Reactive stroma as a prognostic biomarker of metastasis in patients with breast cancer: integration of histopathology and transcriptomic profiling

Background: The tumor stroma plays a pivotal role in cancer progression, but its prognostic relevance remains underexplored in clinical settings. This study evaluated whether histologically defined reactive stroma, a fibrotic and collagen-rich microenvironment, serves as a prognostic marker for overall survival (OS) and metastasis-free survival (MFS) in patients with breast cancer, aiming to establish it as a clinically actionable biomarker. Methods: We retrospectively analyzed 182 formalin-fixed, paraffin-embedded (FFPE) breast cancer samples from patients diagnosed between 2006 and 2020 at three Chilean institutions. Stromal content was digitally quantified on H&E- and Masson’s trichrome-stained sections usingQuPath software and validated by expert pathologists. Optimal cutoffs for total and reactive stroma were identified via maximally selected rank statistics. Survival outcomes were assessed using Kaplan‒Meierand Cox regression models adjustedfor clinical covariates. A subset of samples was subjected to RNA sequencing to explore transcriptomic programs associated with stromal phenotypes. Results: A lower total stromal content (<71.8%) was significantly associated with poor OS in the univariate analysis but not in the multivariate model. Conversely, a high reactive stroma content (>53.2%) was independently associated with poor MFS (HR = 3.76, p < 0.001), regardless of the molecular subtype or other clinicopathological variables. Transcriptomic profiling revealed the overexpression of ECM-related and protumorigenicgenes (e.g., FN1, OLR1, and EDN2) and enrichment of genes related to collagen remodeling and the TGF-β signaling pathwayin reactive stroma-high tumors. Conclusions: Reactive stroma, defined through routine histology and digital pathology, is a robust and independent prognostic marker for metastasis in patients with breast cancer. Its clinical integration could enhance risk stratification and support novel stroma-targeted therapeutic strategies.