Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.

Area of Science:

• Oncology
• Immunology
• Biomarker Discovery
• Pathology

Background:

• Precision immuno-oncology relies on predictive biomarkers from spatial cell arrangements and coexpression patterns.
• Multiplexed immunofluorescence (mIF) is a key technology for assessing these biomarkers.
• Standardization of mIF workflows is crucial for multisite clinical trials and laboratory processes.

Purpose of the Study:

• To optimize and validate an automated six-plex mIF assay for the PD-1/PD-L1 axis.
• To assess the intersite and intrasite reproducibility of mIF staining and analysis.
• To evaluate the measurement of immune cell densities, PD-L1 expression, and PD-1/PD-L1 proximity.

Main Methods:

• A six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, CK) was optimized for quantitative equivalence to IHC.
• Six institutions stained serial tissue sections (tonsil, breast, NSCLC) using standardized automated stainers and imaging platforms.
• A locked-down image analysis algorithm was used to assess cell densities and coexpression patterns.

Main Results:

• The optimized mIF assay achieved 90% equivalence to IHC across six markers, with enhanced amplification for low-level expression.
• High intersite and intrasite concordance for cell density assessments were observed (R² values ranging from 0.72 to 0.88).
• Strong concordance was achieved for %PD-L1+ immune cells (R²=0.88) and PD-1/PD-L1 proximity (R²=0.82).

Conclusions:

• The optimized mIF assay provides sensitive and reproducible characterization of the PD-1/PD-L1 axis across multiple sites.
• High concordance across sites supports the use of mIF for assessing immune cell subsets, coexpression, and proximity with single-cell resolution.
• This standardized workflow is suitable for multisite clinical trials in precision immuno-oncology.