Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Feb 15;154(4):679-691.
doi: 10.1002/ijc.34757. Epub 2023 Oct 20.

Plasma cell-free DNA methylation analysis for ovarian cancer detection: Analysis of samples from a case-control study and an ovarian cancer screening trial

Chiara Herzog  1   2 , Allison Jones  3 , Iona Evans  3 , Daniel Reisel  3 , Adeola Olaitan  3 , Konstantinos Doufekas  3 , Nicola MacDonald  3 , Angelique Flöter Rådestad  4 , Kristina Gemzell-Danielsson  4 , Michal Zikan  5 , David Cibula  6 , Lukáš Dostálek  6 , Tobias Paprotka  7 , Andreas Leimbach  8 , Markus Schmitt  8 , Andy Ryan  9 , Aleksandra Gentry-Maharaj  3   9 , Sophia Apostolidou  9 , Adam N Rosenthal  3 , Usha Menon  9 , Martin Widschwendter  1   2   3   4
Affiliations

Plasma cell-free DNA methylation analysis for ovarian cancer detection: Analysis of samples from a case-control study and an ovarian cancer screening trial

Chiara Herzog et al. Int J Cancer. .

Abstract

Analysis of cell-free DNA methylation (cfDNAme), alone or combined with CA125, could help to detect ovarian cancers earlier and may reduce mortality. We assessed cfDNAme in regions of ZNF154, C2CD4D and WNT6 via targeted bisulfite sequencing in diagnostic and early detection (preceding diagnosis) settings. Diagnostic samples were obtained via prospective blood collection in cell-free DNA tubes in a convenience series of patients with a pelvic mass. Early detection samples were matched case-control samples derived from the UK Familial Ovarian Cancer Screening Study (UKFOCSS). In the diagnostic set (ncases = 27, ncontrols = 41), the specificity of cfDNAme was 97.6% (95% CI: 87.1%-99.9%). High-risk cancers were detected with a sensitivity of 80% (56.3%-94.3%). Combination of cfDNAme and CA125 resulted in a sensitivity of 94.4% (72.7%-99.9%) for high-risk cancers. Despite technical issues in the early detection set (ncases = 29, ncontrols = 29), the specificity of cfDNAme was 100% (88.1%-100.0%). We detected 27.3% (6.0%-61.0%) of high-risk cases with relatively lower genomic DNA (gDNA) contamination. The sensitivity rose to 33.3% (7.5%-70.1%) in samples taken <1 year before diagnosis. We detected ovarian cancer in several patients up to 1 year before diagnosis despite technical limitations associated with archival samples (UKFOCSS). Combined cfDNAme and CA125 assessment may improve ovarian cancer screening in high-risk populations, but future large-scale prospective studies will be required to validate current findings.

Keywords: cell-free DNA; diagnosis; methylation; ovarian cancer.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement

Martin Widschwendter, Allison Jones, Iona Evans and Tobias Paprotka are named as inventors on a patent which describes the DNAme markers analysed in this manuscript. Andreas Leimbach and Markus Schmitt are employed by Eurofins Genomics, which has submitted patent applications related to the intellectual property outlined in this manuscript. Kristina Gemzell-Danielsson has served on an ad hoc basis as speaker or expert on meetings organised by Exelgyn/Nordic, Concept Foundation and HRA-Pharma. Aleksandra Gentry-Maharaj has been funded by grants from the Medical Research Council, Cancer Research UK, National Institute for Health Research and The Eve Appeal; she reports funded research collaborations with industry—iLOF (intelligent Lab on Fibre), RNA Guardian, Micronoma, Mercy Bioanalytics and academics—Cambridge University, QIMR Berghofer Medical Research Institute, Imperial College London, University of Innsbruck and Dana Farber, USA. Aleksandra Gentry-Maharaj is a member of ACED Gynaecological Cancer Working Group and is ACED Co-Director Research Domain Trials. Sophia Apostolidou has been funded by grants from the Medical Research Council, Cancer Research UK, National Institute for Health Research and National Institute for Health Research Health Technology Assessment and has been funded by Abcodia until July 2021 (paid to UCL); she reports funded research collaborations with Cambridge University, QIMR Berghofer Medical Research Institute, iLOF (intelligent Lab on Fibre), RNA Guardian, Micronoma, Mercy Bioanalytics, Syntent Biotechnology, Imperial College London, Dana Faber Cancer Institute and National Health and Medical Research Council Australia. Usha Menon reports institutional research collaborations in early detection of ovarian cancer with industry—RNA Guardian, Micronoma, Mercy Bioanalytics, Synteny and research collaborations in early detection of cancer, in particular ovarian cancer with UK, US and Australian academics supported by public and charity funded grants.

