Stage-Dependent Expression of Hormonal Receptors, HER2, Ki-67, and PD-L1 in Breast Cancer: Correlation With Molecular Subtypes and Tumor Microenvironment

Abstract

Tumor stage and molecular phenotype are both powerful predictors of the clinical behavior and therapeutic responsiveness of breast cancer. Although the most commonly used IHC biomarkers, including ER, PR, HER2, Ki-67, and PD-L1, are actively applied in practice, the dynamics of their integration and their connection with the tumor microenvironment (TME) have not been fully described within a single cohort. This retrospective cross-sectional study involved 70 consecutive patients with primary invasive breast carcinoma (Al-Zahraa Teaching Hospital, Iraq; January 2023-December 2024), stratified according to AJCC TNM staging (8th edition). ER, PR, HER2, Ki-67, and PD-L1 (clone 22C3; tumor proportion score [TPS]) IHC were performed on FFPE tissue. Surrogate IHC criteria were used to assign molecular subtypes; TME characteristics (TILs, stromal density, CD163) were evaluated on H&E sections. Inter-observer agreement (Cohen’s κ), chi-square/Fisher’s exact tests, and Cramer’s V effect sizes were applied. ER positivity declined from 85.7% (Stage I) to 28.6% (Stage IV) (P=0.008; V=0.41); PR declined similarly (78.6% to 14.3%; P=0.004; V=0.44). HER2, Ki-67 (>20%), and PD-L1 (TPS≥1%) increased with advancing stage (P=0.042, <0.001, and 0.011, respectively). Spearman’s correlations confirmed significant stage trends: Ki-67 (ρ=0.62; 95% CI: 0.45-0.75; P<0.001), PD-L1 (ρ=0.38; 95% CI: 0.16-0.56; P=0.001), ER (ρ=−0.44; P<0.001), and PR (ρ=−0.46; P<0.001). Stages III-IV were characterized by a predominance of aggressive subtypes (HER2-enriched, TNBC). High TILs were correlated with TNBC and PD-L1-positive tumors; dominance of M2 macrophages was significantly correlated with PD-L1 expression (Fisher’s exact P=0.009). Combinatory biomarker profiling and TME characterization identify stage-specific changes that could be used to improve patient stratification and guide therapeutic choices, especially the choice of immunotherapy in metastatic breast cancer. Future multicentric validation using standardized genomic subtyping is justified.