T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. Relevant questions are whether T cell recognition of unique epitopes occurs in CRC, and whether these epitopes can also target T cell responses against CSCs. To address these, we set up a proof of concept platform to systematically identify unique neoepitopes from somatically mutated CAN-genes expressed by CRC cells and in the derived CSC cultures. The tumour-derived cDNAs encoding the 20 most frequently mutated CAN-genes in CRC were subjected to Roche''s 454 high throughput pyro-sequencing to identify mutations in the expressed genes in a total of 8 samples.