Currently, microsatellite instability (MSI) detection is limited to tissue samples with sufficient tumor content. Detection of MSI from blood has been explored but confounded by low sensitivity due to limited circulating tumor DNA (ctDNA). We developed a next-generation sequencing–based algorithm, blood MSI signature enrichment analysis, to detect MSI from blood. Blood MSI signature enrichment analysis development involved three major steps. First, marker sites that can effectively distinguish high MSI (MSI-H) from microsatellite stable tumors were extracted. Second, MSI signature enrichment analysis was performed based on hypergeometric probability, under the null hypothesis that plasma samples have similar MSI-H and microsatellite stable read coverage patterns for particular marker sites as the white blood cells from the training data set. Finally, enrichment scores of marker sites were normalized, and all markers were collectively considered to determine the MSI status of a plasma sample. In vitro dilution experiments with cell lines and in silico simulation experiments based on mixtures of MSI-H plasma and paired white blood cell DNA demonstrated 98% sensitivity and 100% specificity at a minimum of 1% ctDNA and 91.8% sensitivity and 100% specificity with 0.4% ctDNA. An independent validation cohort of 87 colorectal cancer patients with orthogonal confirmation of MSI status of tissues confirmed performance, achieving 94.1% sensitivity (16/17) and 100% specificity (27/27) for samples with ctDNA >0.4%.

当前，微卫星不稳定性（MSI）检测仅限于具有足够肿瘤含量的组织样品。已经探索了从血液中检测MSI的方法，但是由于循环肿瘤DNA（ctDNA）的限制，其灵敏度低而令人困惑。我们开发了基于下一代测序的算法，即血液MSI签名富集分析，以检测血液中的MSI。血液MSI签名富集分析开发涉及三个主要步骤。首先，提取可以有效区分高MSI（MSI-H）和微卫星稳定肿瘤的标记位点。其次，在血浆样品对特定标记位点具有类似MSI-H和微卫星稳定读取覆盖模式的虚假假设下，基于超几何概率进行MSI签名富集分析，其与来自训练数据集的白细胞类似。细胞系的体外稀释实验和基于MSI-H血浆与配对的白细胞DNA混合物的计算机模拟实验表明，在1％ctDNA和至少91.8％灵敏度和100％特异性的情况下，灵敏度为98％和100％。 0.4％的ctDNA。通过正交确认组织的MSI状态的87名大肠癌患者的独立验证队列证实了其性能，对于ctDNA> 0.4％的样品，灵敏度达到94.1％（16/17），特异性达到100％（27/27）。