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.

该项目旨在确定白种人、西班牙裔和非裔美国女性乳腺癌正常风险和乳腺癌风险增加的乳腺导管和导管上皮细胞的形态、分子和代谢特征。需要这些信息来定义乳腺癌致癌途径的早期变化，开发用于风险评估的癌前乳腺组织的改进分类和分子特征，确定新目标并促进选择和监测女性预防乳腺癌，并确定乳腺癌发展和表现差异的分子基础。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表征高风险乳腺导管上皮（DN Danforth，PI）。 b.) 对乳腺癌正常风险或高风险的正常乳腺组织的分子变化进行全面的文献综述，以定义早期乳腺癌发生的分子变化，并指导从正常情况下收集的女性乳腺上皮细胞的分子特征分析根据方案 02-C-0077，有患乳腺癌的风险或高风险。 c.) 对导致白人、西班牙裔和非裔美国女性乳腺癌发展、表现和结果差异的分子变化进行了全面的文献综述。最近发表了一篇对文献的全面综述，首次提出了一个模型，描述生物和非生物因素与非裔美国人和白人妇女之间乳腺癌主要差异的发生和发展之间的关系（Danforth，DN乳腺癌研究） ，15：208-220，2013）。该模型确定了非裔美国人和白人女性乳腺癌的多种分子差异，这些差异是非裔美国人与白人女性发病年龄、晚期阶段、更具侵袭性的组织学和较差生存率差异的主要驱动因素。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来影响差异的结果。该模型还强调需要定义这些种族群体早期致癌的分子特征。方案 02-C-0077 通过导管灌洗和导管内窥镜检查白种人、非裔美国人和西班牙裔女性的乳腺导管和导管上皮，这些妇女的乳腺癌风险正常，并且风险较高。对 136 名女性进行了研究，其中 64 名高风险受试者和 72 名正常风险受试者。乳房的导管结构特征已被定义并与导管上皮细胞异型性的存在相关。已经开发出一种显着改进的导管上皮细胞取样方法，该方法提供具有高细胞结构的纯（90％）导管上皮细胞的多个样品。这些重要发现最近发表（Danforth, DN et al, Breast Cancer: Basic and Clinical Research, 9:31-40, 2015）。提取单个完整的导管灌洗样本（液体和上皮细胞）或单独的冷冻细胞成分，可提供适合多种下游分子研究的 DNA 和 RNA，包括 miRNA 种类的 RT-PCR、端粒酶基因的 qPCR、全基因组 DNA 扩增、arrayCGH分析和微阵列基因表达谱。该方法显着扩展了我们根据不同乳腺癌风险女性乳腺导管上皮的乳腺癌风险定义分子特征的能力，并且还引入了一种急需的从乳腺癌正常风险女性收集导管上皮细胞的方法，一个关键的对照组，用于定义乳腺癌风险增加的女性的多种分子特征。重要的是，美国 50% - 70% 患乳腺癌的女性没有可识别的危险因素，因此我们的研究结果应该使我们能够进一步描述和识别这一群体中的高危女性，这一群体是美国女性的主要群体。确定白种人、西班牙裔和非裔美国女性正常高危乳腺上皮细胞的数量和结构染色体异常以及基因和 microRNA 表达的分子研究正在进行中。作为该项目的一部分进行的第二次全面文献综述确定了正常高危乳腺上皮中乳腺癌发生的重要早期变化，包括小片段缺失导致的杂合性/等位基因失衡的丧失、关键肿瘤抑制基因（包括 APC）的 DNA 甲基化、 RASSF1A、p16INK4A、BRCA1 和端粒缩短。这些类型的基因组异常的进展和非整倍性的发展发生在乳腺癌高风险的正常乳腺组织中。我们使用 Affymetrix 平台，通过对乳腺癌高危女性与正常乳腺癌风险女性进行基因表达谱分析，对乳腺导管上皮细胞进行了初步研究，主成分分析表明这两组的表达群体不同，并且存在显着的差异表达高风险组中的基因与这些细胞中乳腺癌发生的进展一致。为了进一步确定与乳腺癌风险增加相关的分子特征，我们根据方案 02-C-0077 收集了风险增加女性的乳腺上皮细胞，因为她们的导管灌洗标本中存在非典型导管上皮细胞（RR = 2.0 - 4.0），乳腺癌的另一个重要危险人群。与这些主要乳腺癌风险类别相关的分子特征的识别应该为我们定义乳腺癌途径和开发风险评估分子特征的目标提供关键信息。正在研究乳腺导管液和导管上皮细胞中是否存在 miRNA，即与 mRNA 结合并导致基因沉默的非编码转录物。在从这些受试者收集的乳腺导管液的外泌体中已经鉴定出 miRNA，这表明乳腺癌范围扩大和风险增加以及通过致癌途径增强进展的重要机制。我们还在该项目中开发一种潜在的非常有价值的资源，即从具有不同乳腺癌风险的女性的导管灌洗样本中开发正常乳腺上皮细胞系。我们正在与 Lonza Corp（马里兰州沃克斯维尔）合作开发这些细胞系。每个受试者都可以获得人口统计数据，包括详细的风险评估信息。根据不同的乳腺癌风险开发一组细胞系将为风险特征的分子分析提供丰富的材料，促进代谢特性分析，包括对外源丝裂原和抑制物质的反应，促进根据风险识别信号通路的变化，并允许开发可用于评估化学预防药物的体外模型。白种人和非裔美国人乳腺癌之间特定异常的识别也将为正常和高风险乳腺上皮的分析提供重要的相关信息，并有助于对高危乳腺上皮中可能具有种族特异性的致癌变化进行表征。