用于结直肠癌液体活检的高特异性多重 DNA 甲基化检测。

IF 3.2 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2024-11-02 DOI:10.1016/j.cca.2024.120026

Dewen Zhu , Jinlei Li , Wenwen Zhang , Yishuai Wang , Huidong Wang , Ruoyan Fei , Qian Ye , Danli Peng , Ju Luan , Chang Xu , Xiaoli Wu , Dan Huang , Chunming Ding , Shengnan Jin

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引用次数: 0

摘要

背景：循环肿瘤DNA（ctDNA）已成为癌症检测和预后的有用生物标志物。在这项研究中，我们制定了一种策略，开发了一种高特异性的多重 qPCR 检测方法，用于检测结直肠癌（CRC）患者血液中甲基化的 ctDNA，并研究了该方法在检测和预后 CRC 方面的潜在用途：方法：利用亚硫酸氢盐转换和扩增子测序确认潜在的 CRC 特异性 DNA 甲基化标记物。选定的候选 DNA 甲基化标记通过 qMSP 验证。六个表现最好的标记物被用于开发一种新的单管多重甲基化特异性定量 PCR 检测（mqMSP）。mqMSP 分析法用于分析 114 名 CRC 患者、47 名晚期腺瘤患者、45 名良性息肉患者和 57 名健康对照者的血浆样本。评估了该检测方法的临床表现以及与临床结果的关联：结果：六种DNA甲基化生物标记物被证实在CRC肿瘤组织中存在特异性高甲基化。新开发的 mqMSP 检测方法检测出 CRC 的特异性极高（特异性为 98.2%，灵敏度为 67.5%）。ctDNA的检出率与肿瘤大小和临床分期显著相关，血液中的ctDNA甲基化水平随肿瘤大小增大、分化程度差和分期晚而明显升高。此外，术前高甲基化ctDNA水平与较差的无复发生存率和总生存率相关：我们为设计用于 CRC 液体活检的多重 DNA 甲基化检测方法提供了一种识别多种高度特异性 DNA 甲基化标记物的策略。新开发的检测方法有望用于 CRC 早期检测和预后判断。

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Highly specific multiplex DNA methylation detection for liquid biopsy of colorectal cancer

Background

Circulating tumor DNA (ctDNA) has emerged as a useful biomarker for cancer detection and prognosis. In this study, we developed a strategy for developing a highly specific multiplex qPCR assay to detect methylated ctDNA in the blood of colorectal cancer (CRC) patients and investigated the potential use for the detection and prognosis of CRC.

Methods

Bisulfite conversion and amplicon sequencing were used to confirm potential CRC-specific DNA methylation markers. The selected DNA methylation candidates were validated by qMSP. The six best-performing markers were used to develop a new single-tube multiplex quantitative methylation-specific PCR assay (mqMSP). The mqMSP assay was applied to analyze plasma samples from 114 CRC patients, 47 patients with advanced adenoma, 45 patients with benign polyps, and 57 healthy controls. The clinical performance of the assay and associations with clinical outcomes were assessed.

Results

Six DNA methylation biomarkers were confirmed to be specifically hypermethylated in CRC tumor tissues. The newly developed mqMSP assay detected CRC with extremely high specificity (specificity of 98.2 %, with sensitivity of 67.5 %). The detection rate of ctDNA was significantly correlated with tumor size and clinical stage, with ctDNA methylation levels in the blood markedly increased with larger tumor size, poor differentiation, and advanced stage. Moreover, high preoperative methylated ctDNA level was associated with worse recurrence-free survival and overall survival.

Conclusion

We provided a strategy for identification of multiple highly-specific DNA methylation markers for designing multiplex DNA methylation assays for liquid biopsies of CRC. The newly developed assay has potential for CRC early detection, and prognosis.

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来源期刊

Clinica Chimica Acta 医学-医学实验技术

CiteScore

10.10

自引率

2.00%

发文量

1268

审稿时长

23 days

期刊介绍： The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells. The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.