Technology for sensitive and reliable mutational profiling in pancreatic cancer

描述（由申请人提供）：胰腺癌的突变谱对于这种疾病的早期检测、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，虽然已经存在用于生殖系或流行体细胞突变的可靠筛查方法，但在具有基质污染的异质性多灶性胰腺癌或体液中检测低流行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”，它们的优势无法利用。我们开发了在较低变性温度下的共扩增聚合酶链反应（COLD-PCR），这是一种新形式的PCR，其优先从野生型和含突变序列的混合物中扩增“少数等位基因”，而不管突变位于何处，在PCR期间提供突变序列的10-100倍富集。因为PCR包括遗传分析中普遍存在的第一步，所以COLD-PCR提供了一个通用平台来提高基本上所有诊断测定的灵敏度。在本申请中，我们提出进一步开发、优化和调整COLD-PCR，以提高两种已建立的突变检测方法的灵敏度，使得它们可用于可靠地鉴定异质性多灶性胰腺癌中的临床相关体细胞突变：基质辅助激光解吸电离-飞行时间用于已知突变的MALDI-TOF（基质辅助激光解吸电离-时间飞行时间）和用于体细胞突变的高通量测序的单分子测序。COLD-PCR与这两种技术的组合（每种技术解决突变检测的不同方面）将提高患者特异性突变谱分析的灵敏度，并且适合于应用于胰腺癌。将编制胰腺癌中突变基因的全面清单，并采用所选技术对COLD-PCR进行调整，以平行筛查胰腺手术标本和血浆样本中的体细胞突变。在即将到来的分子医学时代，临床决策将越来越多地依赖于分子肿瘤分析，并且在不同临床标本中识别体细胞突变的可靠性必须很高。该应用解决了异质性癌症中的分子分析问题。我们将新技术集中在胰腺癌上，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.