Identification and Validation of Frameshift Neoantigens for Mismatch-Repair Deficient Lynch Syndrome
Lynch syndrome (LS) patients develop DNA mismatch repair deficient tumors which generate high loads of neoantigens (neoAgs), thus constituting a well-defined population that can benefit from cancer immune-interception strategies, including neoantigen-based vaccines. Using paired whole-exome sequencing and mRNAseq of colorectal cancers (CRC) (n=13) and pre-cancers (n=61) from our LS patient cohort (N=46), we performed in-silico prediction and immunogenicity ranking of highly recurrent frameshift-neoags, followed by their in-vitro validation. We described the somatic mutation landscape in all cancers and pre-cancers, and showed that mutation burden is positively correlated with neoAgs load. Furthermore, our in-vitro validation showed a 65% validation rate of our top 100 predicted neoags. Consistent with neoAgs burden, our transcriptomic results revealed increased infiltration of CD8+ and CD4+ T-cells in microsatellite unstable samples. Overall, our neoAgs catalog and all other findings, improve our understanding of cancer development in LS and guide us towards the advancement of immunoprevention vaccine strategies. Overall design: All patients included in this study had a confirmed diagnosis of Lynch Syndrome (n = 46) and provided written informed consent at The University of Texas MD Anderson Cancer Center (MDACC). All samples were obtained from study participants through an Institutional Review Board (IRB) approved protocol at MDACC (Protocol PA12-0327). A set of 74 flash-frozen or formalin-fixed paraffin embedded (FFPE) tissue biopsies from polyps or tumors of the lower gastrointestinal tract, with matching normal mucosa and peripheral blood, were collected from 46 LS patients who came to MDACC for a standard of care surveillance colonoscopy. The pathological diagnosis of all tissue samples was confirmed by a gastrointestinal pathologist (M.W.T) at MDACC. Samples were classified as inflammatory polyps (IP), hyperplastic polyps (HP), sessile serrated adenomas (SSA), adenomatous polyps (AP), which included tubular as well as tubullovillous adenomas, and adenocarcinomas, based on pathological diagnosis. Depending on the level of high-grade dysplasia samples were classified into different tissue categories that included pre-cancers, defined as pre-malignant lesions with low or no level of dysplasia, advanced pre-cancers, defined as pre-malignant lesions with high level of dysplasia, and cancers.  DNA and RNA were extracted from all flash-frozen and FFPE tissue samples using the Quick-DNA/RNA Miniprep Kit (Zymo Research, CA) and AllPrep DNA/RNA FFPE Kit (Qiagen, MD), respectively. Genomic DNA was obtained from peripheral blood using Gentra Puregene Blood Kit (Qiagen).