Abstract A097: Analyzing immunotherapy resistance in microsatellite instable colorectal FFPE samples using RNA sequencing

Abstract INTRODUCTION: Immunotherapy outcomes have been underwhelming in most gastrointestinal malignancies. Microsatellite instable (MSI-H) colorectal cancers (CRC) are an exception. Immune checkpoint blockade (ICB) is standard of care for MSI-H CRC. However, a portion of MSI-H CRCs do not respond well to ICB, insufficiently explained by tumor mutational burden or clinical factors. We sought to understand transcriptional pathways of ICB resistance in MSI-H CRC through whole transcriptome analysis of archival formalin-fixed, paraffin-embedded (FFPE) samples. METHODS: Cases of MSI-H CRC were identified from the Duke Pathology lab and Molecular Registry of Tumors from 2017 to 2022. 12 durable responders (MSI-H-R) and 9 non-responders (MSI-H-N) were selected for analysis. 15 locally advanced MSI-H CRC (MSI-H-loc) samples, with a near 100% response rate to ICB in other settings, and 10 locally advanced MSS CRC samples were used as controls. 46 total samples (12 MSI-H-R, 9 MSI-H-N, 15 MSI-H-loc, and 10 MSS) were selected for FFPE-based whole transcriptome analysis using MACE-Seq (GenXPro), a 3’mRNA-sequencing approach. RESULTS: 44 samples (96%) were suitable for analysis with an average of 18,378 genes counted. The tumor suppressor GPRC5A was upregulated in the MSI-H-R group compared to MSI-H-N. MSI-H-N samples exhibited higher expression rates of genes coding for the extracellular matrix and mesenchymal stem cells (KLF2, Desmin, MUC1), hypoxia-induced genes including those related to metabolic processes (LDHD, PDK4) and the beta-globin subunit of hemoglobin (HBB), and terminal T cell exhaustion (TOX). Hypoxic states and LDHD have been shown to correlate inversely with immune cell infiltration and ICB response. HBB overexpression has been shown to suppress reactive oxygen species-mediated apoptosis. Gene set enrichment analysis using Gene Ontogeny (GO) Pathways demonstrated upregulation in neutrophil pathways and developmental signaling, such as bone morphogenesis (BMP), in the MSI-H-N group. We did not observe differences in T-cell markers between MSI-H groups, raising the possibility that non-T-cell mediated mechanisms may dictate response to ICB. MSI-H-R expression profiles resembled the MSI-H-loc group, apart from differences in PLA2G2A, ASS1, AGR2, and SPINK1, which may be markers of metastasis propensity. Gene expression profiling revealed substantive differences between MSI-H-N and MSS groups, indicating their resistance mechanisms to ICB may differ and supporting their fundamental differences. CONCLUSION: Given the rarity of MSI-H CRC and limited available data to predict response to ICB, additional studies are imperative to improve understanding of the therapeutic landscape for MSI-H CRC. MACE-seq is feasible on FFPE specimens and well suited for transcriptional comparisons between populations. Our study revealed a cohort of hypoxia-related genes to correlate with non-response to ICB in metastatic MSI-H CRC. Additional data collection is ongoing, and future studies will expand tumor types and implement mouse models to test genes of interest. Citation Format: Cameron Oswalt, Ashley Moyer, Y-Van Nguyen, Brent Hanks, John Strickler, David Hsu, William Jeck, Nicholas DeVito. Analyzing immunotherapy resistance in microsatellite instable colorectal FFPE samples using RNA sequencing [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A097.