Technology for sensitive and reliable mutational profiling in pancreatic cancer

胰腺癌敏感且可靠的突变分析技术

• 批准号: 7626951
• 负责人: G. Mike Makrigiorgos
• 金额: $ 22.7万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7626951
• 关键词: Address; Alleles; Betaine; Biological Assay; Biological Markers; Blood; Buffers; Cancer Etiology; Cell Line; Clinical; Data; Detection; Diagnostic; Dimethyl Sulfoxide; Disease; Disease Management; Early Diagnosis; Exons; Generations; Genes; Glycerol; Grant; Human; Individual; Liquid substance; Low Prevalence; Malignant Neoplasms; Malignant neoplasm of pancreas; Methods; Methylation; Minority; Molecular; Molecular Analysis; Molecular Medicine; Mutate; Mutation; Mutation Detection; Operative Surgical Procedures; Organic solvent product; Pancreas; Patients; Pharmaceutical Preparations; Plasma; Polymerase; Polymerase Chain Reaction; Population; Positioning Attribute; Public Health; Published Comment; Reaction; Resolution; Role; Sampling; Screening procedure; Somatic Mutation; Specimen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Steam; Stromal Cells; System; Technology; Temperature; Testing; Therapeutic; Time; United States National Institutes of Health; cancer therapy; clinically relevant; cost; design; flexibility; formamide; genetic analysis; improved; melting; mutant; new technology; outcome forecast; prognostic; response; single molecule; tumor

DESCRIPTION (provided by applicant): Mutational profiling of pancreatic cancer holds major promise for early detection, prognosis and therapeutic management of this disease. However, as with many other cancers, while reliable screening methods for germline or prevalent somatic mutations already exist, detection of low-prevalence somatic mutations in heterogeneous, multifocal pancreatic cancers with stromal contamination, or in bodily fluids remains problematic. Thus, for a substantial fraction of clinical pancreatic cancer samples, the new powerful mutation detection technologies "lose steam" and their advantages cannot be exploited. We developed co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), a new form of PCR that amplifies preferentially the "minority alleles" from mixtures of wild-type and mutation-containing sequences, irrespective of where the mutation lies, providing a 10-100-fold enrichment of the mutated sequences during PCR. Because PCR comprises the ubiquitous first step in genetic analysis, COLD-PCR provides a general platform to improve sensitivity for essentially all diagnostic assays. In this application we propose to develop further, optimize and adapt COLD-PCR for increasing the sensitivity of two established mutation detection methods, such that they can be applied for reliable identification of clinically-relevant, somatic mutations in heterogeneous, multifocal pancreatic cancers: matrix assisted laser desorption ionization-time of flight (MALDI-TOF) for known mutations, and single molecule sequencing for high-throughput sequencing of somatic mutations. The combination of COLD-PCR with these two technologies, each tackling a different aspect of mutation detection, will boost the sensitivity of patient- specific mutational profiling, and is suited for application to pancreatic cancer. A comprehensive list of genes mutated in pancreatic cancers will be compiled and COLD-PCR will be adapted for parallel screening of somatic mutations in pancreatic surgical specimens and plasma samples using the selected technologies. In the forthcoming era of molecular medicine, clinical decisions will increasingly rely on molecular tumor profiling, and the reliability of identifying somatic mutations in diverse clinical specimens must be high. This application tackles the problem of molecular analysis in heterogeneous cancers. We focus the new technology on pancreatic cancer, a heterogeneous cancer that currently has very low cure rates and for which molecular biomarkers can make a difference.

描述（由申请人提供）：胰腺癌的突变分析对该疾病的早期发现，预后和治疗管理具有主要的希望。但是，与许多其他癌症一样，虽然已经存在用于种系或普遍的体细胞突变的可靠筛查方法，但在异质，多灶性多灶性胰腺癌中检测低偏见的体细胞突变，具有基质的污染物，或者在身体流体中仍然存在问题。因此，对于很大一部分临床胰腺癌样品，新的强大突变检测技术“失去了蒸汽”，并且无法利用它们的优势。我们在较低的变性温度聚合酶链反应（Cold-PCR）上开发了共扩张，这是一种新形式的PCR，优先从野生型和含有突变的序列的混合物中放大“少数等位基因”，而不管突变所在的位置，在其中提供了10-100范围的突变序列。由于PCR构成了遗传分析中普遍存在的第一步，因此COLD-PCR提供了一个一般平台，以提高基本所有诊断测定的灵敏度。 In this application we propose to develop further, optimize and adapt COLD-PCR for increasing the sensitivity of two established mutation detection methods, such that they can be applied for reliable identification of clinically-relevant, somatic mutations in heterogeneous, multifocal pancreatic cancers: matrix assisted laser desorption ionization-time of flight (MALDI-TOF) for known mutations, and single molecule sequencing for体细胞突变的高通量测序。 Cold-PCR与这两种技术的结合，每种技术应对突变检测的不同方面，将提高患者特异性突变分析的敏感性，并适用于胰腺癌。将编译胰腺癌中突变的基因的全面列表，并将对Cold-PCR进行调整，以平行筛选胰腺手术标本中的体细胞突变，并使用选定的技术进行血浆样品。在即将到来的分子医学时代，临床决策将越来越依赖分子肿瘤分析，并且在各种临床标本中鉴定体细胞突变的可靠性必须很高。该应用解决了异质癌中分子分析的问题。我们将新技术集中在胰腺癌上，胰腺癌是一种目前的治愈率非常低，分子生物标志物可以有所作为。