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 and revealed benign intraductal abnormalities, primarily intraductal fibrous webbing suggesting chronic inflammation, in 40.4% of HR and 5.6% of NR vs. subjects (P2 = 0.0004), respectively. Chronic inflammation may increase the risk of cancer, and its presence in these HR ducts may have significant implications for understanding the high-risk state for breast cancer. 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, 2021, 21, Pages 63-73). Gene expression profiling by microarray of ductal cells is being studied and has indicated comparable gene expression profiles without enriched expression of previously defined oncogenic signatures in subjects with cellular atypia compared to those without atypia, and in HR subjects compared with NR subjects (FDR0.5). These findings indicate the important observation that cytologic ductal atypia in normal risk subjects does not appear to be of clinical significance. Atypia in women at high risk may be associated with benign and malignant breast ductal abnormalities which, however, may not be reflected in gene expression profiles. 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 would allow detailed characterization of each ductal cell type 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 Cancer Res Treat, 167:9 2018). An important secondary objective of the study is to determine the presence of mammary system cells in the ductal microenvironment. Mammary stem cells are considered the progenitors of all ductal cell types. Importantly, mammary stem cells may also be transformed into cancer stem cells, the origin of breast cancer. Identification of MSC or CSC in the breast ductal cellular content could have important implications for understanding early breast carcinogenesis. Recent evidence also indicates the presence of a microbiome in breast tissue. Characterization of the microbiome in our ductal samples would 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. We recently performed a pilot study in which we analyzed 8 previously collected frozen ductal samples 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, with the distribution of these microbia being distinct from control samples. This confirmed the feasibility of studying the microbiome in our normal risk and high risk breast ductal samples. Expansion of these studies are in progress. 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 women. The ductal sampling method which we have developed is applicable to all ethnic groups and both normal risk and high-risk women, and thus represents an excellent means to further define the molecular differences in breast tissue associated with the disparities among ethnic groups at risk for breast cancer.