While the PBMCs are differentiating and through the use of comparative DNA isolated from PBMCs, the researchers will identify all nonsynonymous mutations through whole exome sequencing of biopsied pancreatic cancer cells (Cullinan et al. 2018). Comparing normal exons to those found in pancreatic tumors not only allows for scrutinization of potential neoantigens, but also allows for more treatment flexibility as whole exome sequencing is less expensive than whole genome sequencing. Whole exome sequencing also allows for the inference of HLA types from exon sequences (Cullinan et al. 2018). Expression of identified nonsynonymous mutations can then be verified with cDNA capture. From these expressed mutations, potential neoantigen sequences will be further scrutinized through multiple MHC class I epitope prediction algorithms. The NetMHC algorithm has been previously utilized in analysis of Panc02 tumor cells to predict neoantigens (Kinkead et al. 2018). A Random Forest-based computational algorithm has also demonstrated an immunogenicity prediction accuracy of 83% and an HLA binding prediction accuracy of 97.4% (Wilson et al. 2018). Therefore, the researchers will use the NetMHC and Random Forest algorithms to screen for tumor-specific neoantigens in individual patients. Additional filters can be applied to these algorithms to eliminate epitopes predicted to be poorly processed by the immunoproteasome and those with lower binding affinities than the corresponding wild-type sequences (Gubin et al. 2014). These filters will discern any potential neoantigens that would not be broken down effectively to be displayed via MHC complexes. These filters also eliminate any neoantigens outcompeted to bind to MHC complexes by the wild-type sequences and assure that resulting candidate neoantigens are sufficiently different from self-antigens (Hopkins and Jaffee, 2018). A common method for evaluating immunogenicity and further scrutinizing final candidate neoantigens is interferon-gamma enzyme-linked immunospot assay (Cullinan et al. 2018). This assay allows for the quantification of cytokine secretion [interferon-gamma] within a specific cell. The researchers will perform this assay in undifferentiated PBMCs taken from the patient with the candidate neoantigens to determine immunogenicity.
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