Temperature-Tolerant COLD-PCR enables mutation-enriched targeted re-sequencing

描述(申请人提供)：癌症是由于DNA损伤剂导致细胞转化为肿瘤细胞而导致的关键体细胞突变的终生积累。这些与肿瘤相关的低水平体细胞DNA突变可能对转移的发展、预后、治疗选择、随访或早期癌症检测具有深远的影响。除非它们被有效地检测到，否则这些低水平的突变可能会误导患者的管理决策，或者成为个性化药物的错失机会。广泛使用的技术，如测序，当这些突变的百分比与正常DNA相比非常低时，不足以检测到它们。同样，下一代测序技术(NGS)也是很有前途的技术进步，可以有效地检测靶向基因面板中普遍存在的体细胞突变；然而，由于大多数患者样本中的DNA数量有限，以及在分析血液时正常DNA的丰富，NGS失去了动力，它与临床实践的结合存在问题。对于丰度在~2-5%或更低的突变，NGS会产生与测序深度无关的假阳性(噪声)；然而，这些通常是导致药物治疗耐药的临床相关突变。用于癌症基因面板定向重新测序的商业样品制备试剂盒已经出现1-3个，然而它们一致无法检测到低于2%丰度水平的突变。因此，尽管靶向重测序为NGS与临床肿瘤学的整合提供了机会，但该技术在检测异质性癌症或循环DNA中的DNA突变方面无效。我们打算使用冷-聚合酶链式反应，这是一种新的方法，在聚合酶链式反应扩增过程中，使含有未知突变的序列比野生型、正常等位基因丰富。我们已经能够显示出低至0.02%丰度的突变序列。然而，在目前的形式下，这种方法只能在每个反应中使用一个扩增子，限制了它与NGS的有效结合。在这个项目中，我们提出了一种简单而强大的修改，使冷-聚合酶链式反应能够在一次反应中应用于数百或数千个DNA靶标，从而能够在NGS之前在癌症特异性基因面板中进行突变浓缩。这将把罕见的突变转化为高丰度的突变，克服“噪音”，并避免在NGS期间进行重复序列读取的昂贵需求。这种方法被称为耐温-冷-PCR(TT-COLD-PCR)，将被开发成癌症特异性基因小组的试剂盒，以放大多个DNA靶标中的罕见突变，从而使靶向重新测序在异质性癌症或循环DNA中的应用得以扩大。该项目符合国家癌症研究所的既定目标之一，即支持开发新的诊断方法，以检测、发现和验证用于癌症检测、诊断和预后的生物标志物。

DESCRIPTION (provided by applicant): Cancers develop from the life-long accumulation of critical somatic mutations due to DNA-damaging agents that lead to cells transforming into tumor-forming cells. These low-level tumor-associated somatic DNA mutations can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up or early cancer detection. Unless they are effectively detected, these low-level mutations can misinform patient management decisions or become missed opportunities for personalized medicine. Widely-used technologies such as sequencing are not sensitive enough to detect these mutations when they are at very low percentages compared to normal DNA. Likewise the next generation sequencing technologies (NGS) are promising technology advances that can effectively detect prevalent somatic mutations in targeted gene panels; however due to the limited quantity of DNA in most patient samples and the abundance of normal DNA when analyzing blood, NGS 'loses steam' and its integration with clinical practice is problematic. For mutations at an abundance of ~2-5% or below, NGS generates false positives ('noise') independent of sequencing depth; yet these are often the clinically relevant mutations causing resistance to drug treatments. Commercial sample preparation kits for targeted re-sequencing of cancer gene panels have emerged1-3, however they are uniformly unable to detect mutations below a 2% abundance level. Thus, while targeted re-sequencing provides an opportunity for integration of NGS with clinical oncology, the technology is ineffective in detecting DNA mutations in heterogeneous cancers or in circulating DNA. We intend to use COLD-PCR, a new method that enriches unknown mutation-containing sequences over wild-type, normal alleles during PCR amplification. We have been able to show sequencing of mutations down to 0.02% abundance. However in its current form this method only can be used with single amplicon per reaction, limiting its efficient combination with NGS. In this project we propose a simple and powerful modification that enables COLD-PCR to be applied on hundreds or thousands of DNA targets in a single reaction, thus enabling mutation enrichment in cancer- specific gene panels prior to NGS. This would convert the rare mutations to high abundance mutations, overcoming the 'noise' and avoid the costly need for repeated sequence reads during NGS. This method, known as temperature-tolerant-COLD-PCR (TT-COLD-PCR), will be developed into kits for cancer-specific gene panels, to magnify rare mutations in multiple DNA targets thus enabling expanded application of targeted re-sequencing for heterogeneous cancers or circulating DNA. This project meets one of the stated aims of the NCI to support the development of new methods of diagnosis for the detection, discovery and validation of biomarkers for cancer detection, diagnosis and prognosis.