Overview of next-generation sequencing, neoantigen prediction, and functional T-cell analyses. Overview of next-generation sequencing, neoantigen prediction, and functional T-cell analyses. Whole-exome sequencing was performed on the pretreatment and postprogression tumor and matched normal samples. Exome data were applied in a neoantigen prediction pipeline that evaluates antigen processing, MHC binding, and gene expression to generate neoantigens specific to the patient's HLA haplotype. Truncal neoantigens were identified by correcting for tumor purity and ploidy, and the TCR repertoire was evaluated at baseline, at the time of response, and upon emergence of resistance. Putative eliminated neoantigens at the time of resistance were used to generate peptides and stimulate autologous T cells, followed by TCR next-generation sequencing. PBL, peripheral blood lymphocyte. Valsamo Anagnostou et al. Cancer Discov 2017;7:264-276 ©2017 by American Association for Cancer Research