Development of a novel liquid biopsy test to assist targeted MSI-H cancer treatment

摘要 该SBIR项目的目标是开发一种创新的SA-PCPE-PCR技术， 在野生型DNA的巨大背景中对多个突变的单核苷酸短链重复序列进行基因分型， 用于开发用于高频微卫星不稳定性（MSI-H）癌症的液体活检的测定。 MSI-H的标志是短串联重复序列（STR）的广泛不稳定性，这会改变重复序列的数量。 目前建议对五个monopoltide STR进行检查，以进行MSI分类。 靶向癌症治疗正在成为癌症护理的有效方法，因为它允许医生 根据对肿瘤的遗传学理解，在正确的时间为正确的患者选择正确的治疗方法。 由于MSI-H与微卫星稳定（MSS）肿瘤具有不同的特征，因此其治疗不同于 对方.最近，FDA批准了针对MSI-H治疗的前两种药物。比如说， FDA加速批准Keytruda用于治疗MSI-H患者。在此之前，FDA 根据癌症在体内的起始部位（例如，肺癌或乳腺癌）批准的癌症治疗 癌的Keytruda是第一种基于肿瘤生物标志物（MSI-H）而不考虑肿瘤生物标志物的药物。 肿瘤的原始位置 液体活检是一种使医生能够通过血液来发现有关肿瘤的一系列信息的方法。 样品，并检测无细胞DNA（cfDNA）中的突变正在成为选择液体样品的方法。 活检靶向治疗是其最重要的应用领域之一。 因为它的价值观是有针对性的 在癌症治疗中，作为靶向治疗的伴随测试的各种液体活检测定已经或 正在开发中。例如，FDA已经批准了用于靶向EGFR治疗的液体活检测试。 显然，液体活检对靶向MSI-H治疗同样有价值。然而，没有液体活检测试是 由于缺乏可以检测MSI-H癌症的技术， 因此，本发明提供了一种以敏感、特异和成本有效的方式检测MSI-H的生物标志物的方法。 在此，我们提出了一种称为SA-PCPE-PCR的新方法来解决这一未满足的需求。该方法 解决了cfDNA中突变STR检测的两个基本问题。首先，它丰富了 在PCR之前同时检测多个突变STR。这解决了PCR滑动的问题，使其 在野生型DNA的大背景下使用片段分析对突变的STR进行基因分型是可能的。 其次，它通过引入大小标签来操纵扩增子的片段大小，从而产生足够的差异 扩增子的长度。这解决了cfDNA所施加的大小限制问题，使其成为可能。 基于每个扩增子的独特大小对多个STR进行基因分型。SA-PCPE-PCR检测试剂盒包括 三步首先，将DNA样品进行SA-PCPE，其优先产生可降解的（长） 来自突变等位基因的延伸产物（在PCR之前富集突变体），同时引入大小标签以 这些延伸产品。第二，用延伸产物作为模板进行多重PCR，但 只有长延伸产物被扩增。最后，大小调整的扩增子通过PCR进行片段分析。 常规DNA测序仪来鉴定突变的STR。在本研究中，我们将首先建立一个SA-PCPE-PCR 在大量野生型DNA背景中同时检测五个突变的单核苷酸STR的测定。 然后，我们将评估检测方法的检测限和重现性。最后，我们将评估临床 检测灵敏度和特异性。显然，这个项目的成功可以提供一个液体活检 这可能会改变个性化MSI-H癌症医学的前景。

Abstract The goal of this SBIR project is to develop an innovative SA-PCPE-PCR technology that can simultaneously genotype multiple mutated mononucleotide STRs in a vast background of wild-type DNA, which will be employed to develop an assay for liquid-biopsy of high-frequency microsatellite instability (MSI-H) cancer. The hallmark of MSI-H is extensive instability in short tandem repeat (STR), which alters the repeat number. Examination of five mononucleotide STRs is currently recommended for the MSI classification. Targeted cancer treatment is emerging as an effective approach to cancer care because it allows doctors to select the right treatment for the right patient at the right time based on a genetic understanding of tumor. Because MSI-H has different profiles than microsatellite stable (MSS) tumors, their treatment differs from each other. Recently, FDA approved the first two drugs that are tailored to MSI-H treatment. For example, FDA granted an accelerated approval of Keytruda to a treatment for patients with MSI-H. Until then, FDA approved cancer treatments based on where in the body the cancer started—for example, lung or breast cancers. Keytruda is the first approved drug based on a tumor's biomarker (MSI-H) without regard to the tumor's original location. Liquid biopsy is a method enabling doctors to discover a range of information about a tumor through a blood sample, and detecting mutations in cell-free DNA (cfDNA) is emerging as the method of choice of liquid biopsy. Targeted treatment is one of its most important application areas. Because of its values to targeted cancer treatment, various liquid biopsy assays as the accompany test for targeted treatment have been or are being developed. For example, FDA has approved liquid biopsy tests for targeted EGFR treatment. Clearly, liquid biopsy will be equally valuable to targeted MSI-H treatment. However, no liquid biopsy test is available for targeted MSI-H cancer treatment because of the lack of technologies that can detect the biomarker of MSI-H in a sensitive, specific, and cost-effective manner. Herein, we propose a novel method termed SA-PCPE-PCR to address this unmet need. This method solves two of the fundamental problems plaguing the detection of mutated STRs in cfDNA. First, it enriches multiple mutated STRs simultaneously prior to PCR. This solves the problem of PCR slippages, rendering it possible to genotype mutated STRs in a vast background of wild-type DNA using fragment analysis. Second, it manipulates fragment size of amplicons by introducing size-tags, creating a sufficient difference in length of the amplicons. This solves the size constraint problems imposed by cfDNA, making it possible to genotype multiple STRs based on the unique size of each amplicon. A SA-PCPE-PCR assay consists of three steps. First, a DNA sample is subjected to SA-PCPE, which preferentially produces amplifiable (long) extension products from mutant alleles (enriching mutants prior to PCR), while introducing size-tags to these extension products. Second, multiplexed PCR is performed with extension products as templates, but only long extension products are amplified. Finally, size-adjusted amplicons are fragment analyzed by a conventional DNA sequencer to identify mutated STRs. In this study, we will first develop a SA-PCPE-PCR assay to detect five mutated mononucleotide STRs simultaneously in a large background of wild-type DNA. Then, we will assess the detection limit and reproducibility of the assay. Finally, we will assess the clinical detection sensitivity and specificity of the assay. Clearly, success of this project can provide a liquid biopsy assay that may transform the landscape of personalized MSI-H cancer medicine.