Abstract 2813: A multiplexed, multispectral approach to analyzing the immune microenvironment of oral potentially malignant lesions
Authors: Lin Iris , Noureddine Kouther , Gallagher Paul , Guillaud Martial , Zhang Lewei , Rock Leigha , Rosin Miriam , Laronde Denise 
Published: July 2021

Abstract
Objectives: Tissue sections of oral potentially malignant lesions (OPML) can be used not only to provide pathological assessment (diagnosis) but could also be used to analyze the interactions between cellular populations, signaling molecules, and structural proteins that impact the clinical course of disease. However, much of this information remains unpacked due to methodological limitations. Traditional immunohistochemistry (IHC) limits the number of proteins able to be simultaneously analyzed within a tissue section and is also prone to human error in its analysis. Newer multiplexed IHC (mIHC) methods involving repeated cycles of staining allow for quantification of a greater number of markers; however, tissue integrity may be compromised, and complexity is added to the interpretation of the results. There is a need to develop an immunostaining method that overcomes these barriers.
Hypothesis: mIHC and an in-house Hyperspectral Cell Sociology (HCS) platform will allow for robust and detailed investigation of the immune microenvironment of OPML compared to traditional IHC staining and scoring techniques.
Methods: Automated mIHC staining with a seven immune marker panel was completed on annotated formalin-fixed paraffin-embedded OPML tissue. A cocktail of three primary antibodies was applied, then antigens and chromogens stripped using SDS-glycine before second round staining with four antibodies and a hematoxylin counterstain was completed. Slides were then digitally imaged, regions of interest selected in conjunction with an oral pathologist and staining quantified computationally using the HCS platform. Traditional IHC was completed on a randomized subset of cases using sequentially cut tissue sections and a double-staining technique. Scoring was completed by two blinded clinicians.
Results: One cycle of multiplexed staining and de-staining allowed for the detection of seven markers on one section while minimizing loss of tissue integrity. The HCS platform captures a single tissue section at multiple wavelengths, enabling the unmixing of multiple overlapping, colocalized chromogens. The resulting set of images displayed each stain separately, allowing for nuclei segmentation and the generation of a map of the epithelium and underlying connective tissue. True positive cells for each stain were demarcated on this map, allowing for investigation of marker positivity, co-positivity, cell to cell spatial relationships, and layer-based analysis, compared to cell count and density analyses obtained with traditional IHC.
Conclusion: The immune microenvironment contains a wealth of information pertaining to the biology, pathogenesis, and outcome of disease. Multiplexed staining and imaging methods to analyze and unpack this information is of great clinical utility.
Citation Format: Iris Lin, Kouther Noureddine, Paul Gallagher, Martial Guillaud, Lewei Zhang, Leigha Rock, Miriam Rosin, Denise Laronde. A multiplexed, multispectral approach to analyzing the immune microenvironment of oral potentially malignant lesions [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 2813.