Ovarian cancer causes the majority of cancer-related deaths from gynecological cancers and represents the third most frequent gynecological cancer worldwide . Epithelial ovarian cancer is the main type, characterized by histological and molecular heterogeneity and is considered as a highly aggressive neoplasia. It is often diagnosed at an advanced stage and little progress has been achieved in standard chemotherapy treatment and overall survival (OS) during the last 3 decades . Primary disease is treated with surgical removal of the tumor, followed by standard adjuvant chemotherapy, a combination of platinum and taxane-based treatment , . However, in more than half of the cases, chemoresistance and recurrent disease are observed , . New therapeutic concepts now include targeted therapy applying bevacizumab or the PARP inhibitor olaparib in certain clinical situations , .
Metastasis in ovarian cancer occurs via two main routes characterized by different molecular mechanisms, the transcoelomic passive dissemination of tumor spheroids in the peritoneal fluid and ascites, and the hematogenous metastasis of cancer cells in blood circulation and their preferred seeding to the omentum. Circulating tumor cells (CTCs) contribute to the hematogenous metastatic route , . Generally, in solid malignancies, CTCs are exceedingly rare, and in most cases, the amount of the available peripheral blood sample is limited. The development of different analytical systems for the detection, enumeration and molecular characterization of CTCs has expanded the field of liquid biopsy, providing information on patients clinical outcome and treatment efficacy .
Cell-free DNA (cfDNA) circulates at high concentrations in peripheral blood of cancer patients and can be used for the detection of several molecular alterations related to cancer development . Circulating tumor DNA (ctDNA) represents a small percentage of cfDNA that is shed in circulation by tumor cells and carries all these molecular alterations including tumor specific mutations, microsatellite instability (MI) , loss of heterozygosity (LOH) , and DNA methylation . ctDNA is a very promising non-invasive diagnostic, prognostic and predictive tool, as it provides an easily accessible source of DNA derived from the tumor .
In this review, we will give an overview of the published data on CTCs and ctDNA in ovarian cancer (Figure 1). We also focus on the possible role of liquid biopsy approaches in early diagnosis, prognosis of clinical outcome and the prediction of chemotherapy response or the development of chemoresistance in ovarian cancer patients.
Circulating tumor cells (CTCs)
Recent studies attempted to show the prognostic and predictive value of CTCs as tumor biomarkers in patients with ovarian cancer , and three meta-analyses report these associations using the appropriate methods for the results statistical analysis , , . Different detection methods were used, mainly based on immunocytochemistry (microscopic detection or the FDA-approved CellSearch® system), RT-PCR (AdnaTest, QIAGEN, Hilden, Germany), and RT-qPCR for the quantification of CTCs levels , . The time point of blood collection also differed, however, in the majority of studies the peripheral blood samples were obtained before surgical removal of the tumor. An overview of all research studies on CTCs in ovarian cancer patients is presented in Table 1.
CTCs in ovarian cancer.
|Author||Year||Sampling time||Ovarian cancer patients||CTCs isolation||CTCs detection||Targeted antigen/gene||OS||PFS|
|Chebouti et al. ||2016||Before surgery (BS) and after chemotherapy (AC)||65||AdnaTest Ovarian CancerSelect||AdnaTest Ovarian CancerDetect/RT-PCR||EpCAM, MUC1, MUC16, ERCC1||p=0.0008 (AC)||p=0.0293 (AC)|
|Blassl et al. ||2016||Before surgery||10 (3 pts: single cell analysis)||AdnaTest Ovarian CancerSelect/ AdnaTest EMT-1/StemCellSelect||Multiplex-RT-PCR/AdnaTest Ovarian CancerDetect||Three multi-marker panels for epithelial, EMT and stem cells associated transcipts||NR||NR|
