Abstract 4989: Introduction of a robust workflow for the whole-side acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay

Background: Profiling multiple cell types in tissue samples requires more markers than can be supported by conventional approaches to immunohistochemistry. InSituPlex® multiplex immunofluorescence (mIF) technology enables biomarker multiplexing in tissue samples with an automated workflow to support discovery of immune cell signatures in the tumor immune microenvironment (TiME). Here we have designed and tested an 8-plex immunophenotyping panel using the InSituPlex approach to detect and classify T cells, macrophages, and tumor cells in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) FFPE tissue. Methods: An 8-plex UltiMapper™ immunofluorescence was developed to stain FFPE tissue, including colorectal cancer, human tonsil, and non-small cell lung cancer. Each sample was stained for the following 8 markers: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, and pan-CK. Whole-slide imaging was performed in two imaging rounds on the RareCyte CyteFinder II HT slide scanner, and same-slide image alignment and phenotypic analysis was performed using the HALO Highplex FL module from Indica Labs. Results: Co-registration of two 4-plex imaging rounds and fusing into a single image file allowed for the seamless cell phenotyping of markers across imaging rounds. The 8-plex assay and workflow is compatible across multiple tumor sample types. The samples were characterized based on binned expression and co-expression of biomarkers. The following major cell phenotypes were detected: PD-L1 checkpoint expression, T cells (CD3+), cytotoxic T cells (CD3+/CD8+), T-helper cells (CD3+/CD4+), regulatory T-cells (CD3+/CD4+/FoxP3+), exhausted T cells (CD3+/PD-1+), macrophages (CD68+), and tumor cells (pan-CK+). Conclusions: The 8-plex assay workflow involving InSituPlex mIF, CyteFinder HT scanner, and HALO analysis supports the ability to measure biomarkers in situ in streamlined and scalable approach. Citation Format: Anne E. Hellebust, Kyla Teplitz, Katir K. Patel, Karan Sharma, Amanda Bares, Mael Manesse, Mark Burton, Bonnie Phillips, Sean Downing, Kate Lillard. Introduction of a robust workflow for the whole-side acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4989.