ABSTRACT The human breast is a highly organized, complex organ that consists of an epithelial tissue surrounded by stroma that regulates its proliferation, differentiation, and survival. Stroma is responsible for sustaining normal breast tissue structure and function via a variety of signaling mechanisms that control and regulate normal processes and suppress malignant transformation. The role of stroma in breast tumor initiation, progression, and responsiveness to treatment as well as potential utility for targeted therapies has been widely discussed in recent reviews. While cancer tissue stroma has been widely explored, the evidence of stromal contributions to early stages of carcinogenesis is extremely limited. To fill these gaps, we propose a conceptually and methodologically novel investigation that will focus on benign breast disease (BBD) and high mammographic breast density (MBD), strong risk factors both independently associated with increased breast cancer (BCa) risk, which presents a unique opportunity for studying early changes in the breast and elucidating underlying molecular mechanisms. The study will be conducted by an interdisciplinary team of experts in BCa epidemiology, breast pathology/image analysis, BCa biology, and biostatistics, with a history of long and productive collaboration. We will use data and breast biopsy samples from three established prospective cohorts (Nurses’ Health Study, Nurses’ Health Study II, and Women’s Health Repository) to address the following aims: (1) prospectively examine associations of reproductive risk factors (e.g., parity, age at first birth) with expression of stromal markers (αSMA, FAP, MMP14, TNC, and S100A6) in benign biopsy samples from cancer-free women (n~1,350); (2) examine associations of stromal markers with MBD (n~1,350); and (3) examine associations of stromal markers in women with a previous benign biopsy and the risk of subsequent BCa in a nested case-control design (~400 cases/~975 controls). This proposal leverages established tissue resources, use of validated multiplex immunoflourence for stromal markers, and automated image analysis for MBD assessment. Understanding the associations of BCa risk factors with stromal markers will advance our knowledge on its role in breast carcinogenesis in epidemiologic studies. We will generate the first comprehensive data on the stromal markers’ expression in non-cancer breast and will identify markers that could significantly advance future risk prediction. Stromal activity is potentially modifiable via a variety of targeted therapies; if our project successfully demonstrates an association between stromal markers in benign breast tissue and increased BCa risk and/or high MBD, these findings could eventually translate into pharmaceutical interventions aimed at primary BCa prevention in high-risk women with high MBD and/or BBD. Importantly, these findings would apply to a large segment of women undergoing routine biopsies and those with high MBD in whom novel prevention strategies, improved risk prediction, and tailored clinical management are urgently needed.

PROJECT NARRATIVE Breast tissue stroma plays an important role in sustaining normal breast tissue structure and function and regulating activity of the breast epithelium. While stroma in breast tumors has been widely explored, the stromal contributions to early stages of breast tumor development are poorly understood. To fill this gap, we propose a highly novel investigation that will shed light on associations of selected stromal markers with reproductive breast cancer risk factors and will explore how specific markers could be used to improve breast cancer risk prediction in a large segment of high-risk women undergoing routine breast biopsies and/or in women with high density of breast tissue on the mammograms which could pave a way for novel personalized breast cancer prevention and surveillance strategies.