Ovarian cancer remains a leading cause of cancer-related morbidity and mortality. It is one of the few malignancies for which a protein-based biomarker, CA-125, is routinely used for clinical monitoring. However, the limited sensitivity and specificity of CA-125, particularly in early-stage or low-volume disease, restrict its broader clinical utility and highlight the need for more accurate biomarkers. The emergence of circulating tumour DNA (ctDNA) offers potential applications across multiple points in the disease course through its sensitivity, specificity, and ability to genotype tumours non-invasively. ctDNA enables real-time molecular profiling, allowing dynamic assessment of tumour burden, treatment response, and resistance evolution. The critical evaluation of its clinical performance and potential integration with existing protein-based assays is essential to determine its added value. Increasingly, combined protein and ctDNA data highlight a growing trend towards multi-omic analyses, requiring advanced analytical and bioinformatic approaches. This review examines the current landscape, technical challenges, and translational potential of ctDNA and multi-omic assays in ovarian cancer, outlining key steps towards clinical implementation, drawing on lessons from established circulating biomarkers to guide future applications in precision oncology.

1. Cancer Research UK. Ovarian Cancer Statistics. Available from: https://www.cancerresearchuk.org/health/professional/cancer/statistics/statistics/by/cancer/type/ ovarian/cancer [Last accessed on 2025 Nov 07].

2. Bast RC Jr., Klug TL, St John E, et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med. 1983;309(15):883-887. doi: 10.1056/NEJM198310133091503

3. Ledermann JA, Matias-Guiu X, Amant F, et al. ESGO-ESMO-ESP consensus conference recommendations on ovarian cancer: Pathology and molecular biology and early, advanced and recurrent disease. Ann Oncol. 2024;35(3):248-266. doi: 10.1016/j.annonc.2023.11.015

4. Santoro A, Travaglino A, Inzani F, et al. Prognostic value of chemotherapy response score (CRS) assessed on the adnexa in Ovarian high-grade serous carcinoma: A systematic review and meta-analysis. Diagnostics (Basel). 2022;12(3):633. doi: 10.3390/diagnostics12030633

5. Cohen PA, Powell A, Böhm S, et al. Pathological chemotherapy response score is prognostic in tubo-ovarian high-grade serous carcinoma: A systematic review and meta-analysis of individual patient data. Gynecol Oncol. 2019;154(2):441-448. doi: 10.1016/j.ygyno.2019.04.679

6. Moss EL, Hollingworth J, Reynolds TM. The role of CA125 in clinical practice. J Clin Pathol. 2005;58(3):308-312. doi: 10.1136/jcp.2004.018077

7. Liao XY, Huang GJ, Gao C, Wang GH. A meta-analysis of serum cancer antigen 125 array for diagnosis of ovarian cancer in Chinese. J Cancer Res Ther. 2014;10(Suppl 3):C222-C224. doi: 10.4103/0973-1482.145884

8. Buys SS, Partridge E, Greene MH, et al. Ovarian cancer screening in the Prostate, Lung, Colorectal and Ovarian (PLCO) cancer screening trial: Findings from the initial screen of a randomized trial. Am J Obstet Gynecol. 2005;193(5):1630-1639. doi: 10.1016/j.ajog.2005.05.005

9. Mandel P, Métais P. Nucleic acids in human blood plasma. C R Seances Soc Biol Fil. 1948;142(3-4):241-243.

10. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977;37(3):646-650.

