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

描述（由申请人提供）：由于DNA损伤剂导致细胞转化为肿瘤形成细胞，癌症是由关键体细胞突变的终生积累发展而来的。这些低水平的肿瘤相关体细胞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意义。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。 广泛使用的技术，如测序是不够敏感，以检测这些突变时，他们在非常低的百分比相比，正常的DNA。同样，下一代测序技术（NGS）是有前途的技术进步，其可以有效地检测靶向基因组中的普遍体细胞突变;然而，由于大多数患者样品中DNA的数量有限以及分析血液时正常DNA的丰度，NGS“失去动力”并且其与临床实践的整合是有问题的。 对于丰度为~2-5%或更低的突变，NGS产生与测序深度无关的假阳性（“噪音”）;然而这些通常是引起对药物治疗的抗性的临床相关突变。已经出现了用于癌症基因组靶向再测序的商业样品制备试剂盒1 -3，然而它们均不能检测低于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，该方法使含有未知突变的序列比野生型、正常等位基因丰富。我们已经能够显示低至0.02%丰度的突变测序。然而，在其当前形式中，该方法仅可与每个反应的单个扩增子一起使用，限制了其与NGS的有效组合。 在该项目中，我们提出了一种简单而强大的修改，使COLD-PCR能够在单个反应中应用于数百或数千个DNA靶标，从而使NGS之前的癌症特异性基因组中的突变富集成为可能。这将把罕见突变转化为高丰度突变，克服“噪音”，避免在NGS期间重复序列读取的昂贵需求。这种被称为耐温冷PCR（TT-COLD-PCR）的方法将被开发成癌症特异性基因组的试剂盒，以放大多个DNA靶点中的罕见突变，从而扩大针对异质性癌症或循环DNA的靶向重测序的应用。 该项目符合NCI的既定目标之一，即支持开发新的诊断方法，用于检测，发现和验证癌症检测，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.