Abstract 5378: Spatially resolved human leukocyte antigen (HLA) expression correlates with motif neoepitope expression and durable response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC)

Abstract Background: We previously showed electrostatic interactions enhance binding between “motif” neoepitopes and HLA and predict advanced NSCLC ICB benefit, suggesting optimal neoantigen presentation drives effective anti-tumor immune response (Cummings 2020). However, not all cases with motif neoepitopes exhibit favorable ICB outcomes. Methods: As proof-of-concept, participants from our published NSCLC ICB cohort with available pre-treatment archival tissue were selected based on HLA supertype B44, predicted motif neoepitopes, and best response of progressive disease (PD) vs partial/complete response (PR/CR). Whole exome sequencing, HLA typing and neoepitope prediction were previously performed (Cummings 2020). GeoMx slides were prepared, hybridized with RNA probes and stained with antibodies to cytokeratin (CK), nuclear material and CD45 per manufacture instructions (Geiss 2008). Antibody to CD8 (Novus) was optimized at 1:50 dilution. Twelve regions of interest (ROI) were selected per sample including the following compartments: CK+CD8+, CK+CD8-, CK-CD8+ and CK-CD8- (negative control). Each ROI included ~150 nuclei selected using GeoMx Digital Spatial Profiler. Sequencing was performed on Illumina Novaseq 6000 (50bpx2) demultiplexed using Illumina Bcl2fastq v2.19.1.403. Raw reads were normalized and compared using paired student’s T-tests with Tukey’s correction via R v4.1. Neoepitope (non/motif) comparisons used matched numbers per sample selected by highest expression. Results: Seven participants (3 PD, 3 PR, 1 CR) were included, each exhibiting 1-7 motif neoepitopes without HLA loss of heterozygosity. 84 ROI included 24 CK+CD8+, 24 CK+CD8-, 24 CK-CD8+ and 12 CK-CD8- with at least one ROI per category per participant. PD samples had fewer CD8+ ROI (12 vs 36) and lower CXCL9 (p=0.011) and CD274 (p=0.014). HLA-A/B/C expression followed a gradient with highest expression in CK+CD8+ ROI with limited detection in CK-CD8- ROI. Among HLA, HLA-B had highest median expression (A: 226, B: 650, C: 202; p<0.001). In CK+ ROI, motif neoepitope expression was detectable for all participants but not statistically different from non-motif expression (p=0.23). HLA-A/B/C all exhibited lower expression in PD CK+ ROI (p<0.001). Conclusion: HLA expression may modify prediction of clinically meaningful neoepitopes and response to ICB. Validation in a larger, prospective cohort is planned. Citation Format: Lucas Goldman, Sarah Gorbatov, Collin Shen, Alexa Berezowitz, Edward Garon, Amy Lauren Cummings. Spatially resolved human leukocyte antigen (HLA) expression correlates with motif neoepitope expression and durable response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5378.