The high mortality rate of OC can be largely attributed to the difficulties encountered in early detection [23]. Thus, the majority of patients with HGSOC are diagnosed at an advanced stage where the possibility of a cure is limited [24]. As a result, the early detection of OC appears critical in reducing mortality and morbidity. In clinical practice, serum cancer antigen 125 (CA125) and ultrasonography (US) are widely employed as diagnostic tools for OC but are not useful as screening tests [25]. Last, the UK Collaborative Trial of OC Screening (UKCTOCS) trial did not demonstrate a survival benefit using US and/or CA125 assessments for the early diagnosis of OC [26, 27]. OC is relatively rare with a prevalence of approximately 1% in the population. For this reason, an effective and acceptable screening test should achieve a high sensitivity (> 75%) and very high specificity (99.6%) for early-stage disease [28].

In recent years, liquid biopsy has been investigated as a strategy for early detection of OC. The idea of exploiting liquid biopsy as a non-invasive screening tool is attractive and is based on the observation that cancer patients display higher levels of plasma cfDNA compared to healthy controls [29]. Some authors showed higher cfDNA concentration in patients with OC than in healthy women, and that the quantitative detection of cfDNA could help to discriminate malignant OC from benign ovarian disease [30–33]. While some studies have suggested that the quantitative detection of cfDNA may be more sensitive and specific than conventional tumor markers, a meta-analysis of nine studies showed an acceptable specificity (90%) but an unsatisfactory sensitivity (70%) for the diagnosis of OC [34]. This finding can be explained by the low sensitivity of cfDNA for the detection of early-stage malignancies, which are supposed to be a relevant target of screening programs.

Other studies focused on qualitative changes in ctDNA, including somatic mutations, methylation, and chromosomal aberrations that can be detected with NGS or PCR-based assays. TP53 mutation is highly prevalent in HGSOC, accounting for more than 95% of somatic mutations [35]. Data from several studies indicate a high level of concordance between the detection of TP53 mutation in blood and tissue samples, suggesting its potential role in the diagnosis of OC [36–39]. Calapre et al. [40] demonstrated a concordance rate varying from 60% to 100% depending on the type of NGS panel used.

Several studies have suggested the diagnostic potential of promoter methylation that leads to epigenetic inactivation of tumor suppressor genes [e.g., opioid binding protein/cell adhesion molecule-like (OPCML), slit homologue 2 (SLIT2)] as an early event during OC pathogenesis [41, 42]. Assessment of ctDNA methylation status in plasma samples from OC patients before surgery reported a significant association with abnormal methylation of tumor suppressor genes compared to healthy controls [43–45]. Widschwendter et al. [46] analyzed the methylation status of three epigenetic markers [collagen type XXIII alpha 1 chain (COL23A1), c2 calcium-dependent domain-containing protein 4D (C2CD4D), and wingless-type MMTV integration site family, member 6 (WNT6)] in the early screening setting. This analysis was able to identify OC up to two years before clinical diagnosis with a sensitivity of 58% and a specificity of 88% [46].

Chromosomal instability is another hallmark of OC, linked at least in part, by the early onset of TP53 mutations [35]. Vanderstichele et al. [47] used WGS with low coverage to identify chromosomal instability in cfDNA and differentiated patients with OC from healthy controls with 84% sensitivity and 91% specificity.

An approach involving the analysis of body fluids other than blood might help by increasing the sensitivity of tumor DNA detection. For instance, a tumor-specific liquid biopsy as uterine cavity lavage could provide higher fractions of tumor material [48, 49].

Cohen et al. [50] analyzed circulating protein biomarkers and mutations in cfDNA using a multiomics strategy with a commercial blood test called CancerSEEK. This test used 61 amplicons for massively parallel sequencing to increase sensitivity while maintaining specificity. The sensitivity was 98% in patients with OC, but the detection rate was only 38% in the early stages [50].

Despite the potential application of liquid biopsy in the early diagnosis of OC, it’s far too early to claim its prime time in the clinic. Data currently available in this setting show highly divergent detection rates, small series of patients, significant variability in terminology, timing of sampling, and methodology of analyses, as well as in the histological subtypes of OC detected.

Although most OC patients achieve complete remission after first-line treatment, up to 70% of patients relapse within two years. CA125 and computed tomography (CT) scans are usually used during follow-up. Although CA125 is routinely applied in clinical practice as an OC biomarker, its clinical value is uncertain due to low sensitivity and specificity. In fact, half of the patients with normal CA125 have persistent disease. Additionally, CA125 might be elevated in benign conditions (e.g., endometriosis or pelvic inflammatory disease), limiting its utility in OC follow-up [51–53].

