2320P - Detection of gene fusion-induced neoepitopes in dedifferentiated liposarcoma

Background

Dedifferentiated liposarcomas (DDLS) are malignant soft-tissue tumors that are characterized by focal amplifications on chromosome 12q. They do not respond well to chemotherapy and no approved targeted therapy for DDLS is available. Through whole-exome/genome and RNA sequencing, we found that these structural changes generate open reading frames, leading to potential gene fusions and immunogenic neoepitopes, which modulate immune microenvironment and could serve as therapeutic targets.

Methods

We first analyzed a DDLS cell line (T449). Gene fusions were identified using two variant calling pipelines, Arriba (DKFZ) and EasyFuse (TRON). Fusion-derived (FD) peptides were used as input for HLA binding prediction by NetMHCpan 4.1. For targeted mass spectrometry (MS) analysis, we applied an ultra-sensitive parallel reaction monitoring workflow. For untargeted MS analysis, we combined data-independent acquisition MS with ion mobility separation by high-field asymmetric waveform ion mobility spectrometry. Mass spectra were compared to the human proteome, FD sequences, and libraries of in silico predicted fragment spectra. We used a modified MANAFEST protocol for the expansion of antigen-reactive CD4 and CD8 T cells.

Results

Candidate peptides for the targeted MS approach were selected from overlapping results of both pipelines. For the T449 cell line, we detected 128 fusion events resulting in 493 potential neoepitopes. We selected 46 peptides for targeted MS analysis and identified eight distinct FD neoepitopes. Analysis by untargeted MS yielded 15,578 peptide sequences, including five FD neoepitope candidates. We analyzed tumors from DDLS patients by targeted and untargeted MS analysis as well as their peripheral T-cell repertoire from expansion cultures. In a retrospective analysis of 67 patients, we observed a correlation between the quantity of fusions and specific tumor-infiltrating immune cell fractions.

Conclusions

We show results of our pipeline for the detection of FD neoepitopes using both targeted and untargeted MS approaches and explore the presence of neoepitope-specific T cells in the peripheral blood of DDLS patients. We believe that our work will lead to the clinical development of personalized treatment options for DDLS patients.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Helmholtz Institute for Translational Oncology Mainz.

Disclosure

P. Horak: Financial Interests, Personal, Speaker’s Bureau: Roche, Trillium GmbH; Financial Interests, Personal, Speaker, Consultant, Advisor: Platomics GmbH. All other authors have declared no conflicts of interest.