Mutation Enriched Targeted Re-Sequencing

 描述(由申请人提供)：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，但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，但该技术在检测循环DNA、尿液或异质性癌症中的DNA突变方面无效。我们打算使用冷-聚合酶链式反应，这是一种最近发展起来的方法，在聚合酶链式反应扩增过程中，这种方法可以使含有未知突变的序列超过野生型、正常等位基因。在之前的工作中，我们展示了基于冷-PCR-NGS的突变测序，其丰度低至0.02%。然而，冷-聚合酶链式反应每次只能用一个扩增子，限制了它与NGS的有效结合。这种STTR提出了一种简单而强大的修改，使冷-PCR能够在一次反应中应用于数百或数千个DNA靶标，从而使突变在NGS之前的疾病特异性基因面板中得以丰富。这种新的方法，耐温-冷-聚合酶链式反应(TT-冷-PCR)将罕见的突变转化为高丰度突变，克服了“噪音”，并避免了在NGS期间昂贵的重复序列读取的需要。在第一阶段，我们获得了TT-COLD-PCR的原理证明。在第二阶段，TT-COLD-PCR将被开发成癌症特异性基因面板的试剂盒，以放大多个DNA靶点的罕见突变，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NCI的目标之一，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记物。

 DESCRIPTION (provided by applicant): 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 emerged, 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 circulating DNA, urine, or heterogeneous cancers. We intend to use COLD-PCR, a recently developed method that enriches unknown mutation-containing sequences over wild-type, normal alleles during PCR amplification. In previous work we showed COLD-PCR- NGS-based sequencing for mutations down to 0.02% abundance. However, COLD-PCR was only applicable with a single amplicon per reaction, limiting its efficient combination with NGS. This STTR proposes a simple and powerful modification that enables COLD-PCR to be applied to hundreds or thousands of DNA targets in a single reaction, thus enabling mutation enrichment in disease- specific gene panels prior to NGS. The new approach, temperature-tolerant-COLD-PCR (TT-COLD-PCR) converts the rare mutations to high abundance mutations, overcoming the `noise' and avoiding the costly need for repeated sequence reads during NGS. In Phase I we obtained proof of principle for TT-COLD-PCR. In Phase II, 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 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.