Development of Novel Immunotherapy-Based Therapies in Oesophageal Adenocarcinoma by Harnessing Tumour Neoantigens

Background Outcomes with current therapies for oesophageal adenocarcinoma (OAC) have plateaued. For curative patients, immunotherapy shows promise in the adjuvant setting following chemoradiotherapy, and in the neoadjuvant setting combined with doublet or triplet chemotherapy. However, responses to immunotherapy are heterogenous, with a large proportion of patients failing to show benefit. To overcome this, novel immunotherapy-based approaches are necessary. Tumour neoantigen based therapies have shown promise in melanoma and pancreatic cancer, however in OAC the neoantigen landscape remains unknown. We aimed to profile to the neoantigen landscape and associated immune responses to inform future development of neoantigen based vaccine therapies in OAC. Methods 238 patients with oesophgaeal or gastroesophageal junction adenocarcinoma from three cohorts (the AGITG-DOCTOR trial, the Cancer Evolution Biobank and The Cancer Genome Atlas) were included. Tumour and normal samples were sequenced for WGS or WES as well as RNA sequencing of matched samples. Neoantigens were predicted using the validated pVAC-Seq pipeline. HLA-types were predicted with Optitype and Polysolver and were neoantigen binding affinity was determined using NetMHCpan. A polyfunctional flow cytometry based assay was used to assess for in vitro individual putative neoantigen immunogenicity to autologous peripheral T cells. Analyses were performed in R and FlowJo. Results We found a median of 23 neoantigens per patient. Expressed quality neoantigens were associated with improved survival. RNA-sequencing based deconvolution of the immune microenvironment revealed three distinct clusters. The inflamed cluster comparatively showed higher expressed quality neoantigens, and improved survival, while immune-heterogenous and immune-cold subgroups showed lower neoantigen numbers with impaired outcomes. In vitro flow cytometry-based cytotoxicity assays confirmed the accuracy of our in-silico neoantigen prediction pipeline by revealing polyfunctional cytotoxic T cell responses to synthesised neoantigen peptide sequences. Conclusion Overall development of novel neoantigen-based immunotherapeutic regimens show feasibility in OAC and should be explored. This approach may be further personalised based on the pre-treatment tumour microenvironment.