11. Wan JCM, Sasieni P, Rosenfeld N. Promises and pitfalls of multi-cancer early detection using liquid biopsy tests. Nat Rev Clin Oncol. 2025;22:566-580. doi: 10.1038/s41571-025-01033-x

12. Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: Towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17:223-238. doi: 10.1038/nrc.2017.7

13. Tuna M, Ju Z, Yoshihara K, Amos CI, Tanyi JL, Mills GB. Clinical relevance of TP53 hotspot mutations in high-grade serous ovarian cancers. Br J Cancer. 2020;122(3):405-412. doi: 10.1038/s41416-019-0654-8

14. Diehl F, Li M, Dressman D, et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Nat Acad Sci United States Am. 2005;102(45):16368-16373. doi: 10.1073/pnas.0507904102

15. Swisher EM, Wollan M, Mahtani SM, et al. Tumor-specific p53 sequences in blood and peritoneal fluid of women with epithelial ovarian cancer. Am J Obstet Gynecol. 2005;193(3 Pt 1):662-667. doi: 10.1016/j.ajog.2005.01.054

16. Hou JY, Chapman JS, Kalashnikova E, et al. Circulating tumor DNA monitoring for early recurrence detection in epithelial ovarian cancer. Gynecol Oncol. 2022;167(2):334-341. doi: 10.1016/j.ygyno.2022.09.004

17. Parkinson CA, Gale D, Piskorz AM, et al. Exploratory analysis of TP53 mutations in circulating tumour DNA as biomarkers of treatment response for patients with relapsed high-grade serous ovarian carcinoma: A retrospective study. PLoS Med. 2016;13(12):e1002198. doi: 10.1371/journal.pmed.1002198

18. Phallen J, Sausen M, Adleff V, et al. Direct detection of early-stage cancers using circulating tumor DNA. Sci Transl Med. 2017;9(403):eaan2415. doi: 10.1126/scitranslmed.aan2415

19. Cohen JD, Li L, Wang Y, et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science. 2018;359(6378):926-930 doi: 10.1126/science.aar3247

20. Klein EA, Richards D, Cohn A, et al. Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set. Ann Oncol. 2021;32(9):1167-1177. doi: 10.1016/j.annonc.2021.05.806

21. Li G, Zhang Y, Li K, et al. Transformer-based AI technology improves early ovarian cancer diagnosis using cfDNA methylation markers. Cell Rep Med. 2024;5(8):101666. doi: 10.1016/j.xcrm.2024.101666

22. Cristiano S, Leal A, Phallen J, et al. Genome-wide cell-free DNA fragmentation in patients with cancer. Nature. 2019;570(7761):385-389. doi: 10.1038/s41586-019-1272-6

23. Nguyen CVT, Hanh Nguyen TH, Vo DH, et al. Evaluation of a multimodal ctDNA-based assay for detection of aggressive cancers lacking standard screening tests. Future Oncol. 2025;21(1):105-115. doi: 10.1080/14796694.2024.2413266

24. Medina JE, Annapragada AV, Lof P, et al. Early detection of ovarian cancer using cell-free DNA fragmentomes and protein biomarkers. Cancer Discov. 2025;15(1):105-118. doi: 10.1158/2159-8290.CD-24-0393

25. Shen SY, Singhania R, Fehringer G, et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature. 2018;563(7732):579-583. doi: 10.1038/s41586-018-0703-0

26. Liu L, Toung JM, Jassowicz AF, et al. Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification. Ann Oncol. 2018;29(6):1445-1453. doi: 10.1093/annonc/mdy119

27. Reyes HD, Devor EJ, Warrier A, et al. Differential DNA methylation in high-grade serous ovarian cancer (HGSOC) is associated with tumor behavior. Sci Rep. 2019;9(1):17996. doi: 10.1038/s41598-019-54401-w

28. Liggett TE, Melnikov A, Yi Q, et al. Distinctive DNA methylation patterns of cell-free plasma DNA in women with malignant ovarian tumors. Gynecol Oncol. 2011;120(1):113-120. doi: 10.1016/j.ygyno.2010.09.019

29. Neal RD, Johnson P, Clarke CA, et al. Cell-free DNA-based multi-cancer early detection test in an asymptomatic screening population (NHS-Galleri): Design of a pragmatic, prospective randomised controlled trial. Cancers (Basel). 2022;14(19):4818. doi: 10.3390/cancers14194818

