Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, 10065, USA.
Department of Surgery, Weill Cornell Medicine, New York, NY, 10065, USA.
BMC Cancer. 2020 Jan 31;20(1):85. doi: 10.1186/s12885-020-6574-4.
Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR.
In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood.
Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27).
This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.
在许多研究中，人们通过检测循环肿瘤 DNA（ctDNA）中的特定 CpG 甲基化来进行乳腺癌（BrCa）的早期检测。在这些研究中，许多标志物是基于 BrCa 进展的已知生物学特性确定的，并使用甲基特异性 PCR（MSP）进行检测，该技术涉及亚硫酸氢盐转化、PCR 和 qPCR。
在本报告中，我们展示了一种新的检测方法（多重亚硫酸氢盐 PCR-LDR-qPCR）的开发，该方法通过整合额外的步骤，如连接酶检测反应（LDR）、甲基化 CpG 靶标富集、携带保护（使用尿嘧啶 DNA 糖基化酶）和最小化引物二聚体形成（使用核糖引物和 RNAseH2），可以潜在地改进 MSP。该检测方法是针对通过对 31 种原发性肿瘤（包括 BrCa）的全基因组甲基化数据集进行综合分析而确定的乳腺癌特异性 CpG 标志物设计的，以及匹配的正常组织和外周血。
我们的结果表明，当与过量未甲基化的 CpG 标志物（每个标志物约 3000 个拷贝）混合时，PCR-LDR-qPCR 检测方法能够检测到 3 个 BrCa 特异性 CpG 标志物中的每个约 30 个甲基化拷贝，这是从患者血浆中分离时 BrCa ctDNA 被外周血无细胞 DNA（cfDNA）淹没的合理近似值。生物信息学鉴定的 CpG 标志物位于 NR5A2 和 PRKCB 的启动子区域，以及染色体 1 的非编码区域（EFNA3 上游）。进一步的生物信息学分析表明，这些甲基化标志物与患者的种族和年龄无关，并且与与 BrCa 进展相关的信号通路呈正相关（例如与视黄酸核受体、PTEN、p53、pRB 和 p27 相关的信号通路）。
本报告证明了基于 bisulfite PCR-LDR-qPCR 检测方法和基于生物信息学的生物标志物发现，在基于血液的 BrCa 检测中的潜在应用。
