Pathway impact analysis (PIS) for robust and comprehensive interpretation of differentially expressed genes (DEGs)

Abstract In transcriptome analysis, identifying differentially expressed genes (DEGs) is fundamental for understanding cellular responses and elucidating disease mechanisms. However, conventional DEG selection often relies on arbitrary thresholds for fold change and statistical significance, which are sensitive to experimental noise and sample size, thereby affecting reproducibility and biological interpretability. We present the Pathway Impact Score (PIS), a data-adaptive method that optimizes DEG thresholds by maximizing cumulative pathway enrichment. Unlike fixed cut-offs, PIS systematically determines dataset-specific thresholds that yield the most coherent and enriched pathways. Validation using the MAQC2 benchmark and 16 dataset of pulmonary fibrosis model suggested that PIS can improve the recovery of true DEGs, while maintaining stability under varying noise levels and replicate counts, and increasing pathway enrichment strength. PIS also supported high-resolution analysis of dose-dependent transcriptomic responses to phosphodiesterase (PDE) inhibitors, capturing pathway-level perturbations that appeared to align with each compound’s potency. Such analyses may facilitate more precise characterization of drug-induced transcriptional changes, highlighting the potential of PIS for integrative transcriptomic studies. Overall, PIS provides a practical, biologically informed framework for adaptive DEG selection, particularly suited for meta-analyses and integrative studies where dataset-specific variability needs careful consideration. It is designed for seamless integration into existing transcriptomic analysis pipelines or as a complementary module, requiring minimal additional parameter tuning beyond standard differential expression workflows.