Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer

Division Of Clinical Sciences - Nci

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Abstract

This project is designed to define the cytologic, ductal architectural, and molecular characteristics of breast ducts and ductal epithelial cells of women at normal risk and at high risk for breast cancer, including Caucasian, Hispanic and African American women. This information is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets and to facilitate selection and monitoring of women for breast cancer prevention, and to define the molecular basis for disparities in the development and presentation of breast cancer. This project includes the following clinical and laboratory study: Protocol 02-C-0077, Characterization of High-Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). This protocol examines by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of women at normal risk and at high risk for breast cancer. One hundred fifty-five subjects have been studied, 75 high risk subjects and 80 subjects at normal risk. A significantly improved method of ductal epithelial cell sampling has been developed under this study which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. The details of this sampling method were recently published (Danforth, DN et al, Breast Cancer: Basic and Clinical Research, 9:31-40, 2015). Extraction of a single ductal lavage sample provided DNA and RNA suitable for multiple downstream molecular studies including whole genome DNA amplification, arrayCGH analysis, microarray gene expression profiling, and RT-PCR of miRNA species. This method significantly expands our ability to characterize the molecular characteristics of ductal cells according to breast cancer risk, including women at normal risk for breast cancer, a critical control group for defining the multiple molecular characteristics of women at high risk for breast cancer. To compliment and guide the molecular studies in this protocol, a comprehensive literature review was conducted to identify and define genomic changes in normal breast tissue at normal risk (NR) and at high risk (HR) for breast cancer (Danforth, DN. Breast Cancer: Basic and Clinical Research 10:109, 2016). This indicated that normal risk breast tissues contain evidence of early breast carcinogenesis including loss of heterozygosity, DNA methylation of tumor suppressor and other genes, and telomere shortening. In normal breast tissues at high risk for breast cancer these changes persist and are increased and accompanied by aneuploidy, increased genomic instability, a wide range of gene expression differences, development of large cancerized fields, and increased proliferation. A model for the carcinogenic pathway in normal risk and in high risk normal breast tissue is proposed in this publication. The studies conducted under protocol 02-C-0077 have also provided important clarification of the clinical and prognostic role of cytologic atypia in women at risk for breast cancer. Cytologic studies of ductal cells under this protocol have revealed the presence of atypical epithelial cells in 22.9% of HR and 25.7% of NR subjects. Ductal endoscopy was performed in 89 subjects, with the very important finding that the ducts of 40.4% of women at high risk contained intraductal fibrous webbing suggesting chronic inflammation, compared with only 5.6% of normal risk subjects (P2 = 0.0004). Chronic inflammation has long been recognized as an important factor promoting the development of cancer, as well as promoting expression of immune checkpoint proteins which can inhibit lymphocytes and immunosurveillance. This has been reviewed in a recent publication (Danforth, DN. Cancers, 13:3918, 2021) Immune cells are clearly instrumental in the development of chronic inflammation, and to help define the role of immune cells in normal breast tissue, we have recently published a review of this relationship (SL Goff and DN Danforth. Clinical Breast Cancer, 21:63-73,2021,). Characterization of intraductal chronic inflammatory changes are important topics for future studies. Our studies have further defined the presence of intraductal cytologic atypia. In normal risk subjects cytologic atypia was present in 25.7%, but was not associated with altered gene expression by microarray profiling, or with abnormalities on ductal endoscopy or on breast MRI, or on follow-up, providing the important observation that cytologic ductal atypia in normal risk subjects does not appear to be of clinical significance. In high-risk subjects atypia was present in 22.9%, and follow-up MRI revealed a ductal carcinoma in 13.7% of patients, indicating the importance of ductal evaluation in high-risk subjects. These findings have recently been reported [Danforth, DN., et al. Characteristics of breast ducts in normal risk and high-risk women and their relationship to ductal cytologic atypia. Cancer Prev Res, 2020;13:1027-36]. The ductal samples we are collecting are single cell suspensions of ductal cells, which should be very amenable to separation by flow cytometry. This is being studied and will allow detailed characterization of each ductal cell type, both epithelial and immune, and further define normal risk, high risk, and atypical cell states. To complement these studies a review of the molecular profile of atypical ductal hyperplasia of the breast was recently published (Danforth, DN. Breast Canc Res Treat, 167:9, 2018). An important secondary objective of the study is to determine the presence of mammary stem cells in the ductal microenvironment. Mammary stem cells (MSC) are considered the progenitors of all ductal cell types. Importantly, mammary stem cells may also be transformed into cancer stem cells (CSC), the origin of breast cancer. Identification of MSCs or CSCs in the breast ductal cellular compartment could have important implications for understanding early breast carcinogenesis. Recent evidence has also indicated the presence of a microbiome in breast tissue. We recently performed a pilot study in which 8 frozen ductal samples were analyzed by 16s rRNA sequencing through the CCR Microbiome Core Facility. We identified microbia in all samples with the following relative distribution according to phylum: Bacteroidetes Firmacutes Protobacteria Actinobacteria. This confirmed the feasibility of studying the microbiome in our normal risk and high-risk breast ductal samples. Further characterization of the microbiome in our ductal samples will clarify the relation of the intraductal microbiome to breast cancer risk and as a potential cause of chronic intraductal inflammation which we have observed in the high-risk ducts. Lastly, to complement our studies of the basis of racial disparities, a comprehensive review of the literature has recently been published proposing for the first time a model describing the relationship of biological and nonbiological factors to the initiation and development of the major disparities in breast cancer between African American and Caucasian women (Danforth, DN. Breast Cancer Research, 15:208-220, 2013). This model identified multiple molecular differences in breast cancer between African American and Caucasian women, and these differences are important drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African American vs. Caucasian wom *TRUNCATED*