Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

涉及同时使用多种循环肿瘤DNA（ctDNA）标记物的多重测定可以改善液体活检的性能，因此可以高度预测癌症的复发。我们开发了一种单管甲基化特异性定量PCR分析（mqMSP），它使用10种不同的甲基化标记，能够定量分析血浆样品中的肿瘤DNA仅为0.05％。在来自大肠癌（CRC）患者，腺瘤患者和健康对照的179个血浆样本中，mqMSP测定的灵敏度和特异性分别为84.9％和83.3％。在一项面对面的比较研究中，mqMSP分析在检测早期（I和II期）和恶变前息肉方面也比已发表的SEPT9分析表现更好。在来自82名CRC患者的182个血浆样本（术前，术后和随访）的独立纵向队列中，mqMSP分析检测到73例术前血浆样本中的ctDNA（占89.0％）。ctDNA的术后检测（手术2周内）确定了20例复发患者中的11例，并与较差的无复发生存率相关（危险比为4.20； 3，4，9，12）。P = 0.0005）。通过随后的纵向监测，有14例（70％）患者在复发前具有可检测的ctDNA，中位前置时间比放射影像学提前了8.0个月。mqMSP检测具有成本效益，并且易于实施，可用于CRC复发的常规临床监测，从而可以在术后更好地管理患者。