Westerman, Pria

Abstract Introduction: Pseudoangiomatous stromal hyperplasia (PASH) is a benign breast lesion characterized by stromal myofibroblast accumulation. While most women with PASH do not develop breast cancer, a subset eventually does, often in the same breast where PASH was originally diagnosed, even after surgical removal of the lesion. This pattern suggests the presence of a field effect, where alterations in the PASH microenvironment extend beyond the lesion itself, potentially influencing neighboring breast tissue. This study aims to identify specific fibroblasts within PASH-containing tissues that may create a microenvironment conducive to cancer initiation. Methods: We conducted a retrospective analysis of formalin-fixed, paraffin-embedded breast tissue samples from women diagnosed with PASH, comparing those who later developed breast cancer (cases) to those who remained cancer-free (controls). Using transcriptional profiling with NanoString IO360/BC360 panels, multiplex immunofluorescence (MxIF), and spatial transcriptomics via NanoString CosMX SMI, we investigated molecular and histopathological differences. We focused on the expression of fibroblast activation markers (ITGB1, PDGFRβ) and their spatial relationships with proliferating epithelial cells, as well as changes in extracellular matrix (ECM) composition and stiffness. Results: Transcriptional profiling revealed significant upregulation of proliferation-associated genes in epithelial cells from cases. MxIF showed higher Ki-67 levels in epithelial cells from cases, with proliferating cells forming clustered regions. Activated fibroblasts expressing ITGB1 and PDGFRβ were markedly increased in cases and were located closer to proliferating epithelial cells. Spatial transcriptomics uncovered gene expression changes in fibroblasts indicative of an activated phenotype, and in epithelial cells, consistent with responses to increased tissue stiffness and mechanotransduction signaling. Polarized light imaging with picrosirius red staining revealed enhanced collagen cross-linking and thick fibril formation in cases, indicating greater ECM stiffness. Conclusions: Our findings suggest that a subset of activated fibroblasts in benign breast tissue creates a pro-tumorigenic microenvironment through ECM remodeling and increased tissue stiffness, promoting epithelial proliferation and potentially driving cancer development. The spatial proximity of these fibroblasts to proliferating epithelial cells underscores their possible role in early carcinogenesis. This study introduces the novel concept of cancer-predisposing fibroblasts in benign breast tissue, providing new insights into microenvironmental changes that precede cancer and offering potential targets for early detection and intervention. Citation Format: Nicole Cruz-Reyes, Pria Westerman, Matilde Rossi, Camila Pacheco Erak, Melody Stallings Mann, Bryan McCauley, Robert Vierkant, Stacey Winham, Mark Sherman, Amy Degnim, Derek Radisky. Activated fibroblasts in benign breast tissue are associated with subsequent breast cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2083.