Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer

乳腺癌风险中乳腺导管上皮的分子特征

• 批准号: 9344100
• 负责人: DAVID DANFORTH
• 金额: $ 45.2万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9344100
• 关键词: Address; African American; Age of Onset; Allelic Imbalance; Aneuploidy; Atypia; BRCA1 gene; Basic Research Breast Cancer; Binding; Biological; Breast; Breast Cancer Prevention; Breast Carcinogenesis; Breast Epithelial Cells; CDKN2A gene; Categories; Caucasians; Cell Cycle; Cell Line; Cells; Cellularity; Characteristics; Chemoprevention; Chromosome abnormality; Classification; Clinical Research; Clinical Trials; Control Groups; Copy Number Polymorphism; Cytology; DNA; DNA Methylation; DNA amplification; Data; Development; Doctor of Medicine; Drug Targeting; Duct (organ) structure; Ductal; Ductal Epithelial Cell; Ductal Epithelium; Early identification; Employee Strikes; Endoscopy; Epithelial; Epithelial Cells; Ethnic group; Evaluation; Exhibits; Freezing; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Silencing; Genes; Genomics; Goals; Healthcare; High Risk Woman; Hispanic Americans; Hispanics; Histology; Irrigation; Laboratory Study; Liquid substance; Loss of Heterozygosity; Mammary Gland Parenchyma; Messenger RNA; Metabolic; Methods; MicroRNAs; Microarray Analysis; Mitogens; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Monitor; Outcome; Pathway interactions; Pharmaceutical Preparations; Population; Principal Component Analysis; Property; Protocols documentation; Publishing; RNA; Regulator Genes; Reproductive Health; Resource Development; Resources; Reverse Transcriptase Polymerase Chain Reaction; Review Literature; Risk; Risk Assessment; Risk Factors; Sampling; Signal Pathway; Specimen; Staging; Telomerase; Telomere Shortening; Time; Tissue-Specific Gene Expression; Transcript; Tumor Suppressor Genes; Woman; anticancer research; base; cancer risk; carcinogenesis; caucasian American; design; drug testing; exosome; high risk; improved; in vitro Model; malignant breast neoplasm; mammary epithelium; metabolic abnormality assessment; research and development; response; socioeconomics; whole genome

This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and at increased risk for breast cancer among 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 studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) A comprehensive literature review of the molecular changes in normal breast tissue at either normal risk or at high risk for breast cancer to define the molecular changes in early breast carcinogenesis and to guide the molecular characterization of breast epithelial cells collected from women at normal risk or at high risk for breast cancer under protocol 02-C-0077. c.) A comprehensive literature review of molecular changes contributing to the disparities in development, presentation and outcomes of breast cancer between Caucasian, Hispanic, and African American women. 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 the major drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African American vs. Caucasian women. Multiple socioeconomic, reproductive and health care factors influence the outcome of the disparities through their influence on the breast cancer molecular characteristics. This model also emphasizes the need for defining the molecular characteristics of early carcinogenesis in these ethnic groups. Protocol 02-C-0077 characterizes by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of Caucasian, African American, and Hispanic women at normal risk and at increased risk for breast cancer. One hundred thirty-six