|Kolostova et al. ||2016||Before surgery and longitudinally||56||Size-based (MetaCell®)||Cytomorphological/qPCR||12 gene panel including: EpCAM, MUC1, MUC16, CK18,19, ERCC1||NR||NR|
|Kolostova et al. ||2015||Before surgery||118 (20 pts: gene expression study)||Size-based (MetaCell®)||Cytomorphological/qPCR||EpCAM, MUC1, MUC16, CK18,19||NR||NR|
|Pearl et al. ||2015||Before surgery, before chemotherapy and during a 24 months follow-up||123 (31 pts: monitoring study)||Immunomagnetic CAM+method||ICC||EpCAM, ESA, CA125, DPP4||NR||p<0.00001|
|Pearl et al. ||2014||Before surgery||76||Immunomagnetic CAM+method||ICC||EpCAM, ESA, CA125, DPP4||p=0.0219||p=0.0024|
|Kuhlmann et al. ||2014||Before surgery||143||AdnaTest Ovarian CancerSelect||RT-PCR (AdnaTest)||EpCAM, MUC1, MUC16, ERCC1||p=0.026||p=0.009|
|Liu et al. ||2013||Serial measurements during chemotherapy||78||CellSearch®||CellSearch®||EpCAM, CK8,18,19||NS||NS|
|Obermayr et al. ||2013||Before surgery and after chemotherapy||216||Density gradient centrifugation||RT-qPCR||12 gene panel including: PPIC, EpCAM||p=0.001 (AC)||p=0.001 (AC)|
|Behbakht et al. ||2011||Before and after temsirolimus||54||CellSearch®||CellSearch®||EpCAM, CK8,18,19, M30||NS||NS|
|Aktas et al. ||2011||Before surgery and/or after chemotherapy||122||AdnaTest||RT-PCR (AdnaTest)||EpCAM, MUC1, HER2, CA125||p=0.0054 (BS)|
|Poveda et al. ||2011||After first line chemotherapy||216||CellSearch®||CellSearch®||EpCAM, CK8,18,19||p=0.0017||p=0.0024|
|Fan et al. ||2009||Before surgery||58||Immunomagnetic CAM+method||ICC||EpCAM, ESA, CK4,5,6,8,10,13,18||NS||p=0.042|
|Judson et al. ||2003||Before surgery||53||Immunomagnetic microbeads||ICC||CK7,8,18,20, TFS-2, EGFR||NS||NS|
|Marth et al. ||2002||After surgery/before chemotherapy||90||Immunomagnetic (Dynabeads)||Immunomagnetic beads||MOC-31||NS||NS|
The first studies on CTCs in ovarian cancer were based on the detection of CTCs using specific immunobeads  and an immunocytochemical (ICC) assay , respectively. Marth et al.  found carcinoma cells in the peripheral blood in 12% of ovarian cancer patients with a median follow-up of 25 months. The blood collection took place 7–20 days after surgery and before adjuvant chemotherapy. Judson et al.  detected CTCs in 18.7% of ovarian cancer patients with 18.7 months of a median follow-up time. They observed that most women with CTCs had grade 3 primary ovarian tumor compared to women without CTCs, and this evidence was significantly different. Both studies reported no significant association between the presence of CTCs in the peripheral blood and the clinical outcome of ovarian cancer patients , .
Fan et al.  first reported the prognostic significance of CTCs in primary ovarian cancer. They developed a new method for the detection of CTCs based on the ability of cancer cells to invade and ingest a cell adhesion matrix (CAM). In this study, CTC detection was based on ICC using the epithelial markers epithelial cell adhesion molecule (EpCAM), epithelial specific antigen (ESA) and a panel of seven pan-cytokeratins. They reported that the CAM+CTCs were invasive and their presence significantly correlated with decreased progression-free survival(PFS) (p=0.042) . The same group evaluated the prognostic significance of CTCs in a group of 129 pre-surgery ovarian cancer patients using the same method for the detection and identification of CTCs and observed statistically significant association between the presence of CTCs and both OS (p=0.0219) and PFS (p=0.0024) . The same group also investigated the predictive value of CTC levels in a small group of 31 ovarian cancer patients that received standard taxol/carboplatin chemotherapy, where blood specimens were obtained at different time points, before and after surgery and up to 24 months after chemotherapy treatment. Using the same assay , they showed a statistically significant association between CTC levels and disease progression .
