Global Clinical Biomarkers & Companion Diagnostics, Translational Medicine, Global Development, EMD Serono Research and Development Institute, Inc. a division of Merck KGaA, Billerica, MA, United States of America.
Biosample Informatics and Biobanking, Clinical Trial Management, Global Clinical Operations, Global Development, Merck KGaA, Darmstadt, Germany.
PLoS One. 2021 May 10;16(5):e0250849. doi: 10.1371/journal.pone.0250849. eCollection 2021.
Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768-7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.
开发一种临床适用的基于液体活检的 PD-L1 mRNA 表达检测方法，将有助于为当前的免疫组织化学检测提供补充证据。因此，我们报告了一种使用数字液滴聚合酶链反应 (ddPCR) 检测血液 PD-L1 mRNA 表达的适合目的的检测方法的开发。根据 PD-L1 基因的覆盖范围选择 TaqMan® 检测，并用实时定量 PCR 测试其线性和效率。在阳性对照细胞系（用干扰素 γ [IFN γ] 处理的 A549）基因组 DNA 中分析了四个参考基因。还在 ddPCR 中评估了 PD-L1 引物/探针组的空白限制、检测限和精密度。最后，评估了 35 名健康志愿者样本以建立 PD-L1 表达的基线水平。在 ddPCR 中，空白限制确定为 0 拷贝，检测限确定为 PD-L1 小于或等于 19 拷贝。ddPCR 检测的平均内运行变异系数为 7.44%，平均外运行 CV 为 7.70%。IFN γ 处理 A549 细胞导致 PD-L1 表达增加 6.7 倍（未处理 cDNA 中的 21580 拷贝与处理 cDNA 中的 145000 拷贝）。对健康人样本的分析得出 PD-L1 拷贝/μL 的中位数为 1659 拷贝，范围为 768-7510 拷贝/μL。该检测方法已转移到外部服务提供商，并且我们内部实验和外部实验的结果显示相关性为 0.994。总之，使用 PAXgene RNA 血液样本，开发并验证了一种适合液体活检的、基于 ddPCR 的 PD-L1 mRNA 表达检测方法，该方法具有良好的线性度、可重复性、空白限制和检测限，适合临床应用。这种超灵敏的液体活检 ddPCR 检测方法在筛选、纵向监测和疾病进展检测方面具有潜在的临床应用前景。