women have been studied, 64 high risk subjects and 72 subjects at normal risk. The ductal architectural characteristics of breasts have been defined and correlated with the presence of ductal epithelial cell atypia. A significantly improved method of ductal epithelial cell sampling has been developed which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. These important findings were recently published (Danforth, DN et al, Breast Cancer: Basic and Clinical Research, 9:31-40, 2015). Extraction of a single intact ductal lavage sample (fluid and epithelial cells) or the separate frozen cellular component provided DNA and RNA suitable for multiple downstream molecular studies including RT-PCR of miRNA species, qPCR of the telomerase gene, whole genome DNA amplification, arrayCGH analysis, and microarray gene expression profiling. This method significantly expands our ability to define the molecular characteristics according to breast cancer risk of breast ductal epithelium in women at different risks for breast cancer, and also introduces a much needed method for collecting ductal epithelial cells from women at normal risk for breast cancer, a critical control group for defining the multiple molecular characteristics of women at increased risk for breast cancer. Importantly, 50% - 70% of women in the U.S. who develop breast cancer have no identifiable risk factors, and thus our findings should permit us to further characterize and identify at-risk women in this group, a major population of women in the U.S. Molecular studies to define numerical and structural chromosome abnormalities and gene and microRNA expression of normal at-risk breast epithelial cells from Caucasian, Hispanic and African American women are in progress. The second comprehensive literature review conducted as part of this project has identified important early changes of breast carcinogenesis in normal at-risk breast epithelium, including loss of heterozygosity/allelic imbalance resulting from small segmental deletions, DNA methylation of critical tumor suppressor genes including APC, RASSF1A, p16INK4A, BRCA1, and telomere shortening. Progression of each of these types of genomic abnormalities and the development of aneuploidy occurs in normal breast tissue at high risk for breast cancer. We have conducted preliminary studies of breast ductal epithelial cells by gene expression profiling from women at high risk compared with women at normal risk for breast cancer using the Affymetrix platform, and principal component analysis indicates distinct expression populations for these two groups and a striking differential expression of genes in the high risk group consistent with progression of breast carcinogenesis in these cells. To further define the molecular characteristics associated with increased risk for breast cancer we have collected under protocol 02-C-0077 breast epithelial cells from women at increased risk because of the presence of atypical ductal epithelial cells in their ductal lavage specimens (RR = 2.0 - 4.0), an additional important risk group for breast cancer. Identification of the molecular characteristics associated with these major categories of breast cancer risk should provide critical information toward our objective of defining the breast carcinogenic pathway and for development of a molecular signature for risk assessment. Breast ductal fluid and ductal epithelial cells are being studied for the presence of miRNA, noncoding transcripts which bind to mRNA and result in gene silencing. miRNA has been identified in exosomes of breast ductal fluid