tracers was performed on one freshly resected pediatric ES tumor and surrounding healthy tissue.

Methods: ES patients who gave informed consent for the use of their tissue at the time of surgery and underwent surgical resection between 2018 and 2022 were eligible for this study. Formalin-fixed paraffin-embedded (FFPE) biopsies (n=13) and corresponding whole tissue sections of ES tissue samples with tumor and adjacent healthy tissue (n=8) were selected by a specialized sarcoma pathologist. Immunohistochemistry was performed to assess CD99, CXCR4, CD117, NPY-R-Y1, and IGF-1R expression in ES biopsies and resection specimens. Unfortunately, LINGO-1 and GD2 staining protocols did not work on FFPE tissue. Based expression and clinical availability of a targeted-tracer, anti-CD99, anti-CD117, and anti-GD-2 were further evaluated for binding specificity using flow cytometry and immunofluorescence microscopy. ES cell lines A673 and RD-ES were selected as lines to assess binding of the tracers, while neuroblastoma cell lines SK-N-BE and KCNR, and breast cancer organoid lines BC27T and BC62T served as positive or negative controls for the selected targets. Lastly, as proof of concept, anti-CD99, anti-CD117, and anti-GD2 tracers were topically applied to one freshly resected ES tumor and adjacent healthy tissue.

Results: Immunohistochemistry demonstrated moderate/strong CD99 and CD117 expression in ES-tumor samples, while adjacent healthy tissue had limited expression. Flow cytometry and immunofluorescence microscopy confirmed high CD99 expression, along with low/moderate CD117, and low GD2 expression in ES cell lines. Topical anti-CD99 and anti-GD2 application on ES-tumor showed fluorescence, while anti-CD117 did not show fluorescence for this patient.

Conclusion: Based on the immunohistochemical evaluation, preclinical in vitro experiments, and ex vivo topical application experiment on freshly resected Ewing sarcoma and adjacent healthy tissue, our findings suggest that CD99-targeting tracers hold promise for FGS of ES. CD117 and GD2 tracers could be potential alternatives. The next step towards development of ES-specific FGS tracers could be ex vivo topical application experiments on a large cohort of ES patients.