Increasing evidence from other malignancies supports the clinical application of liquid biopsy to define the risk of recurrence and its earlier detection. ctDNA analysis may serve these purposes by identifying the presence of persisting occult disease after initial treatment, likely responsible for disease relapse and poor prognosis [54–56]. Furthermore, a longitudinal collection of liquid biopsies during follow-up would identify early ctDNA changes resulting in therapeutic anticipation compared to traditional imaging-based follow-up.

In OC evidence suggested that ctDNA analysis could anticipate the detection of relapse in comparison to CA125 and radiological images such as CT and positron emission tomography (PET) scans [57–60].

Pereira et al. [57] used PCR and targeted sequencing to quantify ctDNA levels following surgery in 22 patients with HGSOC. Undetectable levels of ctDNA after adjuvant treatment were associated with significantly improved survival outcomes. The authors also showed that the detection of ctDNA anticipated the diagnosis of recurrence by approximately 7 months compared to CT imaging [57]. Similarly, in a cohort of 48 patients with HGSOC, approximately 80% of patients without residual disease after surgery had detectable ctDNA. Despite being not statistically significant, patients with detectable ctDNA had shorter overall survival [61]. Minato et al. [58] detected ctDNA in all patients (6/11) with recurrent OC using droplet digital PCR, while no ctDNA was detected in the plasma of recurrence-free patients (5/11). In 5/6 cases, the appearance of the ctDNA preceded the increase of the CA125 [58]. Paracchini et al. [59] recently used shallow WGS to calculate the percentage of tumor fraction (TF) in samples from 46 HGSOC patients. In this study, TF calculated at the time of diagnosis was an independent prognostic marker of relapse. Moreover, in longitudinal monitoring, the increase of TF preceded the CA125 by almost 8 months in the detection of disease progression [59].

These studies, although limited by their retrospective nature, small sample sizes, and lack of an independent validation cohort, suggest a potential utility of ctDNA as a predictor of OC recurrence [57–60]. Nevertheless, therapeutic options for recurrent disease are currently limited, and early detection of recurrence may not always impact survival [62]. The clinical value of an early detection of tumor relapse and the earlier start of treatment should be evaluated in well-designed prospective studies using ctDNA detection as a marker of disease recurrence.

Currently, radiological imaging remains the gold-standard to evaluate response to treatment. OC is often associated with peritoneal disease, which is usually non-measurable or hardly measurable by CT scan [63]. The serum marker CA125 has also been used to monitor treatment response, with some disadvantages, such as long half-life and poor tumor-specificity.

To date, evidence of a potential utility of serial ctDNA monitoring to predict response to anticancer treatment in OC is far preliminary. In a retrospective study of 40 patients with relapsed OC, the TP53 mutant allele frequency (TP53MAF) has been qctDNA before, during, and after chemotherapy. Pre-treatment ctDNA TP53MAF concentration correlated with initial tumor volume better than the CA125 value. Of note, patients with a > 60% decrease in TP53MAF after one cycle of chemotherapy had a longer time to progression compared to those with a decrease of less than 60% [41]. Similarly, a study showed that TP53MAF in ctDNA was significantly reduced in 28 patients with OC treated with chemotherapy, but the correlation with clinical outcomes was not reported [39]. These studies also indicate that changes in ctDNA TP53MAF could provide some indications when monitoring treatment response.

Noguchi et al. [64] retrospectively compared the tumor mutation burden in plasma samples utilizing CAPP-seq for plasma ctDNA in 10 patients with OC treated with neoadjuvant chemotherapy (6 chemo-sensitive and 4 chemo-resistant). They found that in 5 responder patients, the variant allele frequency (VAF) of non-synonymous mutations decreased after chemotherapy while it increased in 2 resistant cases, and new mutations emerged following chemotherapy [64]. In a prospective study with cfDNA samples collected from 12 HGSOC patients before, during, and after platinum-based chemotherapy, clinically actionable mutations were detected in 7 (58%) patients. Consistently with previously described studies, the authors observed that responders had a higher number of mutations with decreasing VAF when compared with poor responders. Furthermore, the authors emphasize that in a patient with platinum-resistant disease, trastuzumab was used as treatment following the detection of Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)amplification in ctDNA, leading to a radiological and biochemical response [65].

Rusan et al. [66] assessed the potential role as a biomarker of homeobox A9 (HOXA9) promoter methylation in ctDNA (meth-ctDNA) in patients with platinum-resistant BRCA-mutated OC, treated with a PARP inhibitor. The best clinical outcome was observed in patients with detectable HOXA9 meth-ctDNA at baseline, but subsequent undetectable levels [66].

