Background/Objectives: Circulating tumor DNA (ctDNA) analysis is a powerful tool for non-invasive monitoring of tumor burden and treatment response. Reliable quantification methods are critical for the effective use of ctDNA as a tumor biomarker. Digital PCR (dPCR) offers high sensitivity and quantification, but requires the prior knowledge of tumor-specific genomic alterations. Next-generation sequencing (NGS) provides a more comprehensive approach but is semi-quantitative, relying on variant allelic fraction (VAF), which can be influenced by non-tumor cell-free DNA.Methods: We developed a novel quantitative NGS (qNGS) method for absolute quantification of nucleotide variants, utilizing unique molecular identifiers (UMIs) and of quantification standards (QSs), short synthetic DNA sequences modified to include characteristic mutations for unique identification in sequencing data. We evaluated the performance of this method using plasma samples spiked with mutated DNA and plasma pools from cancer patients. We further applied our technique to plasma samples from four non-small cell lung cancer (NSCLC) patients enrolled in the ELUCID trial.Results: Our qNGS approach demonstrated robust linearity and correlation with dPCR in both spiked and patient-derived plasma samples. Notably, the analysis of clinical samples from the ELUCID trial revealed the ability of our method to simultaneously quantify multiple variants in a single plasma sample. Significant differences in ctDNA levels were observed between baseline and post-treatment samples collected after three weeks of front-line therapy.Conclusions: We introduce a novel qNGS method that enables the absolute quantification of ctDNA, independent of non-tumor circulating DNA variations. This technique was applied for the first time to serial samples from NSCLC patients, demonstrating its ability to simultaneously monitor multiple variants, making it a robust and versatile tool for precision oncology.
背景/目的：循环肿瘤DNA（ctDNA）分析是无创监测肿瘤负荷和治疗反应的重要工具。可靠的定量方法对于ctDNA作为肿瘤生物标志物的有效应用至关重要。数字PCR（dPCR）具有高灵敏度和定量能力，但需要预先了解肿瘤特异性基因组变异。新一代测序（NGS）提供了更全面的检测手段，但其依赖变异等位基因频率（VAF）进行半定量分析，而VAF可能受到非肿瘤来源游离DNA的影响。 方法：我们开发了一种新型定量NGS（qNGS）方法，通过结合独特分子标识符（UMIs）和定量标准品（QSs）——即经修饰后包含特征性突变以便在测序数据中唯一识别的短合成DNA序列——实现核苷酸变异的绝对定量。我们使用掺入突变DNA的血浆样本和癌症患者血浆池评估了该方法的性能，并进一步将其应用于ELUCID临床试验中四名非小细胞肺癌（NSCLC）患者的血浆样本。 结果：我们的qNGS方法在掺入样本和患者来源血浆样本中均表现出良好的线性，并与dPCR检测结果高度相关。值得注意的是，对ELUCID试验临床样本的分析显示，该方法能在单次血浆检测中同时定量多个变异。在基线样本与一线治疗三周后采集的治疗后样本之间，观察到ctDNA水平的显著差异。 结论：我们提出了一种新型qNGS方法，能够实现ctDNA的绝对定量，且不受非肿瘤循环DNA变异的影响。该技术首次应用于NSCLC患者的系列样本，证明了其同时监测多个变异的能力，使其成为精准肿瘤学领域中强大而多功能的工具。
肿瘤(癌症)患者之家