DetermaIO Kit: Decentralizing classification of the tumor microenvironment.

Abstract

e14633 Background: The tumor microenvironment (TME) is critical in modulating cancer progression and therapeutic response, especially for immune checkpoint inhibitors (ICIs). DetermaIO, a 27-gene immuno-oncology signature, has demonstrated clinical utility in predicting immune checkpoint inhibitor responsiveness through classification of the TME. This study focuses on adapting the DetermaIO LDT assay to a research-use-only (RUO) kit for TME classification. 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 IO Score. Methods: DetermaIO is currently run as an LDT using a 2-step RT-qPCR method with 32 assays duplexed in a 384-well plate. To optimize the DetermaIO assay for a kit-based solution, we deployed the following: Transition to one-step RT-qPCR: This eliminated multiple sample-handling steps by using RNA directly into the qPCR reaction. Multiplexing reactions into four per reaction (8 total reactions per sample) to enhance throughput and maximize compatibility with 96-well plate format. Re-evaluating and selecting stable housekeeping genes (HKs) for normalization, using a composite approach that integrates expression stability across diverse cancer tissues, variance filtering, and biological pathway independence. Validating assay reproducibility and concordance with the LDT version, ensuring the kit preserves the DetermaIO established classification of the TME. Results: Workflow improvements significantly reduced hands-on time and minimized opportunities for use error. Reproducibility across intra- and inter-run precision studies were maintained despite the increased multiplexing. Consistent normalization using the 5 HK genes enables accurate quantification of the requisite 27-genes that comprise IO score across various sample types and poor RNA quality FFPE samples. Conclusions: The DetermaIO kit represents a significant step toward democratizing access to tumor microenvironment classification by transforming a validated LDT into a scalable and reproducible RT-qPCR assay. By leveraging a multiplexed, one-step workflow, this kit facilitates broader research applications and harmonization across laboratories, supporting standardized TME classification for precision oncology.