Selected CpG islands.

ObjectiveLiquid biopsy for detection of circulating tumor DNA (ctDNA) offers a promising, non-invasive strategy for real-time monitoring of cancer. We aimed to develop a universal and sensitive one-tube droplet digital PCR (ddPCR) multiplex assay able to detect ctDNA in plasma from metastatic prostate cancer (PCa) patients.MethodsPublicly available array data are applied to identify methylation signatures specific to both normal and cancerous prostate tissue. The assay is based on the highly sensitive ddPCR technology, and tissue and plasma samples are analyzed using the QX600 ddPCR multiplexing platform from BioRad.ResultsWe identified three novel genomic regions, ACTRT2, EVX1, and HOXD13, containing CpG-methylation signatures specific to normal and cancerous prostate tissue. We successfully targeted the three biomarkers in a single-tube methylation-specific multiplex ddPCR (mm-ddPCR) assay along with two previously reported PCa-specific hypermethylated CpG biomarkers, DOCK2 and HAPLN3, and the ALB reference gene. The mm-ddPCR assay consistently detected ACTRT2, EVX1, and HOXD13 across all samples originating from prostate-derived tissue. In contrast, the HAPLN3 and DOCK2 biomarkers were predominantly detected in PCa tissue. We also demonstrate that the five ctDNA biomarkers can be detected in plasma samples from metastatic castration-resistant PCa (mCRPC) patients with a 95% CI (85%, 100%) using the mm-ddPCR assay.ConclusionThis work demonstrates that the mm-ddPCR assay offers a simple, fast, cost-effective, sensitive, and universal tool for the detection of ctDNA biomarkers in plasma from mCRPC patients. Further studies are required to explore its potential for PCa risk stratification and treatment guidance.研究背景：用于检测循环肿瘤DNA（circulating tumor DNA, ctDNA）的液体活检技术，为癌症的实时监测提供了一种极具潜力的非侵入性策略。本研究旨在开发一种通用且灵敏的单管液滴数字PCR（droplet digital PCR, ddPCR）多重检测方法，用于检测转移性前列腺癌（metastatic prostate cancer, PCa）患者血浆中的ctDNA。 研究方法：本研究利用公开获取的微阵列数据，鉴定正常及癌变前列腺组织的特异性甲基化特征。本检测方法基于高灵敏度的ddPCR技术，组织与血浆样本均采用伯乐（BioRad）公司的QX600 ddPCR多重分析平台进行检测。 研究结果：本研究鉴定出三个全新的基因组区域——ACTRT2、EVX1及HOXD13，其包含正常与癌变前列腺组织特异性的CpG甲基化特征。本研究成功在单管甲基化特异性多重ddPCR（methylation-specific multiplex droplet digital PCR, mm-ddPCR）检测体系中，靶向了这三个生物标志物，同时纳入了两个此前已报道的前列腺癌特异性高甲基化CpG生物标志物DOCK2、HAPLN3，以及ALB参考基因。该mm-ddPCR检测方法可在所有前列腺来源组织样本中稳定检测到ACTRT2、EVX1及HOXD13。与之相对，HAPLN3与DOCK2生物标志物主要在前列腺癌组织中被检测到。本研究同时证实，采用该mm-ddPCR检测方法，可在转移性去势抵抗性前列腺癌（metastatic castration-resistant PCa, mCRPC）患者的血浆样本中检测到这5种ctDNA生物标志物，检测置信区间为95%CI（85%，100%）。 研究结论：本研究证实，该mm-ddPCR检测方法可为转移性去势抵抗性前列腺癌患者血浆中的ctDNA生物标志物检测提供一种简便、快速、经济高效且灵敏通用的技术工具。未来仍需开展进一步研究，以探索该方法在前列腺癌风险分层与治疗指导中的应用潜力。