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

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Tumor Biology

Abstract

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.

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Abbreviations

AUC:

Area under the receiver operating characteristic curve

BSP:

Bisulfite sequencing primer

cfDNA:

Cell-free DNA

CI:

Confidential interval

ctDNA:

Circulating tumor DNA

ER:

Estrogen receptor

HER2:

Human epidermal growth factor receptor-2

NGS:

Next-generation sequencing

OR:

Odds ratio

PR:

Progesterone receptor

ROC:

Receiver operating characteristics

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Acknowledgments

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).

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Authors and Affiliations

  1. The State Key Laboratory of Medical Genetics and School of Life Sciences, Central South University, 172 Tongzipo Road, Changsha, Hunan, 410013, China

    Zibo Li, Kun Xia & Jun Wang

  2. Sanway Gene Technology Inc., Changsha, Hunan, 410205, China

    Xinwu Guo, Limin Peng, Ming Chen, Xipeng Luo, Zhongping Deng & Lizhong Dai

  3. Department of Breast Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China

    Lili Tang, Shouman Wang & Zhi Xiao

  4. Research Center for Technologies in Nucleic Acid-Based Diagnostics, Changsha, Hunan, 410205, China

    Zhongping Deng & Lizhong Dai

  5. Research Center for Technologies in Nucleic Acid-Based Diagnostics and Therapeutics, Changsha, Hunan, 410205, China

    Zhongping Deng & Lizhong Dai

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  1. Zibo Li
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  2. Xinwu Guo
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  3. Lili Tang
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  4. Limin Peng
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  5. Ming Chen
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  9. Zhongping Deng
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  10. Lizhong Dai
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  11. Kun Xia
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  12. Jun Wang
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Corresponding author

Correspondence to Jun Wang.

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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.

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Li, Z., Guo, X., Tang, L. et al. Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing. Tumor Biol. 37, 13111–13119 (2016). https://doi.org/10.1007/s13277-016-5190-z

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