Abstract 5270: Novel dual-stain IHC assay enables discovery with enhanced clarity

Abstract Background: Immunohistochemistry (IHC) analysis has become a powerful and integral component in the diagnostic workup of tumors. It has been essential in biomarker discovery, with a growing demand for high-output methods that maximize data from limited tissue samples. Chromoplex II, an advanced technique that enables the simultaneous detection of dual stains on a single slide, permits the selection of primary antibodies to be stained in either color, aiding tissue differentiation while minimizing the interference often seen with endogenous pigments or slide debris. The duplex approach also reduces slide usage compared to single-stains and individual slides. Methods: Using the BOND-III Automated IHC and in-situ hybridization (ISH) stainer from Leica Biosystems, Chromoplex II was evaluated in three dual-marker applications. At least five cases with known tumors of distinct tumor histology/classification were utilized to examine differences between the distinct cellular histology (i.e., adenocarcinoma and squamous carcinoma) as well as classical Hodgkin lymphoma (cHL) versus nodular lymphocyte predominant Hodgkin lymphoma (NLP). Another commonly encountered scenario is the difficulty in separating lung adenocarcinoma and bystander macrophages, so these tissues were tested as well. Stained slides were scanned using the Aperio GT 450 scanner from Leica Biosystems and evaluated by a clinical pathologist for specificity and comparability to single staining. The marker combinations and tissues tested were the following: CD20/CD30: Lymph node CK5/Napsin A: Lung CD163/PD-L1: Lung Results: In all three marker combinations, the Chromoplex II kit produced distinct red and green stains, providing a clear visualization of target markers. Importantly, it minimized background interference, significantly enhancing clarity in challenging tissue sections. The slides exhibited comparable signal and reproducibility compared to single marker staining, and many examples were noted in which the dual stain provided vital context about the nature of the relationship between the two markers studied. Conclusions: Chromoplex II represents a promising tool for high-throughput biomarker studies in research, offering benefits in both clarity of staining and operational efficiency by reducing slide volume. Furthermore, additional studies and/or combinations are necessary to explore expanded marker combinations for broader cancer biomarker research applications. Citation Format: Scott Anderson, Stephen Pyper, Jack Heath, Julio Poveda. Novel dual-stain IHC assay enables discovery with enhanced clarity [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 5270.