Tumor Biology

, Volume 37, Issue 10, pp 13111–13119 | Cite as

Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing

  • Zibo Li
  • Xinwu Guo
  • Lili Tang
  • Limin Peng
  • Ming Chen
  • Xipeng Luo
  • Shouman Wang
  • Zhi Xiao
  • Zhongping Deng
  • Lizhong Dai
  • Kun Xia
  • Jun Wang
Original Article


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.


Breast cancer Plasma Methylation Cell-free DNA Next-generation sequencing Microfludic PCR 



Area under the receiver operating characteristic curve


Bisulfite sequencing primer


Cell-free DNA


Confidential interval


Circulating tumor DNA


Estrogen receptor


Human epidermal growth factor receptor-2


Next-generation sequencing


Odds ratio


Progesterone receptor


Receiver operating characteristics



This work was supported in part by grants from the Natural Science Foundation of China (No. 81272296 and No. 81372228), the Scientific Project of China Hunan Provincial Science and Technology Department (No. 2012SK2013), and the Major Special Projects of the Science and Technology Bureau of Changsha, China (No. K1204017-31 and K1306011-31). Zibo Li was supported by the Hunan Province Postgraduate Student Scientific Innovation Project, China (No. CX2013B087).

Compliance with ethical standards

Ethical standards

We declare that the experiments performed in this study comply with the current laws of the People’s Republic of China.

Conflicts of interest

Xinwu Guo, Limin Peng, Ming Chen, Xipeng Luo, Zhongping Deng, and Lizhong Dai are employees of Sanway Gene Technology Inc.

Supplementary material

13277_2016_5190_MOESM1_ESM.doc (43 kb)
Online Resource Table S1 (DOC 43 kb)
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Online Resource Table S6 (DOC 38 kb)
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Online Resource Figure 1 (DOC 737 kb)


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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Zibo Li
    • 1
  • Xinwu Guo
    • 2
  • Lili Tang
    • 3
  • Limin Peng
    • 2
  • Ming Chen
    • 2
  • Xipeng Luo
    • 2
  • Shouman Wang
    • 3
  • Zhi Xiao
    • 3
  • Zhongping Deng
    • 2
    • 4
    • 5
  • Lizhong Dai
    • 2
    • 4
    • 5
  • Kun Xia
    • 1
  • Jun Wang
    • 1
  1. 1.The State Key Laboratory of Medical Genetics and School of Life SciencesCentral South UniversityChangshaChina
  2. 2.Sanway Gene Technology Inc.ChangshaChina
  3. 3.Department of Breast Surgery, Xiangya HospitalCentral South UniversityChangshaChina
  4. 4.Research Center for Technologies in Nucleic Acid-Based DiagnosticsChangshaChina
  5. 5.Research Center for Technologies in Nucleic Acid-Based Diagnostics and TherapeuticsChangshaChina

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