30. Mouliere F, Chandrananda D, Piskorz AM, et al. Enhanced detection of circulating tumor DNA by fragment size analysis. Sci Transl Med. 2018;10(466):eraat4921. doi: 10.1126/scitranslmed.aat4921

31. Zhou Q, Kang G, Jiang P, et al. Epigenetic analysis of cell-free DNA by fragmentomic profiling. Proc Natl Acad Sci. 2022;119(44):e2209852119. doi: 10.1073/pnas.2209852119

32. Martin De La Fuente L, Veerla S, Li MX, et al. Copy Number Signatures in Cervical Samples Enable Early Detection of High-Grade Serous Ovarian Carcinom [Preprint]; 2025. doi: 10.1101/2025.04.23.25325778

33. Adalsteinsson VA, Ha G, Freeman SS, et al. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. Nat Commun. 2017;8(1):1324-1324. doi: 10.1038/s41467-017-00965-y

34. Dudley JC, Schroers-Martin J, Lazzareschi DV, et al. Detection and surveillance of bladder cancer using urine tumor DNA. Cancer Discov. 2019;9(4):500-509. doi: 10.1158/2159-8290.CD-18-0825

35. Wan JCM, Heider K, Gale D, et al. ctDNA monitoring using patient-specific sequencing and integration of variant reads. Sci Transl Med. 2020;12(548):eaaz8084. doi: 10.1126/scitranslmed.aaz8084

36. Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early stage lung cancer evolution. Nature. 2017;545:446-451. doi: 10.1038/nature22364

37. Paracchini L, Mannarino L, Romualdi C, et al. Genomic instability analysis in DNA from Papanicolaou test provides proof-of-principle early diagnosis of high-grade serous ovarian cancer. Sci Transl Med. 2023;15(725):eadi2556. doi: 10.1126/scitranslmed.adi2556

38. Werner B, Sjoquist KM, Espinoza D, et al. Cell-free DNA in plasma and ascites as a biomarker of bevacizumab response- a translational research sub-study of the REZOLVE (ANZGOG-1101) clinical trial. Transl Oncol. 2024;43:101914. doi: 10.1016/j.tranon.2024.101914

39. Azaïs H, Brochard C, Taly V, et al. Prognostic value of circulating tumor DNA at diagnosis and its early decrease after one cycle of neoadjuvant chemotherapy for patients with advanced epithelial ovarian cancer. An ancillary analysis of the CHIVA phase II GINECO trial. Gynecol Oncol. 2025;192:145-154. doi: 10.1016/j.ygyno.2024.12.004

40. Pereira E, Camacho-Vanegas O, Anand S, et al. Personalized circulating tumor DNA biomarkers dynamically predict treatment response and survival in gynecologic cancers. PLoS One. 2015;10(12):e0145754. doi: 10.1371/journal.pone.0145754

41. Minato T, Ito S, Li B, et al. Liquid biopsy with droplet digital PCR targeted to specific mutations in plasma cell-free tumor DNA can detect ovarian cancer recurrence earlier than CA125. Gynecol Oncol Rep. 2021;38:100847. doi: 10.1016/j.gore.2021.100847

42. Jamieson A, McConechy MK, Lum A, et al. Selective utilization of circulating tumor DNA testing enables disease monitoring in endometrial and ovarian carcinomas. J Gynecol Oncol. 2025;36(1):e5. doi: 10.3802/jgo.2025.36.e5

43. Wan JCM, Mughal TI, Razavi P, et al. Liquid biopsies for residual disease and recurrence. Med. 2021;2(12):1292-1313. doi: 10.1016/j.medj.2021.11.001

44. Kim YM, Lee SW, Lee YJ, Lee HY, Lee JE, Choi EK. Prospective study of the efficacy and utility of TP53 mutations in circulating tumor DNA as a non-invasive biomarker of treatment response monitoring in patients with high-grade serous ovarian carcinoma. J Gynecol Oncol. 2019;30(3):e32. doi: 10.3802/jgo.2019.30.e32

