Technology for sensitive and reliable mutational profiling in pancreatic cancer

描述（由申请人提供）：胰腺癌的突变谱分析对这种疾病的早期发现、预后和治疗管理具有重要的前景。然而，与许多其他癌症一样，虽然已经存在可靠的生殖系或普遍体细胞突变筛查方法，但在异质性、多灶性间质污染胰腺癌或体液中检测低患病率体细胞突变仍然存在问题。因此，对于相当一部分临床胰腺癌样本，新的强大的突变检测技术“失去了动力”，其优势无法发挥。我们开发了低变性温度下的共扩增聚合酶链反应（COLD-PCR），这是一种新的PCR形式，可以优先扩增野生型和含突变序列混合物中的“少数等位基因”，而不管突变位于何处，在PCR过程中突变序列的富集程度可达10-100倍。因为PCR是基因分析中普遍存在的第一步，COLD-PCR提供了一个通用的平台，以提高基本上所有诊断分析的敏感性。在这项应用中，我们建议进一步发展、优化和适应COLD-PCR，以提高两种已建立的突变检测方法的灵敏度，使它们能够用于可靠地鉴定异质多灶胰腺癌中与临床相关的体细胞突变：基质辅助激光脱吸电离飞行时间（MALDI-TOF）用于已知突变，单分子测序用于体细胞突变的高通量测序。COLD-PCR与这两种技术的结合，各自处理突变检测的不同方面，将提高患者特异性突变谱的敏感性，适合应用于胰腺癌。将编制一份胰腺癌突变基因的综合清单，并使用选定的技术将COLD-PCR应用于胰腺手术标本和血浆样本的体细胞突变平行筛选。在即将到来的分子医学时代，临床决策将越来越依赖于分子肿瘤谱分析，在不同的临床标本中识别体细胞突变的可靠性必须很高。这个应用程序解决了异质性癌症分子分析的问题。我们将新技术的重点放在胰腺癌上，这是一种异质性癌症，目前治愈率非常低，分子生物标志物可以发挥作用。

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.