collected from these subjects, suggesting an important mechanism for the expansion of the cancerized field and risk within the breast and enhancement of progression through the carcinogenic pathway. We are also developing in this project a potentially very valuable resource, the development of normal breast epithelial cell lines from the ductal lavage samples from women at different risks for breast cancer. We are developing these cell lines in conjunction with Lonza Corp (Walkersville, MD). Demographic data including detailed risk assessment information is available for each subject. The development of a panel of cell lines according to different risks for breast cancer will provide abundant material for molecular profiling of risk characteristics, facilitate analysis of metabolic properties including response to exogenous mitogens and inhibitory substances, promote identification of alterations in signaling pathways according to risk, and allow development of an in vitro model useful for evaluation of chemoprevention drugs. The identification of specific abnormalities between Caucasian and African American breast cancer will also provide important correlative information for analysis of normal and high risk breast epithelium, and facilitate characterization of carcinogenic changes, potentially ethnic-specific, in at-risk breast epithelium.

该项目旨在定义乳腺导管和导管上皮细胞正常风险的形态，分子和代谢特征，以及高加索人，西班牙裔和非裔美国人妇女中乳腺癌风险的增加。需要此信息来定义乳腺癌的致癌途径的早期变化，以开发出癌性乳房组织的分类和分子特征，以进行风险评估，以识别和促进女性预防乳腺癌预防和监测妇女预防乳腺癌的选择，并在乳腺癌的发育和表现方面定义分子基础。该项目包括以下临床和实验室研究：a。）方案02-C-0077，通过细胞学，乳腺导管内窥镜检查和基因表达谱（DN Danforth，PI）对高风险乳腺导管上皮表征。 b。）对正常乳腺组织的分子变化的全面文献综述，无论是正常风险还是在乳腺癌的高风险下定义早期乳腺癌发生的分子变化，并指导从正常风险或高度风险下，乳腺癌的乳腺上皮细胞的分子表征在乳腺癌的高风险下，方案02-C-0077。 c。）对分子变化的全面文献回顾，导致高加索人，西班牙裔和非裔美国妇女之间乳腺癌的发展，表现和结果的差异。最近对文献进行了全面的综述，首次提出了一个模型，描述了生物学和非生物学因素与非洲裔美国人和高加索妇女之间乳腺癌主要差异的启动和发展的关系（Danforth，DN乳腺癌研究，15：208-220，2013，2013，2013）。该模型确定了非洲裔美国人和高加索妇女之间乳腺癌的多种分子差异，这些差异是发病时代差异，更高级阶段，更积极的组织学和在非裔美国人与高加索妇女中较差的生存率的主要驱动力。多种社会经济，生殖和医疗保健因素通过影响乳腺癌分子特征的影响来影响差异的结果。该模型还强调了在这些族裔群体中定义早期癌变的分子特征的必要性。协议02-C-0077的特征是导管灌洗和导管内窥镜检查是高加索，非裔美国人和西班牙裔妇女正常风险的乳房导管和导管上皮，并且患有乳腺癌的风险增加。已经对一百三十六名妇女进行了研究，有64名高风险受试者和72名受试者正常风险。乳腺的导管结构特征已被定义并与导管上皮细胞的存在相关。已经开发了一种显着改进的导管上皮细胞采样方法，该方法提供了多种纯（90％）导管上皮细胞的样品，具有较高的细胞性。这些重要发现最近发表（Danforth，DN等人，乳腺癌：基本和临床研究，9：31-40，2015）。提取单个完整的导管灌洗样品（流体和上皮细胞）或单独的冷冻细胞成分提供的DNA和RNA提供了适用于多个下游分子研究，包括miRNA物种的RT-PCR，端粒酶基因的QPCR，全基因组DNA扩增，ArrayCGH分析和微生物基因表达。根据乳腺癌不同风险的女性乳腺癌的风险，该方法显着扩大了我们定义分子特征的能力，并且还引入了一种急需的方法，可以从正常的乳腺癌风险中收集导管上皮细胞，这是乳腺癌的正常风险，这是一个关键的对照组，这是一个定义乳腺癌风险增加的妇女的关键分子特征。重要的是，美国患乳腺癌的妇女中有50％ - 70％没有可识别的危险因素，因此我们的发现应该使我们能够进一步表征和识别这一组中的危险女性，这是美国分子研究中的主要妇女，以定义数值和结构性染色体异常和基因的妇女妇女的表达，并且是妇女的正常妇女的表达，并且来自正常的妇女的妇女症状症状，并在较高的妇女中，妇女症状​​症状和非洲妇女症状症状和症状性。作为该项目的一部分进行的第二次综合文献综述确定了正常高危乳房上皮的乳腺癌发生的重要早期变化，包括由于小部分缺失而导致的杂合性丧失/等位基因不平衡，DNA甲基化，关键的肿瘤抑制基因的关键甲基化，包括APC，RASASSF1A，P16ink4a，BREREREN，BROMCA，TELOM，和TELOM，以及TELOM，TELOM和TELOM，TELOM和TELOM和TELOM。这些类型的基因组异常的进展和非整倍性发生在正常的乳腺组织中，乳腺癌的风险很高。与使用Affymetrix平台正常风险的女性相比，我们通过基因表达谱图对乳腺癌的乳腺导管上皮细胞进行初步研究，并且主成分分析表明，这两组的表达群体不同，并且在这些细胞中与乳腺癌发生的高风险群体一致的基因一致。为了进一步定义与乳腺癌风险增加相关的分子特征，我们根据规程02-C-0077乳腺上皮细胞从女性处于风险增加的乳房上皮细胞，因为在其导管灌洗样品中存在非典型导管上皮细胞（RR = 2.0-4.0），这是乳腺癌的其他重要风险组。鉴定与这些主要类别的乳腺癌风险相关的分子特征应提供关键信息，以定义乳腺癌途径和开发分子签名以进行风险评估。正在研究乳腺导管液和导管上皮细胞的存在，以进行miRNA，非编码转录本与mRNA结合并导致基因沉默。在从这些受试者中收集的乳腺导管液的外泌体中已经鉴定出miRNA，这表明了乳腺癌和风险在乳房内扩张的重要机制，并通过致癌途径增强进展。我们还在该项目中开发了一种潜在的非常宝贵的资源，这是来自乳腺癌风险不同的女性导管灌洗样品的正常乳房上皮细胞系的发展。我们正在与Lonza Corp（马里兰州Walkersville）一起开发这些细胞系。人口统计数据在内，包括详细的风险评估信息。根据不同风险的乳腺癌风险的开发，将为风险特征的分子分析提供丰富的材料，促进分析代谢特性，包括对外源性有丝分裂剂的反应和抑制性物质的反应，促进根据风险的信号传导途径变化的鉴定，并允许开发用于评估化学率药物的体外模型。鉴定高加索和非裔美国乳腺癌之间特定异常的鉴定还将提供重要的相关信息，以分析正常和高风险的乳房上皮细胞，并促进在高危乳房上皮中的致癌变化（潜在种族特异性）的表征。