While further studies are necessary to define the optimal timing of sample collection and the most suitable and cost-effective assay for ctDNA measurement, serial ctDNA monitoring has the potential to complement the information available from routine imaging-based disease assessments. Identifying progression prior to the development of clinically evident disease might justify to discontinue treatments that are not providing benefit to the patients, sparing them from clinical and financial toxicity. Moreover, serial monitoring of ctDNA could be used as an early predictor of treatment efficacy to facilitate adaptive clinical trial strategies.

The emergence of platinum-resistant disease is one of the major challenges in the management of OC.

Intratumor heterogeneity and adaptability of the OC genome under the selective pressure of chemotherapy represent one of the main reasons for drug resistance and treatment failure [67, 68]. While a “real-time” molecular characterization of recurrent disease would be crucial for intercepting actionable genetic vulnerabilities or drug resistance mechanisms, longitudinal acquisition of multiple tissue biopsies is clinically impracticable and too invasive for often heavily pre-treated patients. In this scenario, the use of liquid biopsy is a promising option to overcome this issue, allowing for monitoring of tumor evolution over time. Indeed, serial profiling of ctDNA can provide a more comprehensive picture of the tumor heterogeneity, by capturing the dynamic changes in the mutational landscape that may occur under the selective pressure of anticancer treatments [69].

In addition to blood, tumor material can also be searched in ascites [70]. In fact, many patients with recurrent OC present with malignant ascites, and it is not unusual in the clinic that large volumes need to be drained, often repeatedly, to relieve abdominal distension. Sampling ascites might allow for a more comprehensive assessment of the mutational profile at the time of each recurrence or progression [71, 72].

Nearly half of HGSOCs harbor defects in HR, which is a biomarker of platinum-based chemotherapies and PARP inhibitors sensitivity [73]. In the last decade, the introduction of PARP inhibitors has revolutionized the treatment landscape of OC in both first line and relapsed settings [7, 8, 74, 75]. However, acquired mutations that confer resistance to PARP inhibitors have been reported in some patients after long-term exposure to treatment [76, 77].

Germline or somatic pathogenic mutations in BRCA1 or BRCA2 are the best described mechanism of HR deficiency [78]. Accordingly, the most readily conceivable mechanism of PARP inhibitor resistance is the acquisition of reversion mutations, that restore HR function [79–81].

Recent studies have shown the feasibility to detect reversion mutations by cfDNA analysis suggesting its potential clinical use [81–83]. Christie et al. [82] conducted a prospective study in 30 patients with HGSOC carrying a germline BRCA1/2 mutation and detected BRCA1/2 reversion mutations in the tumor in 31.3% of patients treated in the recurrent setting, among which 18.8% also had detectable BRCA1/2 reversions in cfDNA [82].

In a recent larger study, NGS analysis was performed on cfDNA from plasma collected prior to rucaparib treatment in 112 patients with germline or somatic BRCA-mutant HGSOC enrolled in the ARIEL2 trial [81]. BRCA reversion mutations in cfDNA were found in 18% (2/11) of platinum-refractory and 13% (5/38) of platinum-resistant patients, compared with 2% (1/48) of platinum-sensitive patients. Furthermore, patients without BRCA reversion mutations detected in the pre-treatment cfDNA had significantly longer progression-ree survival (PFS) on rucaparib than those with reversion mutations. In addition, the authors also sequenced 78 post-progression cfDNA samples to examine acquired resistance and identified an additional 8 patients with novel BRCA reversion mutations not found in pre-treatment cfDNA, suggesting the ability of cfDNA to monitor dynamic changes in BRCA mutational status over time [81].

Based on these studies, cfDNA sequencing was able to detect reversion mutations, but it was a relatively rare event and thus, liquid biopsy could also be used to identify unknown mechanisms of treatment resistance.

To explore the mutation profile associated with PARP inhibitor resistance, Hu et al. [84] analyzed the cfDNA pre- and post-treatment in 25 patients with platinum-sensitive HGSOC receiving maintenance with olaparib. An increased somatic mutation load in post-treatment samples was detected and it was predictive of poor prognosis. Additionally, patients with MRE11A (a gene involved in the DNA damage repair response) mutations in post-treatment samples had shorter PFS compared to patients without this mutation. Newly acquired mutations in MRE11A were associated with disease progression or resistance to ongoing treatment in more than 90% (12/13) of cases [84].

Understanding the mechanism of treatment resistance is warranted to develop novel therapeutic agents and combination, and to improve patient’s outcomes. Even though the level of evidence in OC is rather preliminary, routine monitoring through liquid biopsy could lead to earlier detection and characterization of drug resistance that may potentially create the opportunity for therapeutic interventions before clinical evident progression. Future clinical trials should implement trial designs that include longitudinal blood sampling at diagnosis, during-treatment, and at the time of each recurrence or progression.