Multimodal molecular profiling of invasive ductal carcinoma: High concordance of qPCR with FISH in HER2 assessment and exomic landscape of high-risk subtypes.

Accurate evaluation of HER2 status is critical for the targeted management of invasive ductal carcinoma (IDC) of the breast; however, resolving equivocal immunohistochemistry (IHC) results remains a significant clinical challenge. This study aims to comparatively evaluate IHC, fluorescence in situ hybridization (FISH), and quantitative polymerase chain reaction (qPCR) for precise HER2 assessment, while mapping the broader genetic landscape of aggressive IDC subtypes using whole-exome sequencing (WES). We comprehensively analyzed 160 histopathologically confirmed IDC samples using IHC, FISH, and qPCR to evaluate hormone receptor and HER2 amplification status. Furthermore, targeted biomarker profiling (Androgen Receptor [AR] and Ki-67) and exploratory WES were performed on a high-risk subset of 10 HER2-positive (IHC 3 +) and triple-negative breast cancer (TNBC) cases to identify underlying somatic mutations and structural variations. qPCR demonstrated a high diagnostic concordance with gold-standard FISH for detecting HER2 gene amplification across all ASCO/CAP classification groups (sensitivity 96.4%, κ = 0.94; p < 0.05). Notably, qPCR successfully resolved the clinically ambiguous IHC 2 + cohort, identifying definitive HER2 amplification in 41.3% of these cases. Biomarker profiling identified AR expression within the TNBC cohort, suggesting the presence of the clinically actionable Luminal Androgen Receptor (LAR) subtype. Furthermore, exploratory genomic profiling via WES revealed profound intratumoral heterogeneity: TNBC samples frequently harbored pathogenic variants in TP53, BRCA1, and MYCN, whereas the HER2 3 + cohort exhibited prominent mutations in PAK1, CUL3, and TP53. Our findings establish qPCR as a highly robust and accurate diagnostic adjunct for resolving clinically equivocal HER2 cases in IDC. Furthermore, while restricted to a limited exploratory subset, the integration of targeted genomic profiling identified complex mutational hubs driving aggressive breast cancer phenotypes. These hypothesis-generating insights provide a vital framework for bridging accurate diagnostic stratification with future precision oncology strategies.