Single-Cell Sequencing of Breast Tumors to Investigate Genome Evolution

通过乳腺肿瘤单细胞测序研究基因组进化

• 批准号: 8657942
• 负责人: Nicholas Navin
• 金额: $ 31.8万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-13 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8657942
• 关键词: Address; Aneuploidy; Antibodies; Basic Research Breast Cancer; Breast Cancer Treatment; Breast Carcinoma; Cancer Patient; Carcinoma; Cell Separation; Cells; Chromosomal Gain; Chromosome Deletion; Chromosomes; Clinical; Clonal Expansion; Complex; DNA Sequence Rearrangement; Data; Defect; Diagnosis; Diploidy; Ductal Carcinoma; Embryo; Evolution; Gene Frequency; Genes; Genome; Genomic Instability; Genomics; Goals; Grant; Heterogeneity; Human; Immune System Diseases; In Situ; Individual; Label; Lead; Malignant Neoplasms; Mammary Gland Parenchyma; Mammary Neoplasms; Methods; Mission; Modeling; Molecular; Morbidity - disease rate; Mutation; Nature; Noninfiltrating Intraductal Carcinoma; Normal Cell; Oncologist; Patients; Point Mutation; Population; Preimplantation Diagnosis; Primary Neoplasm; Process; Prognostic Marker; Public Health; Radio; Recurrence; Reporting; Research; Research Personnel; Resolution; Sampling; Signal Transduction; Staging; Stromal Cells; Surface; Technology; Testing; Therapeutic; Time; Tissues; Trees; United States National Institutes of Health; Variant; Work; base; cancer cell; cell type; clinical Diagnosis; clinically relevant; clinically significant; hormone therapy; human disease; improved; innovation; laser capture microdissection; malignant breast neoplasm; mortality; neoplastic cell; nervous system disorder; next generation sequencing; research study; therapeutic target; tool; triple-negative invasive breast carcinoma; tumor; tumor growth; tumor progression

DESCRIPTION (provided by applicant): One of the major factors confounding both the clinical diagnosis and basic research of breast cancer is the genomic heterogeneity within tumors. This salient feature of cancer has long been observed in cytological studies of breast cancer and is becoming ever-more clear with next-generation sequencing studies of tumor genomes, which show mixtures of allele frequencies that are inconsistent with single clonal subpopulations of tumor cells. Although clinicians and researchers are starkly aware of tumor heterogeneity, this hallmark of cancer is often ignored because it complicates both the diagnosis and treatment of tumors, and because tools do not exist to study it. In this grant we propose to develop and apply an innovative single-cell sequencing method that can fully resolve tumor heterogeneity by profiling genomic copy number in individual tumor cells. Using this technology we will test our central hypothesis that tumor evolution can be reconstructed from genomic heterogeneity. Assuming that mutational complexity increases with time, we can compare copy number profiles of single cells to infer lineages of progression. Our rationale is that while aneuploidy has long been reported in human tumors, very little is known about how genomes evolve these complex rearrangements during primary tumor growth. The objective of this application is to use single cell-sequencing to delineate the clonal substructure of triple-negative (ER-, PR-, Her2-) breast tumors and infer genomic evolution at different stages of primary tumor growth. In aim 1 we will investigate copy number evolution in advanced breast tumors to determine if rearrangements occur gradually or in punctuated bursts of evolution. In aim2 we will determine if invasive subpopulations emerge directly from in situ subpopulations in early stage breast cancers, or alternatively if they are independently evolving subpopulations of cells. In aim3 we will study breast tumor stroma to understand how aneuploidy evolves from normal diploid genomes. Our single- cell sequencing approach is innovative because it can resolve complex populations of cells, while standard bulk genomic methods are limited to reporting an average signal and cannot detect rare tumor clones. This research is significant because achieving these aims will improve our fundamental understanding of clonal diversity in human breast cancer and how aneuploid genomes evolve complex rearrangements during primary tumor growth. Our long-term goal is to use single-cell sequencing methods to study the evolution of different molecular clocks in human tumors, including copy number aberrations, structural variation, indels and point mutations to understand how tumors evolve. Our findings are directly relevant to human cancer, because we conduct all of our experiments on human breast tissues. The proposed research aims are directly aligned with the mission of NIH to reduce mortality in breast cancer. Developing these powerful single-cell sequencing tools will also have a positive impact on human diseases beyond cancer, including neurological disorders, immunological diseases, embryonic defects and improving preimplantation genetic diagnosis.

描述（由申请人提供）：混淆乳腺癌临床诊断和基础研究的主要因素之一是肿瘤内的基因组异质性。癌症的这一显著特征在乳腺癌的细胞学研究中早已观察到，并且随着肿瘤基因组的下一代测序研究变得越来越清楚，这些研究显示等位基因频率的混合物与肿瘤细胞的单克隆亚群不一致。尽管临床医生和研究人员都清楚地认识到肿瘤的异质性，但这种癌症的标志往往被忽视，因为它使肿瘤的诊断和治疗变得复杂，而且还没有研究它的工具。在这项资助中，我们提出开发和应用一种创新的单细胞测序方法，通过分析单个肿瘤细胞的基因组拷贝数，可以完全解决肿瘤的异质性。使用这项技术，我们将测试我们的中心假设，肿瘤的演变可以从基因组异质性重建。假设突变的复杂性随着时间的推移而增加，我们可以比较单细胞的拷贝数谱来推断进展的谱系。我们的理由是，虽然非整倍体在人类肿瘤中的报道由来已久，但对原发性肿瘤生长过程中基因组如何演变这些复杂的重排知之甚少。本申请的目的是使用单细胞测序来描绘三阴性（ER-、PR-、Her 2-）乳腺肿瘤的克隆亚结构，并推断原发性肿瘤生长的不同阶段的基因组进化。在目标1中，我们将研究晚期乳腺肿瘤中拷贝数的演变，以确定重排是逐渐发生的还是间断性爆发的演变。在aim 2中，我们将确定侵袭性亚群是否直接从早期乳腺癌的原位亚群中出现，或者它们是否是独立进化的细胞亚群。在aim 3中，我们将研究乳腺肿瘤间质，以了解非整倍体是如何从正常二倍体基因组进化而来的。我们的单细胞测序方法是创新的，因为它可以解析复杂的细胞群体，而标准的批量基因组方法仅限于报告平均信号，无法检测罕见的肿瘤克隆。这项研究意义重大，因为实现这些目标将提高我们对人类乳腺癌克隆多样性的基本理解，以及非整倍体基因组在原发性肿瘤生长过程中如何演变复杂的重排。我们的长期目标是使用单细胞测序方法研究人类肿瘤中不同分子钟的演变，包括拷贝数畸变、结构变异、indels和点突变，以了解肿瘤如何演变。我们的发现与人类癌症直接相关，因为我们所有的实验都是在人类乳腺组织上进行的。拟议的研究目标与NIH降低乳腺癌死亡率的使命直接一致。开发这些强大的单细胞测序工具还将对癌症以外的人类疾病产生积极影响，包括神经系统疾病，免疫性疾病，胚胎缺陷和改善植入前遗传诊断。