Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, People's Republic of China; Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, People's Republic of China.
Department of Medical Oncology, The First Affiliated Hospital of Soochow University, Suzhou, People's Republic of China.
J Mol Diagn. 2020 Jul;22(7):860-870. doi: 10.1016/j.jmoldx.2020.04.210. Epub 2020 May 16.
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%.
目前，微卫星不稳定性 (MSI) 的检测仅限于具有足够肿瘤含量的组织样本。已经探索了从血液中检测 MSI，但由于循环肿瘤 DNA (ctDNA) 含量有限，检测灵敏度较低，因此受到干扰。我们开发了一种基于下一代测序的算法，即血液 MSI 特征富集分析，用于从血液中检测 MSI。血液 MSI 特征富集分析的开发涉及三个主要步骤。首先，提取可以有效区分高微卫星不稳定性（MSI-H）与微卫星稳定肿瘤的标记物。其次，基于超几何概率进行 MSI 特征富集分析，在血浆样本与训练数据集白细胞具有相似的 MSI-H 和微卫星稳定读覆盖模式的零假设下进行。最后，对标记物的富集分数进行归一化，并综合所有标记物来确定血浆样本的 MSI 状态。基于细胞系的体外稀释实验和基于 MSI-H 血浆和配对白细胞 DNA 混合物的计算机模拟实验表明，在最低 1%的 ctDNA 下，灵敏度为 98%，特异性为 100%，在 0.4%的 ctDNA 下，灵敏度为 91.8%，特异性为 100%。87 例结直肠癌患者的独立验证队列，通过对组织 MSI 状态的正交确认，验证了该方法的性能，在 ctDNA>0.4%的样本中，灵敏度为 94.1%（16/17），特异性为 100%（27/27）。