The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Overview of the study.
High-risk cancers included all grade 2 and 3 cancers.
Figure 2
Figure 2. Cell-free DNA methylation pipeline and quality control metrics.
(A) Overview of sample processing and analysis. (B) Visualisation of mapping rates for samples in the Diagnostic and Early detection sets. (C) Proportion of aligned reads in each of the three targeted regions (EFC 144, EFC 204, EFC 228).
Figure 3
Figure 3. Threshold selection and diagnostic validation.
(A) Values of fully methylated reads (% of all reads in each respective region) for the three targeted regions EFC 144, EFC 204 and EFC 228 in healthy volunteers and women with benign pelvic pathologies or ovarian cancer. (B) Receiver operating characteristic curves for each of the four regions. A specificity cutoff of 97% and the corresponding value of fully methylated reads was set as the threshold. A cfDNAme score (WID-cfOC) was derived from regions EFC 144, EFC 204 and EFC 228. If any of the regions was above the respective threshold, the WID-cfOC was regarded positive. The WID-cfOC was evaluated in (C) all ovarian cancers and (D) high-risk ovarian cancers (see Table 1).
Figure 4
Figure 4. Sensitivity and specificity of the cfDNAme score in an early detection setting.
All measures are demonstrated in all samples, or those with lower or higher gDNA contamination, respectively. gDNA contamination was quantified via cfDNA/gDNA peaks and a ratio was calculated (see Figure S3). Samples with a higher than median cfDNA/gDNA ratio had lower gDNA contamination and vice versa. (A) Specificity of the cfDNAme score in UKFOCSS samples. (B) Sensitivity for the cfDNAme score in all ovarian cancer patients, (C) samples from high-risk ovarian cancer patients, or (D) samples from high-risk ovarian cancer patients collected <1 year from diagnosis. For samples with matched CA125 data, cfDNAme, CA125 and a combined score (positive when either cfDNAme or CA125 were positive) were evaluated in (E) all cancers or (F) high-risk cancers regardless of gDNA contamination. CA125 and combined score overlap, likely due to a limited amount of data based on a small sample size. (G, H) Evaluation of sensitivity and specificity in CA125 negative samples including all or only high-risk cancers.
Figure 5
Figure 5. Outlook on ovarian cancer screening using combined molecular tests.
See this image and copyright information in PMC

References

    1. Labidi-Galy SI, Papp E, Hallberg D, et al. High grade serous ovarian carcinomas originate in the fallopian tube. Nat Commun. 2017;8:1093. doi: 10.1038/s41467-017-00962-1. - DOI - PMC - PubMed
    1. Ovarian cancer survival statistics. 2022. [Internet] https://www.cancerresearchuk.org/health-professional/cancer-statistics/s....
    1. Menon U, Ryan A, Kalsi J, et al. Risk algorithm using serial biomarker measurements doubles the number of screen-detected cancers compared with a single-threshold rule in the United Kingdom collaborative trial of ovarian cancer screening. J Clin Oncol. 2015;33:2062–2071. doi: 10.1200/JCO.2014.59.4945. - DOI - PMC - PubMed
    1. Jacobs IJ, Menon U, Ryan A, et al. Ovarian cancer screening and mortality in the UK collaborative trial of ovarian cancer screening (UKCTOCS): a randomised controlled trial. Lancet Lond Engl. 2016;387:945–956. doi: 10.1016/S0140-6736(15)01224-6. - DOI - PMC - PubMed
    1. Menon U, Gentry-Maharaj A, Burnell M, et al. Ovarian cancer population screening and mortality after long-term follow-up in the UK collaborative trial of ovarian cancer screening (UKCTOCS): a randomised controlled trial. Lancet. 2021;397:2182–2193. doi: 10.1016/S0140-6736(21)00731-5. - DOI - PMC - PubMed