title: Circulating cell-free DNA-based epigenetic assay can detect early breast cancer.
abstract: Circulating cell-free DNA (cfDNA) has recently been recognized as a resource for biomarkers of cancer progression, treatment response, and drug resistance. However, few have demonstrated the usefulness of cfDNA for early detection of cancer. Although aberrant DNA methylation in cfDNA has been reported for more than a decade, its diagnostic accuracy remains unsatisfactory for cancer screening. Thus, the aim of the present study was to develop a highly sensitive cfDNA-based system for detection of primary breast cancer (BC) using epigenetic biomarkers and digital PCR technology.
  
  
  
  Array-based genome-wide DNA methylation analysis was performed using 56 microdissected breast tissue specimens, 34 cell lines, and 29 blood samples from healthy volunteers (HVs). Epigenetic markers for BC detection were selected, and a droplet digital methylation-specific PCR (ddMSP) panel with the selected markers was established. The detection model was constructed by support vector machine and evaluated using cfDNA samples.
  
  
  
  The methylation array analysis identified 12 novel epigenetic markers (JAK3, RASGRF1, CPXM1, SHF, DNM3, CAV2, HOXA10, B3GNT5, ST3GAL6, DACH1, P2RX3, and chr8:23572595) for detecting BC. We also selected four internal control markers (CREM, GLYATL3, ELMOD3, and KLF9) that were identified as infrequently altered genes using a public database. A ddMSP panel using these 16 markers was developed and detection models were constructed with a training dataset containing cfDNA samples from 80 HVs and 87 cancer patients. The best detection model adopted four methylation markers (RASGRF1, CPXM1, HOXA10, and DACH1) and two parameters (cfDNA concentration and the mean of 12 methylation markers), and, and was validated in an independent dataset of 53 HVs and 58 BC patients. The area under the receiver operating characteristic curve for cancer-normal discrimination was 0.916 and 0.876 in the training and validation dataset, respectively. The sensitivity and the specificity of the model was 0.862 (stages 0-I 0.846, IIA 0.862, IIB-III 0.818, metastatic BC 0.935) and 0.827, respectively.
  
  
  
  Our epigenetic-marker-based system distinguished BC patients from HVs with high accuracy. As detection of early BC using this system was comparable with that of mammography screening, this system would be beneficial as an optional method of screening for BC.
authors.affiliation: Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Natsue
authors.initials: N
authors.lastname: Uehiro
authors.affiliation: Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan. fsatoh@kuhp.kyoto-u.ac.jp.
authors.forename: Fumiaki
authors.identifier: 0000-0001-8186-9290
authors.initials: F
authors.lastname: Sato
authors.affiliation: Department of Target Therapy Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Fengling
authors.initials: F
authors.lastname: Pu
authors.affiliation: Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Sunao
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kawashima
authors.affiliation: Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Kawaguchi
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sugimoto
authors.affiliation: Department of Target Therapy Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.|Department of Medical Oncology, Fukushima Medical University, Fukushima, Japan.
authors.forename: Shigehira
authors.initials: S
authors.lastname: Saji
authors.affiliation: Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Toi
mesh_terms: D000328:Adult; D000367:Age Factors; D000368:Aged; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D016022:Case-Control Studies; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D030561:Databases, Nucleic Acid; D055088:Early Detection of Cancer / Q000379:methods; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
keywords: Breast cancer; Circulating DNA; DNA methylation; Early detection; Epigenetics