Identification and characterization of recurrent neoantigens in upper gastrointestinal (GI) cancers.
Authors: Battaglin Francesca , Elliott Andrew , Zhang Jian , Stafford Phillip , Helmstetter Anthony , Oberley Matthew James , Arai Hiroyuki , Jayachandran Priya , Kawanishi Natsuko , Soni Shivani , Lo Jae Ho , Zhang Wu , Weinberg Benjamin Adam , Lou Emil , Shields Anthony Frank , Spetzler David , Goldberg Richard M. , Marshall John , Korn W. Michael , Lenz Heinz-Josef 
Published: January 2022

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Background: Neoantigens are short peptides derived from tumor-specific somatic mutations that can bind to HLA molecules and be presented on the cell surface to activate the immune system. Recognition of neoantigens by autologous T cells promotes sensitivity to immune checkpoint blockade of mismatch repair deficient (MMRd)/microsatellite instability high (MSI-H) tumors. Neoantigen-targeted reactivity by autologous T cells has also been reported in microsatellite stable (MSS) tumors. We aimed to comprehensively assess the spectrum of neoantigens in upper GI cancers and identify recurrent immunogenic candidate neoantigens. Methods: 600 tumor specimens including 268 esophageal (EC), 211 gastric (GC), and 121 gastroesophageal junction (GEJ) cancers tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing (NGS) on DNA (whole exome) and RNA (whole transcriptome) were analyzed. MSI status was determined by immunohistochemistry of MMR protein and/or NGS. Immune epitope prediction was performed on translated peptide sequences harboring detected mutations using the NetMHCpan v4.0 method in the Immune Epitope Database, with HLA genotyping performed using arcasHLA. Immune/stromal cell abundance was quantified using the Microenvironment Cell Populations Counter method. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature, TIS). Results: Overall, the median patient age was 66 years (range 22-90), 72.8% were male, and 33.7% of samples were metastatic site biopsies. MMRd/MSI-H rate was 2.2% in EC, 12.4% in GC, and 2.5% in GEJ. A total of 1172 unique recurrent neoantigens with predicted binding-level affinity for patient specific HLA alleles were identified (317 in EC, 786 in GC, 157 in GEJ), with 442 and 552 neoantigens exclusively associated with MSS and MSI-H tumors, respectively. Across each cancer type, a higher positive TIS score correlated with both immune and stromal cell population abundance in the tumor microenvironment, most notably cytotoxic lymphocyte and monocytic cell types (r > 0.80, P < 0.0001). Recurrent peptides associated with highest average TIS scores resulted from mutation of TP53 (R248W, 2 peptides: 2.8%/3.2% of samples, respectively) and CUX1 (L162F, 1.6%) in MSS EC; REL (D318A, 2.3%) and ERBB2 (V842I, 1.2%) in MSS GC; MSH3 (K383fs, 36%) and ASXL1 (G645fs, 24%) in MMRd/MSI-H GC; and HOXD12 (A70-A71dup , 2.8%) and TP53 (R248Q, 3.7%) in MSS GEJ. Conclusions: This is one of the largest studies to investigate the landscape of recurrent neoantigens in upper GI cancers. We were able to identify candidate recurrent peptides with high HLA binding affinity and an association with a positive TIS signature in both MSI and MSS tumors, supporting the role of recurrent neoantigens as potential cancer immunotherapy targets.