The potential of circulating tumor DNA methylation analysis for the early detection and management of ovarian cancer

Background: Despite a myriad of attempts in the last three decades to diagnose ovarian cancer (OC) earlier, this clinical aim still remains a significant challenge. Aberrant methylation patterns of linked CpGs analyzed in DNA fragments shed by cancers into the bloodstream (i.e. cell-free DNA) can provide highly specific signals indicating cancer presence.Methods: We analyzed 699 cancerous and non-cancerous tissues using a methylation array or reduced representation bisulfite sequencing to discover the most specific OC methylation patterns. A three-DNA-methylation-serum-marker panel was developed using targeted ultra-high coverage bisulfite sequencing in 151 women and validated in 250 women with various conditions, particularly in those associated with high CA125 levels (endometriosis and other benign pelvic masses), serial samples from 25 patients undergoing neoadjuvant chemotherapy, and a nested case control study of 172 UKCTOCS control arm participants which included serum samples up to two years before OC diagnosis.Results: The cell-free DNA amount and average fragment size in the serum samples was up to ten times higher than average published values (based on samples that were immediately processed) due to leakage of DNA from white blood cells owing to delayed time to serum separation. Despite this, the marker panel discriminated high grade serous OC patients from healthy women or patients with a benign pelvic mass with specificity/sensitivity of 90.7% (95% confidence interval [CI] = 84.3–94.8%) and 41.4% (95% CI = 24.1–60.9%), respectively. Levels of all three markers plummeted after exposure to chemotherapy and correctly identified 78% and 86% responders and non-responders (Fisher's exact test, p = 0.04), respectively, which was superior to a CA125 cut-off of 35 IU/mL (20% and 75%). 57.9% (95% CI 34.0–78.9%) of women who developed OC within two years of sample collection were identified with a specificity of 88.1% (95% CI = 77.3–94.3%). Sensitivity and specificity improved further when specifically analyzing CA125 negative samples only (63.6% and 87.5%, respectively).Conclusions: Our data suggest that DNA methylation patterns in cell-free DNA have the potential to detect a proportion of OCs up to two years in advance of diagnosis and may potentially guide personalized treatment. The prospective use of novel collection vials, which stabilize blood cells and reduce background DNA contamination in serum/plasma samples, will facilitate clinical implementation of liquid biopsy analyses

Additional file 1: Figure S1. of The potential of circulating tumor DNA methylation analysis for the early detection and management of ovarian cancer

Design of the nested case-control study based on the UKCTOCS Cohort. Figure S2. DMR discovery with Illumina 450 K methylation arrays. Figure S3. Pattern counts for informative regions. Figure S4. Pattern frequencies for the different regions analyzed in serum set 1 samples. Figure S5. Pattern frequencies for the different regions analyzed in serum set 2 samples. Figure S6. DNA methylation for regions #144, #204, and #228 according to OC stages. Figure S7. Coverage (number of reads) for the three different regions analyzed in serum set 3 samples. Figure S8. CA125 levels measured in NACT serum set samples. Figure S9. Pattern frequencies for the top three reactions measured in NACT serum set samples. Figure S10. Coverage (number of reads) for the top three reactions measured in NACT serum set samples. Figure S11. Average DNA amount extracted correlates with average UK temperature. Figure S12 The fraction (%) of small fragment (50–250 bp) DNA in the serum DNA preparation for 171 UKCTOCS samples analyzed in the study. Figure S13. Box plots comparing the average beta values for 450 k array probes within regions #204 and #228 between each normal (N), cancer (C) group, and white blood cell (WBC) data for OC and other 19 TCGA cancer types. (DOCX 3024 kb

Additional file 1: Figure S1. of Methylation patterns in serum DNA for early identification of disseminated breast cancer

Samples from the SUCCESS trial analyzed within this study. Figure S2. Samples from the UKCTOCS cohort analyzed within this study (nested case/control setting). Figure S3. Absolute pattern counts for all patterns detected in the region of marker EFC#93 in Serum Set 1 samples. Figure S4. Pattern frequency of EFC#93 serum DNAme in two prospectively independently collected cohorts. Figure S5. DNA amount per mL serum in the prospectively collected serum (Set 1 and 2), SUCCESS cohort, and UKCTOCS cohort. Figure S6. Pattern frequency for EFC#93 measured in SUCCESS serum set samples from women with no, 1–4 or ≥ 5 CTCs in the matched blood sample before (A) or after (B) chemotherapy. Figure S7. Impact of the presence (+ve, ≥ 1 cancer cell in blood sample) or absence (-ve) of CTCs on patient outcome. Figure S8. Impact of the presence (+ve, EFC#93 pattern frequency ≥ 0.00008) or absence (-ve) of serum DNA methylation in CTC + ve (≥1 cancer cell in pre-chemotherapy blood sample) or absence CTC-ve patients. Figure S9. Relapse-free and overall survival according to samples taken after chemotherapy. Figure S10. Relapse-free and overall survival according to samples taken after chemotherapy. Figure S11. Average serum DNA amount correlates with average UK temperature. Figure S12. Average serum DNA fragment size correlates with average UK temperature. Figure S13. Correlation of DNA fragment size and DNA amount. Figure S14. Overall survival of women whose samples were taken before and after chemotherapy and before anti-hormonal treatment in hormone receptor-negative and -positive SUCCESS participants. Figure S15. Overall survival of women whose samples were taken before and after chemotherapy and before anti-hormonal treatment in hormone receptor-negative and -positive SUCCESS participants. (PDF 2123 kb

Additional file 3: Table S1. of Methylation patterns in serum DNA for early identification of disseminated breast cancer

Coordinates and primers used to amplify the identified target regions using bisulfite sequencing. (PDF 578 kb