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，该技术可以同时 在野生型DNA的广阔背景下对多个突变的单核苷酸短串联重复序列进行分型，这将是 用于建立高频微卫星不稳定性(MSI-H)癌的液体活组织检查方法。 MSI-H的特点是短串联重复序列(STR)的广泛不稳定，它改变了重复数。 目前建议对5个单核苷酸STR进行MSI分类。 靶向癌症治疗正在成为癌症护理的一种有效方法，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，它们的治疗方法不同于 彼此之间。最近，FDA批准了为MSI-H治疗量身定做的前两种药物。例如, FDA批准Keytruda加速批准用于治疗MSI-H患者。在此之前，FDA 根据癌症起始部位批准的癌症治疗--例如，肺癌或乳腺癌 癌症。Keytruda是第一个被批准的基于肿瘤生物标记物(MSI-H)的药物，而不考虑 肿瘤的原始位置。 液体活组织检查是一种使医生能够通过血液发现肿瘤的一系列信息的方法。 样品和检测无细胞dna(Cfdna)突变的方法正在成为液体的选择方法。 活组织检查。靶向治疗是其最重要的应用领域之一。 因为它的价值是有针对性的 癌症治疗中，各种液体活组织检查作为靶向治疗的伴随试验已经或 正在开发中。例如，FDA已经批准了靶向EGFR治疗的液体活组织检查。 显然，液体活检对于MSI-H的靶向治疗同样有价值。然而，没有液体活组织检查是 可用于靶向MSI-H癌症治疗，因为缺乏可以检测到 以一种敏感、特异和经济的方式作为MSI-H的生物标志物。 在此，我们提出了一种名为SA-PCPE-PCR的新方法来解决这一未得到满足的需求。这种方法 解决了困扰cfDNA中突变STR检测的两个基本问题。首先，它丰富了 在聚合酶链式反应前同时检测多个突变的短串联重复序列。这解决了聚合酶链式反应滑移的问题， 可以利用片段分析在野生型DNA的广泛背景下对突变的STR进行基因分型。 其次，它通过引入大小标签来操纵扩增片段的大小，从而产生足够的差异 在扩增的长度上。这解决了cfDNA强加的大小约束问题，使其成为可能 根据每个扩增子的独特大小对多个STR进行基因分型。SA-PCPE-PCR检测方法包括 三个步骤。首先，DNA样本经过SA-PCPE，它优先产生可扩增的(Long) 突变等位基因的延伸产物(在聚合酶链式反应之前丰富突变)，同时引入大小标签 这些延伸产品。第二，以延伸产物为模板进行多重PCR，但是 只有长延伸产物才会被放大。最后，对大小调整后的扩增器进行片段分析 常规DNA测序仪用于识别突变的STR。在本研究中，我们将首先建立SA-PCPE-PCR 同时检测野生型DNA大背景中5个突变的单核苷酸短串联重复序列。 然后，我们将评估该分析的检测限和重复性。最后，我们将评估临床 该检测方法的检测灵敏度和特异性。显然，这个项目的成功可以提供液体活检 这可能会改变个性化MSI-H癌症药物的面貌。

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.