Aktas et al. investigated the prognostic value of CTCs in a large cohort of 122 ovarian cancer patients, before surgery and/or after platinum-based chemotherapy. They used the commercially available AdnaTest BreastCancer (QIAGEN, Hilden, Germany), for the isolation and detection of CTCs. AdnaTest BreastCancer is based on immunomagnetic enrichment targeting EpCAM and anti-mucin 1 (MUC1), followed by multiplex RT-PCR for EpCAM, MUC1 and human growth factor receptor 2 (HER2/neu). CA-125 transcripts were also analyzed using a singleplex RT-PCR. CTCs were detected in 19% of patients before surgery and in 27% after platinum-based chemotherapy. According to their findings, the presence of CTCs significantly correlated with shorter OS before surgery (p=0.0054) and after chemotherapy (p=0.047) . In a more recent study, Kuhlmann et al. investigated the predictive value of ERCC1-positive CTCs in 143 pre-surgery epithelial ovarian cancer patients. AdnaTest OvarianCancerSelect (QIAGEN, Hilden, Germany) was used for the immunomagnetic tumor cell enrichment in blood samples and AdnaTest OvarianCancerDetect (QIAGEN, Hilden, Germany) for the molecular characterization of CTCs. ERCC1 transcript detection was performed using singleplex RT-PCR. The presence of CTCs was confirmed in 14% of patients and was significantly correlated with OS (p=0.041). ERCC1-positive CTCs (ERCC1+CTC) were detected in 8% of patients and significantly correlated with both OS (p=0.026) and PFS (p=0.009). A very interesting finding in this study was the association of ERCC1+CTC with platinum resistance. The presence of ERCC1+CTC at primary diagnosis independently predicted platinum resistance (p=0.010), although the ICC analysis of ERCC1 expression in primary tumor tissue did not reveal any prognostic or predictive value . In their very recently published study, they were able to show that the additional assessment of ERCC1-transcripts enhances overall CTC detection rate in ovarian cancer patients before surgery and after chemotherapy and defines an additional highly overlapping fraction of ERCC1-expressing CTCs, which is potentially selected by platinum-based chemotherapy. Moreover, we describe that the assessment of CTC-derived ERCC1-transcripts alone is almost equivalently sufficient in detecting ERCC1-expressing prognostic relevant CTCs. We further showed that the presence of ERCC1+CTCs after chemotherapy correlates with post-therapeutic outcome of ovarian cancer and particularly, dynamics of ERCC1+CTCs mirror response to platinum-based chemotherapy .
Poveda et al.  also confirmed the prognostic impact of CTC detection in ovarian cancer after chemotherapy. They reported a correlation of CTC numbers with shorter OS (p=0.0017) and PFS (p=0.0024) in a phase III clinical trial (NCT00113607, www.clinicaltrials.gov) of pegylated liposomal doxorubicin (PLD) with trabectedin versus PLD for relapsed ovarian cancer. They used for the first time the CellSearch® system (Janssen Diagnostics) for CTC isolation and enumeration in 216 ovarian cancer patients. Behbakht et al. also used the CellSearch® system for CTC enrichment and enumeration in a phase II clinical trial (NCT00429793, www.clinicaltrials.gov) for the evaluation of the efficacy of the mTOR inhibitor temsirolimus. Fifty four recurrent ovarian cancer patients were recruited and blood specimens were obtained before and after treatment with temsirolimus. No significant association between the presence of CTCs with PFS and OS was reported . Liu et al.  also used the CellSearch® system in 78 newly diagnosed and recurrent ovarian cancer patients. They performed serial measurements during chemotherapy, but according to their findings, the number of CTCs did not correlate with PFS or OS.
Obermayr et al.  developed a six-marker gene panel for the molecular detection of CTCs on female cancer patients, including ovarian cancer, using a RT-qPCR platform. The multimarker analysis using this novel panel positively identified 19% of the 23 ovarian cancer patients. The same group aimed to identify novel markers for the characterization of CTCs in ovarian cancer, using a density gradient centrifugation-based method for the isolation and RT-qPCR for CTC detection and quantification. They defined a sample as CTC positive if at least one of the 11 gene marker panels was found over-expressed. By using this gene panel, they detected CTCs in 24.3% of the baseline (before primary treatment) and 20.4% of the follow-up (6 months after chemotherapy) samples. In two-thirds of the patients, cyclophilin C gene (PPIC) overexpression was observed, but only a few samples were identified by EpCAM overexpression. PPIC-positive CTCs during follow-up were detected significantly more often in platinum-resistant than platinum-sensitive follow-up patients. This fact also indicated poor outcome independently from other prognostic parameters .
