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

描述（由申请人提供）：癌症是由于dna损伤剂导致细胞转化为肿瘤形成细胞的关键体细胞突变的终生积累而发展起来的。这些低水平肿瘤相关的体细胞DNA突变对转移的发展、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，或成为错失个性化医疗的机会。与正常DNA相比，当这些突变的百分比非常低时，测序等广泛使用的技术不足以检测到这些突变。同样，下一代测序技术（NGS）是有前途的技术进步，可以有效地检测目标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DNA的数量有限，而在分析血液时正常DNA的数量丰富，NGS“失去了动力”，其与临床实践的结合存在问题。对于丰度在~2-5%或以下的突变，NGS产生假阳性（“噪声”），与测序深度无关；然而，这些通常是引起对药物治疗产生耐药性的临床相关突变。用于癌症基因面板靶向重测序的商业样品制备试剂盒已经出现1-3，但是它们一致无法检测丰度低于2%的突变。因此，虽然靶向重测序为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突变方面是无效的。我们打算使用COLD-PCR，这是一种新的方法，在PCR扩增过程中，在野生型、正常等位基因上富集未知的含突变序列。我们已经能够显示突变测序的丰度低至0.02%。然而，在目前的形式下，这种方法只能用于每个反应的单个扩增子，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饰，使COLD-PCR能够在一次反应中应用于数百或数千个DNA靶标，从而在NGS之前实现癌症特异性基因面板的突变富集。这将把罕见的突变转化为高丰度的突变，克服了“噪音”，避免了在NGS过程中重复序列读取的昂贵需求。这种被称为耐温冷- pcr （TT-COLD-PCR）的方法将被开发成用于癌症特异性基因面板的试剂盒，以放大多个DNA靶标中的罕见突变，从而扩大靶向重测序在异质性癌症或循环DNA中的应用。该项目符合NCI的既定目标之一，即支持开发用于癌症检测、诊断和预后的生物标志物的检测、发现和验证的新诊断方法。

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.