Circulating cell-free DNA-based epigenetic assay can detect early breast cancer

Background: 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. Methods: 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. Results: 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. Conclusion: 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

Additional file 3: Figure S1. of Circulating cell-free DNA-based epigenetic assay can detect early breast cancer

Laser microdissection of pan-cytokeratin (AE1/AE3)-immunostained FFPE specimens. Figure S2. A-B Validation analysis using large public datasets. Figure S3. A-L DNA methylation status in genomic region surrounding candidate marker loci, and differentially methylated region. Figure S4. Unmasking of epigenetically silenced genes by demethylating agent and histone deacethylase inhibitor (common BC markers). Figure S5. Unmasking of epigenetically silenced genes by demethylating agent and histone deacethylase inhibitor (luminal-dominant markers). Figure S6. Relationship between ACTB and the new panel of internal control markers. Figure S7. A-C ROC curves and ddMSP data of 12 methylation markers and three parameters. Figure S8. Detection index and age. Figure S9. ROC curves in each stage of BC. Figure S10. Distribution of detection indexes of HER-positive patients by stage. Figure S11. cfDNA concentration in HVs and patients with BC. Figure S12. A-E Determining upper and lower thresholds for positive droplets. (PPTX 12394 kb

Additional file 1: of Circulating cell-free DNA-based epigenetic assay can detect early breast cancer

This file provides detailed materials and methods for this study. (DOCX 66 kb