Identification of Somatic Mutations in Rare Subclones of Solid Tumors

实体瘤罕见亚克隆中体细胞突变的鉴定

基本信息

批准号：
8155075
负责人：
Olivier Harismendy
金额：
$ 12.86万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-22 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8155075
关键词：
Affect Biological Assay Biological Markers Breast Calibration Cancer cell line Cell Fraction Cell Line Cells Clinic Clinical Clinical Trials Clonal Evolution Collaborations Colon Colonic Neoplasms DNA DNA Sequence DNA amplification Data Databases Detection Disease Disease Progression Dissection Drug resistance Emerging Technologies Ensure Formalin Freezing Frequencies Funding Gene-Modified Genome Goals High Prevalence Laboratories Lasers Libraries Ligation Malignant Neoplasms Measures Methods Microfluidics Modeling Mutate Mutation Neoplasm Metastasis Noise Non-Malignant Normal Cell Normal tissue morphology Oncogenes Oncogenic Oncologist One-Step dentin bonding system Paraffin Embedding Performance Pharmaceutical Preparations Phase Preparation Prevalence Research Project Grants Sampling Sensitivity and Specificity Series Side Signal Pathway Signal Transduction Site Solid Neoplasm Somatic Mutation Specificity TP53 gene Technology Tissues Training Translational Research Validation Variant assay development base cancer cell cancer recurrence cancer therapy cost cost effective design improved malignant breast neoplasm meetings neoplastic cell response tumor

项目摘要

DESCRIPTION (provided by applicant): Cells within a tumor sample are known to be heterogeneous, due to the contamination from non-malignant tissue or the presence of multiple sub-clones, each carrying different somatic mutations. The majority of somatic mutations identified to date are clustered (mutational hotspots) in the functional sites of a few "cancer genes" with key roles in cell signaling pathways of proliferation and survival. Somatic mutations in cancer genes modify their oncogenic potential or affect sensitivity to therapy. Currently available assays that are able to detect rare somatic mutations are not comprehensive. They are usually focused on a few commonly mutated loci, and not implemented in clinical setting due to cost or technical reasons. Therefore, a current technological need exists for an assay that can reliably detect and accurately measure the prevalence of multiple somatic mutations present only in a fraction of the cells in a heterogeneous tumor. Such an assay would facilitate translational research to study the selection of tumor sub-clones during disease progression and treatment. Additionally the assay could be used by clinicians to improve tumor characterization and selection of therapy choices during clinical trials. We propose to leverage the emerging technology of targeted high-throughput sequencing to develop a cost- effective assay capable of detecting somatic mutations that are present in e1% of tumor cells. Specifically we will perform ultra-deep targeted sequencing (UDT-Seq) of ~100 kb in each tumor assaying 518 mutational hotspots located in 46 cancer genes. The selected mutational hotspots cover ~87% of all entries in the COSMIC database. We will develop a streamlined sample preparation in collaboration with RainDance Technologies to ensure a straightforward implementation in the clinic. This sample preparation integrates the targeting PCR and the library preparation in one step using chimeric PCR primers. The amplified targeted hotspots (200bp long) will be thus directly sequenced on the Illumina Genome Analyzer (GAII) at a very high coverage (~20,000x). We will then precisely model the sequencing error using calibration samples to filter true mutations from the sequencing noise. Our specific aims are: 1) To calibrate the UDT-Seq assay by analyzing both pooled DNA samples containing precise ratios of known SNPs and DNA samples spiked with low amounts of mutated DNA from cancer cells. For this, we will develop a statistical sequencing error model to detect rare mutations in deep sequence. 2) To evaluate the accuracy of the UDT- Seq assay to detect rare somatic mutations in both frozen and formalin fixed paraffin embedded solid tumors. If the quantitative milestones set for this pilot phase of the UDT-Seq assay development are met, we will apply for R33 funding to further develop and make this assay broadly available to clinical oncologists for their own translational research through a CLIA laboratory. PUBLIC HEALTH RELEVANCE: We propose to develop an assay to analyze somatic mutations in rare subclones of solid-tumors. This assay will feature microfluidic-based sample preparation method and high throughput DNA sequencing. It would be the most comprehensive assay to identify rare somatic mutational hotspots in a clinical setting and clinical oncologists will potentially use this assay to identify new biomarkers for disease progression or predictive of drug response, for a more personalized treatment of cancer.

描述（由申请人提供）：已知肿瘤样品内的细胞是异质的，这是由于非恶性组织的污染或存在多个亚克隆，每个亚克隆携带不同的体细胞突变。迄今为止发现的大多数体细胞突变都聚集在一些“癌症基因”的功能位点（突变热点），这些基因在细胞增殖和存活的信号通路中发挥关键作用。癌症基因的体细胞突变会改变其致癌潜力或影响对治疗的敏感性。目前能够检测罕见体细胞突变的可用测定方法并不全面。它们通常集中于一些常见突变位点，并且由于成本或技术原因而没有在临床环境中实施。因此，当前的技术需要能够可靠地检测和准确测量仅存在于异质性肿瘤的一小部分细胞中的多种体细胞突变的流行率。这种测定将促进转化研究，以研究疾病进展和治疗过程中肿瘤亚克隆的选择。此外，临床医生可以使用该测定来改善临床试验期间的肿瘤特征和治疗选择的选择。我们建议利用新兴的靶向高通量测序技术来开发一种经济高效的检测方法，能够检测 e1% 肿瘤细胞中存在的体细胞突变。具体来说，我们将对每个肿瘤进行约 100 kb 的超深靶向测序 (UDT-Seq)，分析位于 46 个癌症基因中的 518 个突变热点。选定的突变热点覆盖 COSMIC 数据库中所有条目的约 87%。我们将与 RainDance Technologies 合作开发简化的样品制备方法，以确保在临床中直接实施。该样品制备使用嵌合 PCR 引物将靶向 PCR 和文库制备整合在一个步骤中。因此，扩增的目标热点（200bp 长）将在 Illumina 基因组分析仪 (GAII) 上以非常高的覆盖率 (~20,000x) 直接测序。然后，我们将使用校准样本对测序错误进行精确建模，以从测序噪声中过滤出真正的突变。我们的具体目标是：1) 通过分析含有精确比例的已知 SNP 的混合 DNA 样本和掺有少量癌细胞突变 DNA 的 DNA 样本来校准 UDT-Seq 测定。为此，我们将开发一个统计测序错误模型来检测深层序列中的罕见突变。 2) 评估 UDT-Seq 检测在冷冻和福尔马林固定石蜡包埋实体瘤中检测罕见体细胞突变的准确性。如果达到 UDT-Seq 检测开发试验阶段设定的定量里程碑，我们将申请 R33 资金来进一步开发并通过 CLIA 实验室将该检测广泛提供给临床肿瘤学家进行自己的转化研究。公共健康相关性：我们建议开发一种分析方法来分析罕见实体瘤亚克隆的体细胞突变。该检测将采用基于微流体的样品制备方法和高通量 DNA 测序。这将是在临床环境中识别罕见体细胞突变热点的最全面的检测方法，临床肿瘤学家可能会使用该检测方法来识别疾病进展或预测药物反应的新生物标志物，从而实现更加个性化的癌症治疗。