POSTER: Analysis of Multiplexed Immunotherapy Targets and Secreted Ligands Using Computational Tumor Microenvironment Profiling


As the number and diversity of cellular and signaling pathway therapeutic targets in immuno-oncology increases, more therapy combinations are aimed at multiple cell types in the tumor microenvironment (TME). To develop rational combinations of therapies that will be effective in any individual patient, multivariate biomarker assays will be required to understand each patient’s specific tumor and immune profile. Understanding the mechanistic effects of immunotherapy requires identifying the tissue context of direct immunotherapy targets and downstream effector molecules such as secreted cytokines. Although inflammatory response and gene expression of the total tumor can be ascertained through several methods, the spatial arrangements and proximity of cells and colocalization of biomarkers can only be determined with tissue-based assays.

Duplex chromogenic in situ hybridization (CISH) assays using the RNAscope® platform allow for the direct visualization of gene expression of messenger RNA targets for multiple cell types and biomarkers. Computational Tissue Analysis (cTA®) was used to quantify cellular spatial distributions, including regional density, proximity, and biomarker colocalization, in both the tumor nests and surrounding stroma that would be challenging to quantify by visual inspection of duplex RNAscope assays in non–small cell lung cancer (NSCLC) tissue microarrays (TMAs). Oftentimes neither immune effector cells nor the therapeutic target is sufficient in isolation to understand blocked antitumor immune responses and therefore the potential efficacy of a single or combination therapeutic strategy. Multiplex RNAscope assays and cTA in combination allow for the assessment of end points that measure spatial relationships between cell types expressing certain biomarkers. Because these are performed in the context of the tissue microarchitecture within and across multiple tissue sections, they are a significant advancement over traditional tissue-based manual methods.


The use of cTA in multiplex RNAscope assays allows for:

  • Reporting of additional end points not available with traditional immunohistochemistry testing.
  • Quantification of individual and dual positive cells in separate tissue compartments.
  • Spatial measurements to investigate proximity interactions between cells expressing different biomarkers to represent unique biological phenotypes.
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