Li Zibo, Guo Xinwu, Tang Lili, Peng Limin, Chen Ming, Luo Xipeng, Wang Shouman, Xiao Zhi, Deng Zhongping, Dai Lizhong, Xia Kun, Wang Jun
The State Key Laboratory of Medical Genetics and School of Life Sciences, Central South University, 172 Tongzipo Road, Changsha, Hunan, 410013, China.
Sanway Gene Technology Inc., Changsha, Hunan, 410205, China.
Tumour Biol. 2016 Oct;37(10):13111-13119. doi: 10.1007/s13277-016-5190-z. Epub 2016 Jul 23.
Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients' plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.
循环游离DNA（cfDNA）被认为是一种用于非侵入性癌症检测的潜在生物标志物。为了评估血浆cfDNA中六个候选基因（EGFR、GREM1、PDGFRB、PPM1E、SOX17和WRN）的甲基化水平作为乳腺癌早期检测的生物标志物，采用基于微流控PCR的靶向富集和新一代亚硫酸氢盐测序技术，对86例乳腺癌患者和67例健康对照者这些基因的启动子甲基化进行了定量分析。通过ROC曲线下面积（AUC）和比值比（OR）分析，研究了基于候选基因甲基化状态的不同逻辑模型的预测性能。结果显示，EGFR、PPM1E和8个基因特异性CpG位点在癌症患者血浆中表现出显著的高甲基化，且与乳腺癌显著相关（OR范围为2.51至9.88）。这些生物标志物的AUC值范围为0.66至0.75。多个高甲基化基因或CpG位点的组合显著提高了乳腺癌检测的预测性能。我们的研究证明了使用基于微流控PCR的靶向富集和亚硫酸氢盐新一代测序对cfDNA中候选基因甲基化进行定量测量的可行性，这值得进一步验证，并可能在临床肿瘤学实践中有广泛的应用前景。通过新一代测序对血浆cfDNA甲基化模式进行定量分析可能是一种有价值的非侵入性乳腺癌早期检测工具。
