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. 2017 Dec 22;9(1):116.
doi: 10.1186/s13073-017-0500-7.

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

Martin Widschwendter  1 , Michal Zikan  2 , Benjamin Wahl  3 , Harri Lempiäinen  4 , Tobias Paprotka  3 , Iona Evans  5 , Allison Jones  5 , Shohreh Ghazali  5 , Daniel Reisel  5 , Johannes Eichner  4 , Tamas Rujan  4 , Zhen Yang  6 , Andrew E Teschendorff  5   6 , Andy Ryan  5 , David Cibula  2 , Usha Menon  5 , Timo Wittenberger  4
Affiliations

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

Martin Widschwendter et al. Genome Med. .

Abstract

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.

Keywords: Cell-free DNA; DNA methylation; Early diagnosis; Ovarian cancer; Personalized treatment; Screening; Serum DNA.

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Conflict of interest statement

Ethics approval and consent to participate

Samples were collected prospectively at the University College London Hospital in London and at the Charles University Hospital in Prague and the Department of Gynaecology and Obstetrics. The study was approved by the local research ethics committees: UCL/UCLH Biobank for Studying Health & Disease NC09.13) and the ethics committee of the General University Hospital, Prague approval No.: 22/13 GRANT – 7. RP – EPI-FEM-CARE. All patients provided written informed consent. The UKCTOCS study was approved by the local research ethics committees (UCL/UCLH Biobank for Studying Health & Disease NC09.13) and was approved as part of trial approval by the UK North West Multicentre Research Ethics Committees (North West MREC 00/8/34). All patients provided written informed consent and all studies were conducted in accordance with the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

HL is an employee and shareholder of Genedata AG. BW is affiliated with a private company. UM has stock ownership in and research funding from Abcodia Pvt Ltd which has an interest in cancer biomarkers. The remaining authors declare that they have no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Study design. Using two different epigenome-wide technologies, 699 human tissue samples have been analyzed to identify a total of 31 regions whose methylation status has been analyzed in two serum sets consisting of 151 serum samples. Three markers have been validated in three independent settings: serum set 3, which consisted of 250 serum samples, from women with various benign and malignant conditions of the female genital tract. NACT set, consisting of serial samples from women with advanced stage ovarian cancer before and during chemotherapy. UKCTOCS (United Kingdom Collaborative Trial of Ovarian Cancer Screening) set which included serum samples from those 43 of the 101,539 women in the control arm who developed OC within 2 years; for each case, three control women who did not develop OC within 5 years of sample donation have been matched
Fig. 2
Fig. 2
Principles of methylation pattern discovery in tissue and analyses in serum. RRBS was used in tissue samples in order to identify those CpG regions for which methylation patterns discriminate OC from other tissues, in particular blood cells which are the most abundant contaminant of cell-free DNA. An example of region #141 is provided which is a 136-bp region containing seven linked CpGs. The cancer pattern consists of reads in which all linked CpGs are methylated, indicated by “1111111” (a). b The tissue RRBS data have been processed through a bioinformatic pipeline in order to identify the most promising markers. c The principles of the serum DNA methylation assay are demonstrated
Fig. 3
Fig. 3
Serum DNA methylation analysis in women with benign and malignant conditions of the female genital tract. Pattern frequencies for the different regions and CA125 levels analyzed in serum set 3 samples are shown and horizontal red bars denote the mean (a–d; ns not significant; *p < 0.05, **p < 0.01, ***p < 0.001; Mann–Whitney U test compared to HGS; H healthy, BPM benign pelvic mass, BOT borderline tumors, NET non-epithelial tumors, OCM other cancerous malignancies, NHGS non-high grade serous ovarian cancers, HGS high grade serous, OC ovarian cancers)
Fig. 4
Fig. 4
The dynamics of serum DNAme markers and CA125 as a function of exposure to Carboplatin-based chemotherapy. The changes in pattern frequency of the three markers as well as CA125 is shown before being compared after two cycles of chemotherapy (a–d) in the NACT set. Responder: no recurrence within six months after successful completion of NACT and adjuvant chemotherapy and interval debulking surgery; Non-Responder: either no response to chemotherapy or progression on chemotherapy or recurrence within six months after successful completion of NACT and adjuvant chemotherapy and interval debulking surgery
Fig. 5
Fig. 5
Performance of the serum DNAme marker panel in a population-based cohort for early OC diagnosis. Compared to the prospectively collected samples within the EpiFemCare Programme, UKCTOCS samples contained a significantly higher DNA concentration (a) and larger average DNA fragment size (b). As a result of this, we had to lower the cut-off for the three markers by a factor three (i.e. pattern frequency cut-off for #141, #204, and #228 is 0.00027, 0.00001, and 0.0000033, respectively). For OC Set 2, we only display the result of the 50 samples for which we have analyzed both DNA amount and fragment size (for 42 samples, we only analyzed DNA amount). In addition, in one UKCTOCS sample, the fragment size analysis failed. ***p < 0.001
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