Technology for sensitive and reliable mutational profiling in pancreatic cancer

描述(由申请人提供)：胰腺癌的突变特征对这种疾病的早期发现、预后和治疗管理具有重要的希望。然而，与许多其他癌症一样，尽管已经存在可靠的胚系或普遍存在的体细胞突变的筛查方法，但在有间质污染的异质性、多灶性胰腺癌中或在体液中检测低患病率的体细胞突变仍然是有问题的。因此，对于相当一部分临床胰腺癌样本来说，新的强大的突变检测技术失去了动力，其优势无法发挥。我们开发了在较低变性温度下进行共扩增的聚合酶链式反应(COLD-PCR)，这是一种新的PCR形式，它优先从野生型和含有突变的序列的混合物中扩增“少数等位基因”，无论突变位于哪里，在聚合酶链式反应过程中提供10-100倍的突变序列。因为聚合酶链式反应是遗传分析中无处不在的第一步，所以冷聚合酶链式反应提供了一个通用的平台来提高基本上所有诊断分析的灵敏度。在这一应用中，我们建议进一步开发、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，以便它们可以用于可靠地鉴定异质性、多灶性胰腺癌的临床相关的体细胞突变：用于已知突变的基质辅助激光解吸电离飞行时间(MALDI-TOF)，以及用于高通量体细胞突变测序的单分子测序。冷-聚合酶链式反应与这两种技术相结合，各自处理突变检测的不同方面，将提高患者特异性突变图谱的敏感性，适合应用于胰腺癌。将编制一份完整的胰腺癌突变基因清单，并将采用所选技术对胰腺手术标本和血浆样本中的体细胞突变进行并行筛选。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图谱，在不同的临床标本中识别体细胞突变的可靠性必须很高。这一应用解决了异质性癌症的分子分析问题。我们将这项新技术集中在胰腺癌上，这是一种目前治愈率非常低的异质性癌症，分子生物标记物可以对其产生影响。

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.