Abstract 153: Quantifying tumor heterogeneity and mapping complex immune cell interactions with high-throughput, 7-color multispectral slide scans
Authors: Coltharp Carla , Johnson Kent , Zhang Wenliang , Wang Chichung , Roman Kristin , Eversole Daniel , Hoyt Clifford , Miller Peter 
Published: August 2019

Abstract
Introduction
As the need for multiple biomarker assessment in immune-oncology has become more clear, multiplex fluorescent immunohistochemistry (fIHC) techniques have become integral to immune oncology research.
Applying multispectral approaches to fIHC improves the quantitative performance by ensuring removal of autofluorescence signals and ensuring signal specificity by removing bleed-through between spectrally-adjacent dyes.
We have recently demonstrated a novel high-throughput multispectral scanning approach that allows acquisition of a 7-color multispectral slide scan in 5 - 10 minutes.
Here, we demonstrate the spatial measurements that have now been made possible by this high-throughput, translational workflow. These measurements span the scales related to tumor biology from the distance between nearest cell neighbors to the extent of the invasive margin.
Methods
Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents. Tissue sections from primary tumors (lung, melanoma, colorectal, lymphoma)were stained against 5 markers of key interest in the field of immune oncology (CD8, FoxP3, PD-1, PD-L1, CD68) along with a tumor marker (Cytokeratin, Sox10&S100, PAX5 or PAX8) and DAPI counterstain, resulting in 7 colors on each slide.
Conventional and multispectral digital scans were acquired on a Vectra Polaris® automated imaging system and analyzed with inForm® and R software using the phenoptr and phenoptrReports packages.
Results
Multispectral scans showed a wide variety of immunological states among samples and within samples. We observed differences in the overall composition and density of immune cells in the tumor microenvironment, and further distinctions when looking at the local proximities of cell types and cells expressing either PD-1 or PD-L1, based on marker combinations that could not be observed with a conventional 3- or 4-color slide scan.
We developed spatial analyses to visualize and quantify this observed heterogeneity, both across the entire tissue and along the invasive margin. These tools revealed hot spots of immune activity and were further used to compare and categorize the properties of different tumors.
Furthermore, when compared with results from analogous scans analyzed without spectral unmixing, the unmixed imagery showed a marked improvement in the dynamic range of detected positive cells.
Conclusion
These tools for spatial analyses of whole-section 7-color multispectral slide scans provide translational researchers the opportunity to discover and validate biomarkers that capture immuno-biological interactions at microscopic and macroscopic scales, to better understand drug method-of-action and why some patients respond and some don’t.
Citation Format: Carla Coltharp, Kent Johnson, Wenliang Zhang, Chichung Wang, Kristin Roman, Daniel Eversole, Clifford Hoyt, Peter Miller. Quantifying tumor heterogeneity and mapping complex immune cell interactions with high-throughput, 7-color multispectral slide scans [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 153.