Combining FAIMS-DIA and targeted immunopeptidomics to enhance neoepitope discovery

Liposarcomas are malignant soft-tissue tumors of which only a subset responds to conventional cytostatic drugs and there is currently no approved targeted therapy available. One of the most common histologic subtypes, dedifferentiated liposarcoma (DDLS), is characterized by focal amplifications on chromosome 12. We hypothesized that these structural changes generate open reading frames and thus presumably transcribed and translated chimeric genes. The encoded neoepitopes are promising targets for epitope-centric individualized therapies if they are presented by HLA class I molecules.

Using two different variant calling pipelines – Arriba and EasyFuse – we identified gene fusions from whole genome/exome and RNA sequencing data of eight DDLS patients. Next, we performed a comprehensive mass spectrometry (MS) analysis of the patient's tumor immunopeptidome by combining untargeted and targeted methods in order to identify fusion-derived neoepitopes. First, we performed data-independent acquisition with ion mobility separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS-DIA) on an Orbitrap Exploris 480 connected to a Ultimate3000 RSLCnano system. MS raw data was analyzed using the Spectronaut 17 software combining library generation directly from DIA data with PROSIT in silico predicted libraries for putative neoepitopes. We identified up to 16500 unique peptides per patient, including several neoepitope candidates. Next, we utilized our recently established optiPRM workflow. Here, candidate neoepitopes identified by FAIMS-DIA as well as additional candidates from variant calling with excellent predicted binding properties were synthesized as stable isotope-labeled (SIL) variants and assay-relevant parameters such as normalized collision energy (NCE) and exact retention times were determined relative to iRT peptides. Targeted analysis by PRM not only confirmed several of the FAIMS-DIA candidates but also yielded additional detections in 5 out of 6 analyzed patients.

Using our complimentary FAIMS-DIA and PRM immunopeptidomics workflows, we have identified fusion-derived neoepitopes in 5 out of 8 DDLS patient samples. Importantly, the identifications contained both neoepitopes which would have been missed by the less sensitive FAIMS-DIA approach or which would have been excluded from the targeted analysis due to their poor predicted binding properties, highlighting the advantage of the combined approach.

Currently, we are testing all identified fusion-derived neoepitopes in epitope-specific expansion cultures (ESPEC) from the respective patients for reactive T cell populations. Additionally, we plan to perform TCR repertoire analysis to identify fusion neoepitope-reactive T cell receptors. Both detected neoepitopes and cognate T cell receptors identified will provide starting points for the clinical development of personalized treatment options in liposarcoma such as cancer vaccines or TCR-based therapies.