Kolostova et al.  developed a novel size-based method (MetaCell®, MetaCell s.r.o., Ostrava, Czech Republic) for the enrichment and separation of viable CTCs, followed by in vitro CTCs culturing and cytomorphological analysis and finally, CTC molecular characterization by gene expression studies using qPCR. They isolated and cultivated CTCs in 77 (65.2%) of 118 pre-surgery advanced-stage ovarian cancer patients. Gene expression analysis was performed in 20 selected positive samples by cytomorphological analysis. They looked at possible associations between CTC presence and clinicopathological characteristics of the patients, mainly with the CA-125 status. Based on their results, they proposed a new and independent prognosis staging information. They also suggest that hematogenous metastasis route is represented by CTCs and elevated CA-125 levels indicate lymphogenic dissemination . Using the same methodology, this group aimed to isolate and identify CTCs in 56 ovarian cancer patients. In this study, gene expression analysis was performed in all samples found positive by cytomorphological analysis. They reported that EpCAM relative expression is elevated in CTC-enriched fractions compared to whole peripheral blood sample and that this expression grows with in vitro cultivation time. They suggested that a seven-gene panel, including EpCAM and MUC16, could better confirm the presence of CTCs in peripheral blood of ovarian cancer patients, than a one-marker test . Both studies did not provide any information on the patients clinical outcome with regard to OS and/or PFS data , .
A very recent study on CTCs in ovarian cancer proposed a multi-marker gene panel for gene expression profiling of single CTCs . Blassl et al. used the AdnaTest OvarianCancerSelect (QIAGEN, Hilden, Germany) and/or the AdnaTest EMT-1/StemCellSelect (QIAGEN, Hilden, Germany) for CTC isolation and enrichment in peripheral blood samples of 10 pre-surgery epithelial ovarian cancer patients. CTCs were detected and characterized by using the AdnaTest OvarianCancerDetect (QIAGEN, Hilden, Germany) and the AdnaTest EMT-1/StemCellDetect. They isolated single cells using CellCelector (ALS GmbH, Jena, Germany) from only three ovarian cancer patients. Single CTCs were characterized by multiplex-RT-PCR, followed by capillary electrophoresis. The multiplex-RT-PCR gene panel included stem cell (CD44, ALDH1A1, Nanog, Oct 4) and EMT (N-cadherin, Vimentin, Snail2, CD117, CD146) markers. They observed inter-cellular and intra/inter-patient heterogeneity and co-expression of epithelial, mesenchymal and stem cell transcripts on the same CTC simultaneously .
Cell-free DNA (cfDNA)
A sufficient number of studies on cfDNA in patients with ovarian cancer pursued to clarify its clinical value . For this purpose, they quantified total cfDNA and/or the circulating cell-free mitochondrial DNA (mtDNA) levels in some cases, or aimed at the detection of different genetic and epigenetic alterations, such as chromosomal abnormalities and specific tumor LOH, cancer-related somatic gene mutations and aberrant DNA methylation. Additionally, in a recent case study, Martignetti et al.  detected the FGFR2-FAM76A tumor-specific fusion in cfDNA of an advanced stage serous epithelial ovarian cancer patient.
However, in some cases, the results are still controversial. The discrepancies probably occur due to the different methods and pre-analytical conditions, the use of serum instead of plasma by some researchers and the different volumes of plasma/serum for cfDNA extraction. Many studies focused on the potential use of cfDNA as a diagnostic, prognostic and predictive biomarker in ovarian cancer and a recent meta-analysis by Zhou et al. attempted to evaluate the role of cfDNA in ovarian cancer diagnosis . An overview of the research studies on cfDNA in ovarian cancer is summarized in Table 2.
cfDNA in ovarian cancer.