45. Kfoury M, Bonnet C, Delanoy N, et al. Dynamic changes in TP53 mutated circulating tumor DNA predicts outcome of patients with high-grade ovarian carcinomas. Int J Gynecol Cancer. 2024;34(11):1836-1839. doi: 10.1136/IJGC-2024-005581

46. Gale D, Heider K, Ruiz-Valdepenas A, et al. Residual ctDNA after treatment predicts early relapse in patients with early-stage non-small cell lung cancer. Ann Oncol. 2022;33(5):500-510. doi: 10.1016/j.annonc.2022.02.007

47. Garcia-Murillas I, Chopra N, Comino-Méndez I, et al. Assessment of molecular relapse detection in early-stage breast cancer. JAMA Oncol. 2019;5(10):1473-1478. doi: 10.1001/jamaoncol.2019.1838

48. Swanton C. Intratumour heterogeneity: Evolution through space and time. Cancer Res. 2013;72(19):4875-4882. doi: 10.1158/0008-5472.CAN-12-2217

49. Jamal-Hanjani M, Wilson GA, Horswell S, et al. Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer. Ann Oncol. 2016;27(5):862-867. doi: 10.1093/annonc/mdw037

50. Murtaza M, Dawson SJ, Pogrebniak K, et al. Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer. Nat Commun. 2015;6:8760. doi: 10.1038/ncomms9760

51. Lin KK, Harrell MI, Oza AM, et al. BRCA reversion mutations in circulating tumor DNA predict primary and acquired resistance to the PARP inhibitor rucaparib in high-grade ovarian carcinoma. Cancer Discov. 2019;9(2):210-219. doi: 10.1158/2159-8290.CD-18-0715

52. Roussel-Simonin C, Blanc-Durand F, Tang R, et al. Homologous recombination deficiency (HRD) testing on cell-free tumor DNA from peritoneal fluid. Mol Cancer. 2023;22(1):178. doi: 10.1186/s12943-023-01864-1

53. Abbosh C, Frankell AM, Harrison T, et al. Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA. Nature. 2023;616(7957):553-562. doi: 10.1038/s41586-023-05776-4

54. Patch AM, Christie EL, Etemadmoghadam D, et al. Whole- genome characterization of chemoresistant ovarian cancer. Nature. 2015;521(7553):489-494. doi: 10.1038/nature14410

55. Oikkonen J, Zhang K, Salminen L, et al. Prospective longitudinal ctDNA workflow reveals clinically actionable alterations in ovarian cancer. JCO Precis Oncol. 2019;3:1-12. doi: 10.1200/PO.18.00343

56. Turnbull C, Wald N, Sullivan R, et al. GRAIL-Galleri: Why the special treatment? Lancet. 2024;403(10425):431-432. doi: 10.1016/S0140-6736(23)02830-1

57. Sasaroli D, Coukos G, Scholler N. Beyond CA125: The coming of age of ovarian cancer biomarkers. Biomark Med. 2009;3(3):275-288. doi: 10.2217/bmm.09.21

58. Heider K, Wan JCM, Gale D, et al. ctDNA Detection by Personalized Assays in Early-Stage NSCLC. medRxiv [Preprint]; 2021. doi: 10.1101/2021.06.01.21258171

59. Wan JCM, Eldarwi A, Diaz LA, Jr. RARE-seq: An inflection point in cfRNA liquid biopsy. Trends Pharmacol Sci. 2025;46(9):823-825. doi: 10.1016/j.tips.2025.08.001

60. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6(224):224ra24. doi: 10.1126/scitranslmed.3007094

61. Barbieri CE, Bangma CH, Bjartell A, et al. The mutational landscape of prostate cancer. Eur Urol. 2013;64(4):567-576. doi: 10.1016/j.eururo.2013.05.029

62. Yu J, Yang C, Zhu X, et al. A DNA alteration and methylation co-detection method for clinical purpose. EMBO Mol Med. 2025;17:1825-1841. doi: 10.1038/s44321-025-00259-7