Erak, Camila Pacheco

Abstract Purpose: Black women are disproportionately affected by aggressive breast cancers compared to white women, suggesting underlying biological differences in normal breast tissue. This study aims to identify molecular disparities in normal breast tissue between premenopausal Black and white women that may contribute to increased breast cancer risk. Experimental Procedures: Visium spatial transcriptomic analysis was performed on a pilot cohort of normal breast tissue samples from premenopausal Black and white women. Tissue sections were spatially profiled, and capture spots were classified based on predominant cell types: epithelial, stromal, or endothelial. Differential gene expression analysis between racial groups was conducted for each cell type classification. Multiplex immunofluorescence (mIF) was used to validate selected markers in the pilot cohort. Meta-analysis compared differentially expressed genes with published gene sets associated with breast cancer subtypes. Results: Significant racial differences in gene expression profiles were identified in both epithelial and stromal spots. In epithelial spots, genes upregulated in Black women significantly overlapped with over 20 published gene sets associated with estrogen receptor-negative (ER−) breast cancer, including key genes involved in Wnt signaling inhibition (e.g., WIF1) and basal epithelial markers (e.g., KRT14, KRT15). This suggests alterations in pathways linked to more aggressive breast cancer phenotypes. In stromal spots, differentially expressed genes related to extracellular matrix remodeling and fibroblast activation (e.g., PI16, DPT, EFEMP1) were observed, indicating potential differences in stromal-epithelial interactions. Ongoing deconvolution and spatial analyses are being performed to further elucidate these findings. Preliminary mIF validation confirmed increased expression of PI16 in samples from Black women. Validation of additional markers is underway in a larger cohort of premenopausal Black and white women. Conclusions: Spatial transcriptomic profiling of normal breast tissue reveals distinct molecular differences between Black and white women, particularly in pathways associated with aggressive breast cancer subtypes and stromal interactions. These findings suggest that inherent variations in the normal breast microenvironment may contribute to the increased risk and aggressiveness of breast cancer in Black women. Further validation and analysis may identify potential biomarkers for risk assessment and inform targeted prevention strategies. Citation Format: Camila Pacheco Erak, Nicole Cruz-Reyes, Pria Westerman, Joshua Ogony, Jean R. Clemenceau, Tae Hyun Hwang, Sarah McLaughlin, Derek Radisky. Spatial transcriptomic profiling of normal breast tissue reveals potential mechanisms underlying increased breast cancer risk in Black women [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 765.

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.

Abstract Introduction: Fibroadenoma is the most common type of benign breast lesion and is not traditionally considered an indicator of increased breast cancer risk. This study aimed to identify biomarkers within fibroadenoma associated with subsequent breast cancer development using patient samples from the Mayo Clinic Benign Breast Disease (BBD) cohort. Methods: We analyzed formalin-fixed paraffin-embedded (FFPE) breast biopsy samples from patients diagnosed with non-proliferative fibroadenoma who either developed breast cancer later (n=10) or remained cancer-free (n=5). NanoString RNA profiling identified differentially expressed genes in fibroblasts associated with subsequent cancer development. Immunohistochemistry was first done to compare overall expression of PI16 in this cohort. Opal multiplex immunofluorescence staining (mIF) was then performed to evaluate the expression of PI16 in fibroblasts (Vim+PanCK-) and assess immune cell infiltration (CD45+) in different tissue compartments. Fibroblasts were identified by vimentin positivity (Vim+), and epithelial cells were marked by pan-cytokeratin positivity (PanCK+). Results: Compared to patients who remained cancer-free, those who developed breast cancer showed significantly increased expression of PI16 in fibroblasts (PI16+Vim+PanCK-, 3.25-fold increase, p=0.0011). Additionally, the number of immune cells (CD45+) was significantly decreased (0.52-fold, p=0.03) in the fibroadenoma lesions of patients who later developed cancer. Conclusions: Our findings suggest that elevated expression of PI16 in fibroblasts within fibroadenoma may serve as potential biomarkers for predicting breast cancer risk for women with fibroadenoma. PI16 encodes Peptidase Inhibitor 16, which is implicated in modulating fibroblast activity and extracellular matrix remodeling—processes that can contribute to a tumor-supportive microenvironment. Elevated levels of this protein in fibroblasts suggest an altered microenvironment that may foster tumor initiation and progression. The decreased immune cell infiltration further implies an immunosuppressive microenvironment that may facilitate cancer progression. As these markers can be detected in FFPE samples, our results could lead to the development of a readily applicable clinical test to identify fibroadenoma patients at increased risk for breast cancer, enabling earlier intervention and surveillance. Citation Format: Pria Westerman, Nicole Cruz-Reyes, Camila Pacheco Erak, Robert Vierkant, Bryan McCauley, Stacey Winham, Mark Sherman, Amy Degnim, Derek Radisky. Elevated PI16 expression in fibroadenoma fibroblasts is 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 2077.