Objective:

To explore the potential of virtual biomarker staining as a scalable alternative to multiplex immunofluorescence (IF) for characterizing the tumor microenvironment.

Key Findings:

• Models demonstrated strong per-cell discrimination with AUCs ranging from 0.90 to 0.93.
• Pearson correlations exceeded 0.70, indicating reliable cell-level signal.
• Virtual stains highlighted expected cell populations and tissue compartments as confirmed by pathologists.
• Predictions maintained spatial context of biomarker expression.

Interpretation:

The findings support the feasibility of generating biologically meaningful virtual biomarker images from routine H&E whole-slide images, enhancing tumor microenvironment analysis.

Limitations:

• Dependence on the quality of training data from clinical-grade IHC labels.
• Potential variability in model performance across different tissue types.

Conclusion:

Virtual biomarker staining could revolutionize tumor microenvironment analysis in NSCLC by making it more practical, scalable, and accessible, potentially aiding in precision oncology workflows.