Single‑Nucleus Genomic Profiling of HR+/HER2– Breast Tumors Revealed Limited Intratumoral Genetic Heterogeneity in Early‑Stage Disease

Hormone receptor-positive, HER2-negative (HR+/HER2-) breast cancer is the most common breast cancer subtype, yet the level of intratumoral heterogeneity, subclonal architectures and temporal ordering of mutations driving its progression remain poorly understood. To resolve these evolutionary trajectories, we utilized targeted single-nucleus DNA sequencing on 228 early-stage, primary HR+/HER2- breast tumors, capturing over 757,000 individual nuclei. We developed a novel pipeline integrating somatic varians, copy-number alterations (CNAs) and germline variants, and identified detectable intratumoral heterogeneity (ITH) in 25% of the cohort. Most polyclonal tumors exhibited simple architectures consisting of two subclones, with divergence driven primarily by CNAs rather than driver mutations. Evolutionary timing analyses demonstrated that major arm-level events (such as 1q gain and 16q loss) and TP53mutations were predominantly early, truncal events. We also observed hotspot-specific clonality for PIK3CA mutations, where kinase-domain mutations (H1047R) were mostly clonal, and helical-domain mutations (E545K) were frequently subclonal. Additionally, PIK3CA driver mutations were detected in normal diploid cells in 3.9% of the cohort. While ITH was positively associated with tumor size and negatively with the tumor grade, it was not significantly associated with overall survival. Importantly, minor subclonal ESR1 mutations were detected in treatment-naive tumors, indicating that endocrine resistance mechanisms can arise prior to therapeutic pressure. Together, these findings reveal that functionally significant clonal diversity is established in quarter of tumors early during HR+/HER2- breast cancer evolution.