|cfDNA||Author||Year||Source||Ovarian cancer patients||Targeted gene||Early detection||Prognosis||Response to treatment|
|DNA amount (cfDNA)||Kamat et al. ||2006||Plasma||19||GADPH, β-actin, β-globin||Yes|
|Capizzi et al. ||2008||Plasma||22||hTERT||Yes|
|Kamat et al. ||2010||Plasma||164||GADPH, β-actin||Yes|
|No et al. ||2012||Serum||36||B2M, RAB25, CLDN4, ABCF2||Yes|
|Steffensen et al. ||2014||Plasma||144||Cyclophilin A||Yes|
|Shao et al. ||2015||Serum||36||NR (bDNA technique)||Yes|
|Mitochondrial (mtDNA)||Zachariah et al. ||2008||Plasma/serum||21||MTATP8||Yes|
|Choudhuri et al. ||2014||Plasma||100 (20 follow-up)||MTATP8||Yes|
|Chromosomal abnormalities/LOH (ctDNA)||Kuhlmann et al. ||2012||Serum||63||-||Yes|
|Harris et al. ||2016||Plasma||10||-||NR||NR||NR|
|Cohen et al. ||2016||Plasma||32||-||Yes|
|Vanderstichele et al. ||2016||Plasma||57||-||Yes|
|Somatic mutations (ctDNA)||Otsuka et al. ||2004||Plasma||27||TP53||Yes|
|Swisher et al. ||2005||Plasma/serum||69||TP53||Yes|
|Dobrzycka et al. ||2011||Plasma||126||KRAS||Yes|
|Forshew et al. ||2012||Plasma||46||TP53, PTEN, EGFR, BRAF, KRAS, PIK3CA||Yes|
|Murtaza et al. ||2013||Plasma||3||RB1, ZEB2, MTOR, CES4A, BUB1, PARP8||Yes|
|Bettegowda et al. ||2014||Plasma||7||Panels including: TP53, PIK3CA, BRAF, EGFR||Yes||Yes|
|Pereira et al. ||2015||Serum||22||Panels including: TP53, PIK3CA, MET, PTEN, KRAS, BRAF, FBXW7||Yes|
|Aberrant methylation (ctDNA)||Gifford et al. ||2004||Plasma||138||hMLH1||Yes|
|Ibanez et al. ||2004||Plasma/serum||50||BRCA1, RASSF1A||Yes|
|Melnikov et al. ||2009||Plasma||33||BRCA1, HIC1, PAX5, PGR-PROX, THBS1||Yes|
|Liggett et al. ||2011||Plasma||30||RASSF1A, CALCA, EP300, PGR-PROX, BRCA1, CDKN1C||Yes|
|Bondurant et al. ||2011||Serum||106||RASSF1A||Yes|
|Giannopoulou et al. ||2017||Plasma||59||RASSF1A||Yes|
|Dong et al. ||2012||Serum||36||SLIT2||Yes|
|Zhang et al. ||2013||Serum||87||APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5, OPCML||Yes|
|Wu et al. ||2014||Plasma||47||RASSF2A||Yes|
|Zhou et al. ||2014||Serum||45||OPCML||Yes|
|Wang et al. ||2015||Serum||114||RUNX3, TFPI2, OPCML||Yes|
The first studies on ovarian cancer circulating DNA attempted to quantify the total cfDNA amount, or the nuclear and mitochondrial DNA amounts separately, in plasma or serum of ovarian cancer patients. One of the first studies on cfDNA in ovarian cancer screening aimed to quantify plasma cfDNA using a real-time PCR assay for three reference genes and to determine the number of genome equivalents (GE) using a standard curve. Kamat et al.  reported that cfDNA levels in advanced ovarian cancer samples were elevated when compared to controls. A more recent study on ovarian cancer screening using cfDNA quantification showed a significant increase in serum cfDNA of advanced stage ovarian cancer patients compared to early stage (p<0.01). Shao et al.  also reported a correlation between serum cfDNA levels and ovarian cancer occurrence using receiver operating characteristic (ROC) curves and a branched DNA (bDNA) technique for cfDNA quantification.
Kamat et al. also investigated the prognostic value of cfDNA in epithelial ovarian cancer. They quantified plasma cfDNA levels in 164 epithelial ovarian cancer patients using real-time PCR for β-globin and determined the number of GE. They reported a significant association of cfDNA>22,000 GE/mL with decreased PFS (p<0.001) and this association was shown as an independent prognostic value (p=0.02) after adjusting for other clinical characteristics . On the contrary, No et al.  examined the prognostic value of cfDNA and reported no significant difference between cfDNA levels of cancer patients and patients with benign disease. They recruited 36 epithelial ovarian cancer samples and 16 benign tumor samples and used commercially available copy number assay kits to measure cfDNA levels of four selected genes, but they used serum as cfDNA source instead of plasma.
In a more recent study, Steffensen et al. measured plasma cfDNA levels of 144 multiresistant epithelial ovarian cancer patients treated with bevacizumab using real-time PCR for cyclophiline A gene. They found a statistically significant correlation between cfDNA levels and both PFS (p=0.0004) and OS (p=0.005) in both univariate and multivariate survival analyses. Thus, they concluded that plasma cfDNA is an independent prognostic factor in platinum-resistant ovarian cancer patients treated with bevacizumab .
