Mutation Enriched Targeted Re-Sequencing

突变富集靶向重测序

• 批准号: 9195704
• 负责人: G. Mike Makrigiorgos
• 金额: $ 71.65万
• 依托单位: TRANSGENOMIC, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195704
• 关键词: Alleles; Blood; Cancer Detection; Cancer Etiology; Clinical; Clinical Oncology; Collection; Colon; DNA; DNA Sequence Alteration; Detection; Development; Diagnosis; Disease; Drug resistance; Engineering; Future; Gene Targeting; Genes; Genomic DNA; Genotype; Heterogeneity; Liquid substance; Malignant Neoplasms; Methods; Modification; Molecular Profiling; Mutation; Neoplasm Metastasis; Noise; Nucleotides; Oncogenes; Operative Surgical Procedures; Patients; Pharmacotherapy; Phase; Plasma; Positioning Attribute; Preparation; Process; Public Health; Reaction; Reproducibility; Role; Sampling; Screening for cancer; Sensitivity and Specificity; Small Business Technology Transfer Research; Somatic Mutation; Steam; Technology; Temperature; Testing; Time; Tube; Urine; Validation; Work; base; cancer biomarkers; cancer diagnosis; cancer therapy; circulating DNA; clinical practice; clinically relevant; clinically significant; cost; cost effective; design; follow-up; individual patient; liquid biopsy; melting; minimally invasive; mutation screening; new technology; next generation sequencing; novel strategies; outcome forecast; personalized medicine; prevent; public health relevance; response; screening; treatment choice; tumor

 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.

 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产生深远的影响。除非被有效检测到，否则这些低水平突变可能会误导患者管理决策或错失个性化医疗的机会。 当这些突变与正常 DNA 相比比例非常低时，测序等广泛使用的技术不够灵敏，无法检测到这些突变。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组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，而在分析血液时正常 DNA 却很丰富，NGS“失去了动力”，其与临床实践的结合也存在问题。对于丰度约为 2-5% 或更低的突变，NGS 会产生与测序深度无关的假阳性（“噪音”）；然而，这些通常是临床相关的突变，导致对药物治疗产生耐药性。用于癌症基因组靶向重测序的商业样品制备试剂盒已经出现，但它们普遍无法检测丰度水平低于 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，但该技术在检测循环DNA、尿液或异质癌症中的DNA突变方面无效。 我们打算使用 COLD-PCR，这是一种最近开发的方法，可在 PCR 扩增过程中比野生型正常等位基因富集包含未知突变的序列。在之前的工作中，我们展示了基于 COLD-PCR-NGS 的突变测序，丰度低至 0.02%。然而，COLD-PCR 每次反应仅适用于单个扩增子，限制了其与 NGS 的有效组合。 该 STTR 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，从而在 NGS 之前实现疾病特异性基因组的突变富集。新方法耐温 COLD-PCR (TT-COLD-PCR) 将罕见突变转化为高丰度突变，克服了“噪音”并避免了 NGS 期间重复序列读取的昂贵需求。 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组的试剂盒，以放大多个DNA靶标中的罕见突变，从而扩大针对异质癌症或循环DNA的靶向重测序的应用。 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、发现和验证癌症检测、诊断和预后的生物标志物。