Multiplex IHC Detection of Immune Checkpoint Receptors in the Tumor Microenvironment
Jennifer Ziello
Friday, May 12, 2017
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Chapters
Introduction
Agenda
Lots of Biomarkers, Limited Biopsy Material
Multiplexing the Complex Tumor Microenvironment
The Exhausted T Cell Phenotype
Immune Cell Checkpoint Receptors
Modalities of Multiplexing
Tyramide Signaling Amplification (TSA)
Multispectral Imaging and Tissue Analysis
Breast Adenocarcinoma
Antibody Validation for Immunohistochemistry
Tyramide Staining is Reflective of the Chromogenic
The mIHC Workflow
Post-Chromogenic mIHC Panel Validation
Optimized Conditions: The T Cell Exhaustion Panel
Human Tonsil
Immune Checkpoint Receptor and Ligand Expression in Lung, Breast and Ovarian Tumors
Non-inflamed “Cold” Tumors vs. Inflamed “Hot” Tumors
Increased PD-1 and TIM-3 Expression on CD8+ T cells in Hot Tumors
Non-inflamed “Cold” Tumors vs. Inflamed “Hot” Tumors
Visualization of T cell Checkpoint Receptors and Ligands in Various Tumor Types
TIM-3 frequently expressed on CD8- Cells
Immune Checkpoint Receptors Expressed on T Cells or CD8- Cells
Immune Checkpoint Receptors: Targets of Immunotherapy
PD-L1 Expression on the Tumor and/or TME
Increased PD-L1 Expression on Immune Infiltrate in Hot Tumors
PD-1:PD-L1 Interactions are Visualized within the TME
Utility of mIHC to Demonstrate Proximity
Phenotypes Present in Cold and Hot Tumors
Unique Expression of Immune Checkpoint Receptors Among Patient Tumors: Breast 389
Unique Expression of Immune Checkpoint Receptors Among Patient Tumors: Breast 396
Unique Expression of Immune Checkpoint Receptors Among Patient Tumors: Lung 987
Unique Expression of Immune Checkpoint Receptors Among Patient Tumors: Ovary 120
Conclusions
Next Steps
Optimized Panels with IHC Validated Antibodies
Optimized Panels with IHC Validated Antibodies
CST: Rooted in Scienece
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Maintaining spatial localization and proximity of multiple biomarkers is critical to visualizing the tumor microenvironment. Fluorescent multiplex immunohistochemistry (mIHC) enables detection of 6+ proteins in FFPE tissue and is a valuable tool for investigating therapeutic targets and predictive biomarkers in limited patient samples. The fundamental building blocks of a successful mIHC assay are highly specific, application validated antibodies.
Here we explore the protocol and technical considerations for selecting and using antibodies in mIHC to assess immune checkpoints and T cell exhaustion in FFPE tissue samples.