Ten years ago, Kamat et al.  proposed the potential use of tumor-specific cfDNA levels in predicting tumor response to chemotherapy, by using an orthotopic mouse model. Capizzi et al. further investigated the predictive value of cfDNA in ovarian cancer patients. They quantified plasma cfDNA levels before and after chemotherapy in 22 epithelial ovarian cancer patients of a prospective nonrandomized clinical study and found a significant discrimination between patients and healthy controls and a correlation of cfDNA amounts with response to standard chemotherapy .
Altered circulating cell-free mtDNA content may serve as a potential cancer biomarker in many solid malignancies . In ovarian cancer, only two studies include the determination of circulating cell-free mtDNA levels. Zachariah et al. quantified nuclear cfDNA and circulating cell-free mtDNA levels using a multiplex qPCR assay, in serum and plasma of patients with epithelial ovarian cancer, benign epithelial tumors and endometriosis, and a healthy control group. They found a significant increase in nuclear cfDNA and circulating cell-free mtDNA amounts in ovarian cancer patients compared to both healthy group and benign epithelial tumor patients. Interestingly, they reported a significant difference between ovarian cancer patients and the endometriosis group circulating cell-free mtDNA, but not in nuclear cfDNA . More recently, Choudhuri et al. investigated whether nuclear cfDNA and circulating cell-free mtDNA levels can be used for advanced epithelial ovarian cancer diagnosis and for the prediction of treatment response. They recruited 100 patients and measured both levels before treatment, but in only 20 patients after the completion of chemotherapy. A significant difference was reported in nuclear cfDNA levels of the follow-up patients before and after treatment, but not in circulating cell-free mtDNA levels .
Circulating tumor DNA (ctDNA)
Circulating tumor DNA (ctDNA) constitutes a tiny subgroup of total cfDNA in the peripheral blood of cancer patients . The following studies refer on specific aberrations characterizing ctDNA shed in the circulation from the primary ovarian tumor. They are classified according to specific genetic or epigenetic alterations detected only in ctDNA, shown as below.
It is well known that ovarian cancer and in particular the high-grade serous ovarian cancer (HGSC) subtype, is characterized by frequent chromosomal instability . Recent studies aimed to detect copy number variations (CNV)  and to quantify specific LOH  or aberrant somatic chromosomal rearrangements  in ctDNA of ovarian cancer patients. Kuhlmann et al. quantified cfDNA of 63 primary epithelial ovarian cancer patients before surgery and after chemotherapy. They used a PCR-based fluorescence microsatellite analysis in order to measure the LOH in two fractions of cfDNA, the high- and low molecular-weight fraction (HMWF and LMWF, respectively). They reported that LOH at two markers can predict tumor grade (p=0.033) and FIGO stage (p=0.004) in the LMWF cfDNA. Remarkably, a LOH at another marker can significantly predict patients OS (p=0.030) in both HMWF and LMWF .
Harris et al. introduced an algorithm for the quantification of cfDNA using a qPCR assay in order to predict relapse and treatment efficacy. They identified aberrant chromosomal junctions in primary tumors of 10 ovarian cancer patients and detected them in plasma ctDNA of eight patients before surgery. In three cases, ctDNA was also detected after surgery, indicating the presence of the disease, but in the remaining five cases, ctDNA was absent after surgery, indicating the consequential absence of the disease .
The first study on ovarian cancer screening using CNV detection in cfDNA was elaborated by Cohen et al. . They applied a well-established non-invasive prenatal testing (NIPT) commercial platform in cfDNA of 16 pre-surgery early and 16 advanced HGSC patients. The obtained sequencing data were analyzed for the detection of subchromosomal changes and the determination of whole chromosome gains or losses. They detected 40.6% of all HGSC cases, and more specifically, 38% of early stages, indicating a potential utility for early HGSC screening in plasma cfDNA based on specific multiple segmental chromosome gains and losses . However, more validation studies along with the improvement of pre-analytical conditions and the examination of paired tumor DNA are needed before the routine application of this approach .
