Abstract 2049: The 27-gene IO Score on the Ion Torrent Platform for classification of the tumor immune microenvironment

Abstract Background: The tumor immune microenvironment (TIME) plays a critical role in modulating cancer progression and therapeutic response, especially for immune checkpoint inhibitors (ICIs). DetermaIO, a 27-gene signature assay, has demonstrated clinical utility in predicting immune checkpoint inhibitor responsiveness through classification of the TIME. This study focuses on adapting DetermaIO to the Ion Torrent platform as a research-use-only (RUO) kit, OncoTIME. This assay enables precise and reproducible gene expression profiling of the immune system, tumor biology, and surrounding stromal biology in solid tumors, using this to derive the OncoTIME Score. Method: OncoTIME was designed as a multiplexed targeted RNA sequencing assay using Ion AmpliSeq technology to quantify the expression of 27 immune-related genes and 5 housekeeping (HK) genes. Target regions for the 27-gene panel guided by prior design of the DetermaIO assay. HK genes were identified through a composite approach, combining three methodologies: Expression Stability: Evaluated consistency across tissue types and tumor states using datasets from The Cancer Genome Atlas (TCGA) and internal NGS datasets. Variance Filtering: Selected genes with minimal inter-sample variance within experimental datasets. Biological Context: Ensured HK genes were biologically non-variable and minimally involved in immune-related pathways. These methods ensured robustness of normalization across diverse sample types and RNA input qualities. The assay design used Thermo Fisher's Ion Torrent GeneStudio platform, leveraging its efficient library preparation and sequencing workflow. Results: The OncoTIME panel demonstrated: High specificity and coverage of target regions for all 27 immune-related genes. Reproducibility assessed across intra- and inter-run precision studies. Consistent normalization using the 5 HK genes, enabling accurate quantification of immune signatures across a dynamic range of tumor microenvironment profiles. Preliminary studies confirmed that OncoTIME recapitulates the DetermaIO Score. Conclusion: OncoTIME democratizes access to TIME classification for research use and provides a scalable, reproducible, and cost-effective assay for the Ion Torrent platform. This RUO kit recapitulates DetermaIO while enabling broader utility in characterizing the TIME and its potential biological associations. Citation Format: Tyler J. Nielsen, Jim Fee, Geoff Bien, Brock Schweitzer. The 27-gene IO Score on the Ion Torrent Platform for classification of the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2049.