Abstract 2784: Automated detection of IFN-γ, ERBB2, IgG-Kappa mRNA and protein in FFPE tissues
Authors: Masabanda Julio S. , Figueroa Sara , Vargas Joseph , Ramos Jason 
Published: July 2021

Abstract
Introduction: In recent years, the detection of RNA molecules has become increasingly important for the analysis of the physiological status of cells within tissues. This has complemented the established immunohistochemistry (IHC)/immunofluorescent (IF) technologies for detection of protein targets on cells and tissues. Combined detection of RNA and protein targets is more informative and is providing better insights into the physiological status of cells and tissues than the individual tests alone. However, the sequential detection procedures applied in IHC/IF and RNA in situ hybridization (RISH) can be tricky when performed manually. Thus, continuous development of automation platforms is geared toward simplifying the application of these technologies. In this study, we report the automated co-detection of ERBB2, IgG-Kappa and INF Gamma (IFN-γ) protein and mRNA on the ONCORE Pro platform.
Materials & Methods: Tissues used in this study were FFPE and sectioned at 5 microns. Slides were deparaffinized, dehydrated and peroxidase blocked before performing HIER or enzymatic digestion with pronase. Then, for ERBB2 and IG-Kappa, the mRNA detection was followed by IF detection of the protein target. For IFN-γ, this order was reversed. The Advanced Cell Diagnostics (ACD) recommended protocol for detection of RNA was modified for fluorescent detection and adapted for application on the ONCORE Pro. mRNA was visualized using Opal 520 with green fluorescence and protein targets with mouse or goat antibody-CF543 orange fluorescence. Antibodies and reagents were provided by Biocare Medical and Abcam, in situ probes and detection by ACD, and Opal 520 by Akoya Biosciences.
Results: In our experiments, for the detection of mRNA of ERBB2 and IG-Kappa, we used pronase, and this seemed not to have any effect on the further detection of their protein targets. However, for IFN-γ, we observed that pronase seemed to destroy the target protein, and when the detection order was reversed (i.e. the protein first and then the mRNA), both targets could be seen under the microscope. The sequential approach worked well with generating a clear and consistent staining of 3+ on a staining strength scale of 1-3 for IF and ISH, while the negative controls displayed no specific staining and were clean. Detection of IFN-γ protein was not trivial, we observed certain tissue-specific patterns that were identified when detecting IFN by IHC versus the mRNA in certain tissues indicating the possibility of a non-perfect correlation. Indeed, co-application of RISH simplified the identification of the true positive cells and target areas on tissues.
Conclusions: Application of the combined ISH-IF detection approach is beneficial. When added this to the apparent advantages of full automation (ONCORE Pro), this blend makes these technologies more attractive for studying the physiological status of immuno-oncology markers in target tissues and organs.
Citation Format: Julio S. Masabanda, Sara Figueroa, Joseph Vargas, Jason Ramos. Automated detection of IFN-γ, ERBB2, IgG-Kappa mRNA and protein in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2784.