Abstract 7244: Detection of gene fusion-induced neoepitopes and cognate T cell receptors in liposarcoma

Abstract Introduction: Dedifferentiated liposarcomas (DDLS) are malignant soft tissue tumors characterized by focal amplifications on chromosome 12q. Using whole genome and RNA sequencing, we found that these structural alterations generate open reading frames that may lead to potential gene fusions and immunogenic neoepitopes. These may serve as potential therapeutic targets. We present the results of the detection and validation of fusion-derived neoepitopes in eight liposarcoma patient samples. In addition, we investigate the patients' T cell repertoires against the identified fusion-derived (FD) neoepitopes. Methods: Two variant calling pipelines were employed to identify gene fusions. The FD peptides were used as input for HLA binding prediction. We used mass spectrometry (MS)-based immunopeptidomics for HLA presentation analysis. We performed untargeted MS analysis using data-independent acquisition MS combined with ion mobility separation by high-field asymmetric waveform ion mobility spectrometry. The mass spectra were compared with the human proteome, FD sequences, and libraries of in silico predicted fragment spectra. Targeted MS analysis was performed using the ultra-sensitive parallel reaction monitoring workflow (optiPRM). All identified FD neoepitopes were subjected to Epitope-SPecific Expansion Cultures (ESPEC) testing and the TCR repertoire of expanded T-cells analyzed by deep sequencing for candidate peptide selection. Results: We identified 17 to 123 fusion events on chromosome 12, resulting in 122 to 1126 neoepitope candidates per patient by sequence analysis. The MS workflow successfully identified neoepitopes in six of the eight samples tested. All neoepitope candidates identified by the untargeted approach were subjected to targeted analysis, regardless of their predicted HLA binding ability. In addition, we targeted the top 20 FD candidates from each patient based on the in silico binding prediction. The targeted analyses confirmed the results of the untargeted approach and provided additional detections in five of the six patients analyzed. The validated FD peptides were used to stimulate autologous peripheral blood mononuclear cells from four patients in ESPECs to identify reactive TCRs. T cell expansion was observed in ESPEC cultures against one to 15 peptides per patient, which were subjected to TCR repertoire analysis by deep sequencing. Conclusions: We detected bona fide HLA-presenting FD neoepitopes and identified neoepitope-specific T cells in the peripheral blood of DDLS patients. These data may further aid the process of identifying and selecting candidate immunogenic peptides, thereby facilitating the clinical development of personalized treatments for DDLS patients, including cancer vaccines and TCR-based therapies. Citation Format: Peter Horak, Jonas P. Becker, Sven Blobner, Julian Hlawatsch, David Weber, Jonas Förster, Annika Baude, Johanna Wagner, Isabel Poschke, Inka Zörnig, Michael Volkmar, Michael Platten, Dirk Jäger, Priya Chudasama, Stefan Fröhling, Angelika Riemer. Detection of gene fusion-induced neoepitopes and cognate T cell receptors in liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7244.