Abstract 3971: Rapid antibody drug conjugate markers and RNA biomarkers in solid tumors using a hybrid Ultivue InSituPlex and Protease-Free RNAscope Workflow

Abstract Introduction: The spatial relationships between tumor cells, immune infiltrates, and the surrounding stroma—characterized by both protein expression and transcriptional activity—are critical determinants of therapeutic response and disease prognosis. Traditional spatial profiling approaches often require serial sections, compromising exact cellular co-localization, or utilize harsh processing steps that degrade target integrity. There is an urgent need for robust, high-throughput, single-slide methods to co-detect protein and RNA with high fidelity. Methods: We developed and validated a novel, streamlined hybrid workflow combining Ultivue’s highly multiplexed InSituPlex technology (protein detection) with Advanced Cell Diagnostics’ (ACD) newly developed protease-free capability with RNAscopeTM in situ hybridization (ISH) workflow. This pairing is crucial: the protease-free ISH step prevents the degradation of critical cell surface epitopes required for robust downstream multiplex immunofluorescence (mIF) protein staining. A comprehensive panel was rapidly assembled, including key immuno-oncology protein markers for antibody drug conjugates (ex. HER2) via InSituPlex, co-detected with relevant transcriptional targets (e.g., cytokine mRNA, therapeutic target transcripts) via ISH. The assay was applied to FFPE tissue sections from Non-Small Cell Lung Cancer (NSCLC), Triple-Negative Breast Cancer (TNBC), and Gastric Cancer (GC) to demonstrate broad applicability. Results: We successfully achieved simultaneous, high-resolution co-localization of up to 5 protein markers and 3 RNA transcripts within a single tissue section across all three heterogeneous cancer types. The protease-free pre-treatment maintained optimal tissue and epitope morphology, enabling exceptional signal-to-noise ratios for both protein and RNA channels. Critically, this combined multi-omic readout allowed for the precise spatial phenotyping of cell populations (e.g., CD8+ T cells co-expressing specific cytokine mRNA) and the assessment of spatial proximity between cells defined by combined protein/transcriptional signatures. The modularity of the Ultivue assay allowed for rapid assembly and substitution of antibody panels, enabling high-speed assay optimization. Conclusion: This innovative, combined Ultivue InSituPlex and protease-free ACD ISH assay provides a powerful, single-slide platform for spatially resolved multi-omic analysis. It overcomes the technical limitations of traditional sequential staining, offering a high-throughput, high-fidelity tool that can be rapidly deployed for complex biomarker validation, detailed tumor microenvironment characterization, and improved patient stratification strategies in translational oncology and clinical trials. Citation Format: Patrick Savickas, AKASH PARVATIKAR, Jack Casey, Anushka Dikshit, AMAN TYAGI. Rapid antibody drug conjugate markers and RNA biomarkers in solid tumors using a hybrid Ultivue InSituPlex and Protease-Free RNAscope Workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3971.