Abstract 4555: Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays
Authors: Boisvert Heike , Wong Alexis , Chatterjee Gourab , Mohammed Abdul , Wood Douglas , Manesse Mael 
Published: August 2019

Background: In recent years, identifying and characterizing biomarkers within the tumor microenvironment (TME) has become a major focus for immune-oncology research. The growing field of immuno-oncology necessitates the identification of more biomarkers that help characterize the TME, and development of complimentary processes to rapidly analyze precious tissue samples. Ultivue® has developed UltiMapper™ assays that provide numerous advantages over alternative multiplexed IHC approaches, including high multiplexing in situ, sample preservation, streamlined workflows with staining completed in the same day, and easy integration into any histology lab. In this study we demonstrate a 10-plex immuno-oncology assay in FFPE tumor tissues by combining markers that profile the T-cell infiltrate, antigen presenting cells (APC), and checkpoint expression along the PD-L1/PD-1 axis. This 10-plex is achieved with a single antibody staining step for a fast workflow that is independent of staining order. The combination of 10 different biomarkers allows for characterization of the TME and investigation of PD-L1 expression on tumor and antigen presenting cells on a single slide. Methods: A 10-plex immunofluorescence assay was carried out using the UltiMapper approach on multiple deidentified FFPE tissue samples, including lung, melanoma, colon, and breast. Each sample was stained for the following 10 markers: CK or SOX10, PD-L1, PD-1, CD3, CD8, CD11c, CD20, CD68, CD163, and MHCII. Staining was performed in a single step using the Leica® Bond® Rx autostainer. Whole slide imaging was performed on various tissue scanners including the Zeiss® Axio Scan.Z1 slide scanner, and image analysis was performed using HALO® software from Indica Labs. Results: Cells were characterized and phenotyped based on their expression of specific biomarkers and the abundance of PD-L1 on macrophages, dendritic cells, B cell, cytotoxic T-cells, and tumor cells as measured. PD-L1 expression was observed on both immune and tumor cells with a range of different expression levels. Spatial analysis was employed to measure the distances between immune cells with different phenotypes and tumor cells to further characterize tumors as hot or cold. Conclusions: The multiplexed UltiMapper IHC approach allows for fast and comprehensive analysis of tumor samples necessary to research complex cancer biology. This approach achieves high quality multiplexed data on various tissue types and preserves precious tissue samples. The ability to interrogate a 10-plex I/O panel on a single slide with whole slide imaging presents a significant leap forward for IHC analysis, providing a faster and more thorough approach to characterize the TME and analyze PD-L1 expression on antigen presenting cells and tumor cells.

Citation Format: Heike Boisvert, Alexis Wong, Gourab Chatterjee, Abdul Mohammed, Douglas Wood, Mael Manesse. Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4555.