Vanderstichele et al. reported for the first time the potential of using cfDNA for primary HGSC diagnosis. They recruited 68 patients with an adnexal mass, including 57 diagnosed with invasive or borderline carcinoma and 11 with benign disease. They measured specific patterns of chromosomal instability in plasma cfDNA of all patients and reported a significantly higher quantitative measure of chromosomal instability in ovarian cancer patients compared to patients with benign disease or healthy individuals .
Few studies attempted to detect tumor-specific somatic mutations in ctDNA of epithelial ovarian cancer patients. Otsuka et al.  first identified TP53 mutations in only two/12 pre-surgery plasma cfDNA of patients with ovarian cancer. A tumor-specific TP53 mutation was also detected in 21 out of 69 cfDNA samples of epithelial ovarian cancer patients in a study by Swisher et al. The presence of ctDNA characterized by this mutation was significantly associated with decreased survival (p=0.02) . Mutations of KRAS gene were investigated by Dobrzycka et al. in plasma cfDNA of 126 epithelial ovarian cancer patients. They detected KRAS mutations in 43.7% of patients and reported a significantly decreased OS for patients with serous ovarian tumors and detectable cfDNA (p=0.022) .
The development of very sensitive novel technologies for ctDNA detection overcomes the issue of the extremely low concentrations of ctDNA out of the total cfDNA. Based on this concept, Forshew et al. proposed a different approach for the detection and identification of cancer-specific mutations in plasma ctDNA. They established a novel method for targeted deep sequencing (Tam-Seq) of mutations at low allele frequencies (AF) with increased sensitivity and specificity, and measured mainly the frequencies of TP53 mutant alleles at ctDNA of 46 advanced stage HGSC patients. Remarkably, an EGFR mutation was detected in one ctDNA sample but not in the initial ovarian tumor tissue. All results were confirmed using digital PCR .
Murtaza et al. performed whole exome sequencing in plasma ctDNA of three ovarian cancer patients. Serial sample measurements and quantification of allele fractions in ctDNA led to the identification of specific gene mutations related to acquired resistance to treatment. The genes with significantly increased mutant AFs are shown in Table 2. All results were confirmed using both digital PCR and Tam-Seq assay .
Another study by Bettegowda et al. accomplished the detection of ctDNA using digital PCR-based assays for mutation analyses in a large cohort of patients with different malignancies, including seven patients with advanced stage ovarian cancer. They detected ctDNA in most metastatic cancer patients and quantified the mutant fragments for the determination of cfDNA concentration. They reported a high mutant allele fragments (approximately 10,000 per 5 mL) for advanced ovarian cancer patients .
In a more recent study, Pereira et al. recruited patients with gynecological malignancies, including 22 ovarian patients, and identified specific cancer-related mutations using whole exome and targeted sequencing. They also measured and quantified ctDNA levels using droplet digital PCR (ddPCR). The detectable ctDNA after treatment significantly predicted survival for eight ovarian cancer patients, indicating a possible role of ctDNA measurements in personalized medicine .
Epigenetic alterations hold an important role in cancer initiation and progression and aberrant DNA methylation patterns, mainly characterized by promoter hypermethylation, are a frequent event in most human cancers . Epigenetic inactivation of a tumor suppressor gene often results from its promoter methylation and is considered as an early event during carcinogenesis . Many studies have reported methylation changes in ovarian cancer  and a recent review summarizes the differences in the observed methylation patterns in the main histological subtypes of the disease, including HGSC . DNA methylation changes have the potential to serve as biomarkers for early diagnosis of gynecological malignancies . This is also observed in Table 2; only one study by Gifford et al.  aimed to show the prognostic value of ctDNA methylation in ovarian cancer.
In this study, the researchers investigated hMLH1 methylation status in plasma cfDNA of 138 epithelial ovarian cancer patients enrolled in a phase III clinical trial (NCT00003998, www.clinicaltrials.gov), before carboplatin/taxoid chemotherapy and at relapse. They reported an increase in hMLH1 methylation at relapse and the remarkable presence of cfDNA methylation at 25% of relapse patients that was not detected before chemotherapy. This acquired methylation provided significant clinical information for patients OS (p=0.007) .
Ibanez et al. examined RASSF1A and BRCA1 hypermethylation in cfDNA of 50 epithelial ovarian cancer patients and first confirmed the detection of methylation in early stage (stage I, II) patients, using methylation specific PCR (MSP). They also observed a concordance between tumor and plasma/serum DNA methylation patterns in 82% of matched samples .
A microarray mediated methylation assay (MethDet test) was developed by Melnikov et al.  and its application in 33 serous ovarian cancer patients led to the characterization of a five genes panel for ovarian cancer detection. The same group used this assay in three cohorts of serous ovarian cancer patients, benign ovarian disease patients and healthy controls. Liggett et al.  now reported the distinctive promoter methylation of all three groups according to the methylation status of six selected genes.
A larger study by Bondurant et al. quantified RASSF1A promoter methylation in 106 serous ovarian cancer cfDNA samples, using a novel quantitative real-time PCR assay. They found RASSF1A promoter methylation in about half of ovarian cancer patients and observed agreement in the methylation status of 20 available paired tumor/serum samples. Interestingly, they measured RASSF1A methylation in nine patients over the course of treatment and found a concordance between cfDNA methylation changes and disease progression for eight patients, suggesting a possible role of cfDNA methylation in ovarian cancer prognosis .
Our group also reported RASSF1A promoter methylation in plasma ctDNA of 15/59 patients with high-grade serous ovarian cancer using a real-time MSP assay. We performed the first comparison study on RASSF1A promoter methylation in primary tumors, adjacent tissues and plasma samples in HGSC patients and we observed an agreement between primary tumor samples and corresponding plasma in 62.3% of cases studied .
Zhang et al. developed a multiplex-MSP assay for the early detection of ovarian cancer. They recruited 87 epithelial ovarian cancer patients and examined the serum cfDNA methylation status of seven selected genes simultaneously. A sample was characterized as positive, if at least one gene was found methylated . In a more recent study by Wang et al., a multiplex-nested MSP was also developed for the detection of three genes methylation in 114 serum cfDNA of epithelial ovarian cancer patients. cfDNA methylation levels were significantly increased in ovarian cancer patients compared to benign disease patients and healthy control groups .
Furthermore, studies on SLIT2 , OPCML  and RASSF2A  promoter methylation in cfDNA of epithelial ovarian cancer patients demonstrate the frequently aberrant methylation status of these genes and suggest a possible role for ovarian cancer early detection.
Methylation patterns in whole-blood DNA and white blood cell (WBC) DNA in ovarian cancer patients have been also examined using methylation arrays and bisulfite pyrosequencing. Teschendorff et al.  performed a methylation study in peripheral blood DNA of pre- and post-treatment ovarian cancer patients and they observed a significantly different methylation pattern in blood DNA of epithelial ovarian cancer patients compared to healthy controls. Flanagan et al.  investigated WBCs DNA methylation status in 880 epithelial ovarian cancer patients enrolled in a phase III clinical trial (NCT00003998, www.clinicaltrials.gov), using bisulfite pyrosequencing and reported a significant correlation between mean SFN methylation and PFS (p=0.016). The same group analyzed blood DNA methylation patterns in 247 ovarian cancer patients enrolled in the previous clinical trial. They identified specific CpGs alterations in blood DNA at relapse after platinum-based chemotherapy and found an independent significant association with survival (p=2.8×10−4) .
The development of a cancer biomarker and its implementation in the clinical routine requires a multistage procedure and constitutes the final result of multiannual and toilsome research approaches. However, multiple pre-analytical, analytical and post-analytical issues should be overcome and studies on the assay validations with regard to repeatability and reproducibility are also necessary . The lack of effective biomarkers for early detection, prognosis of clinical outcome and response to treatment contributes to the maintenance of low survival rates for ovarian cancer patients, despite the numerous research studies on the field, the last decades. Liquid biopsy procedures are minimally invasive and allow for the easily tolerated serial sample measurements during the course of treatment. This can help towards the establishment of more efficient personalized therapeutic algorithms and real-time therapy monitoring. Nevertheless, specific challenges should be taken into account for CTCs and ctDNA analyses, including pre-analytical issues about the sample volume, the proper tubes for sample collection, the samples storage and the time of the analysis, quality control and analytical validation of the assays.
The clinical significance of both CTCs and ctDNA has been revealed in many types of cancer , including ovarian cancer. However, no standard methods are used for the isolation and detection in the bloodstream and few studies recruited large cohorts of ovarian cancer patients. Further studies towards the validation, standardization and quality control of the assays used are a matter of utmost importance before the implementation of liquid biopsy approaches in the clinical routine.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
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