Cancer antigen 125 or carcinoma antigen 125, also known as MUC16, is a protein encoded by the MUC16 gene [73]. Clinically, it is used as a diagnostic test to measure the amount of the protein CA125 in the serum. In most laboratories, the normal value for CA125 is 0 to 35 units/mL.

Up to 80% of women diagnosed with late-stage epithelial ovarian cancer have elevated CA125 levels in their serum [22]. Unfortunately, CA125 has limited usefulness in detecting ovarian cancer in the early stages, as only 50% of these cases had elevated CA125 levels [23]. In addition, many other conditions can also cause the elevation of CA125 levels, including endometriosis, liver cirrhosis, normal menstruation, pelvic inflammatory disease and uterine fibroid. Therefore, this antigen lacks the specificity and sensitivity to be considered a reliable biomarker for the early detection of ovarian cancer [43]. CA125 is found to be more sensitive and specific in postmenopausal women than in premenopausal women. Serum CA125 is incorporated into the Risk Malignancy Index (RMI) algorithm. The RMI is widely used as a risk assessment of ovarian malignancy in clinical practice. The RMI score is generated by the simplified serum CA125 level regression equation, the menopausal status score and the ultrasound features score (RMI = ultrasound findings × menopause status × CA125 U/mL). The use of the RMI has a higher sensitivity of 87% and a specificity of 97% for ovarian cancer detection compared to CA125 alone [74].

In a recent study using a training and confirmation cohort, four existing clinical tests available for the diagnosis of ovarian cancer (RMI score and ROMA, CA125 and HE4) and a panel of 28 immunosoluble biomarkers from 66 patients undergoing surgery for suspected ovarian cancer were assessed through a multiplex immunoassay. Using a two-step triage model for women with presumed ovarian mass, IL-6 > 3.75 pg/mL was established as the main triage, supplemented by standard testing (CA125 or RMI score) for ovarian cancer in patients greater than CA125 or RMI alone (misclassification rate 4.54–3.03 percent vs. 9.09–10.60). Therefore, in conjunction with traditional studies, IL-6 can be a beneficial therapeutic biomarker for the triage of patients with potential malignant ovarian mass [26]. The reproducibility of IL6 measurement may be a challenge in clinical practice as IL6 level can increase if infections and or inflammatory conditions occur.

Osteopontin is an adhesive glycophosphoprotein secreted by activated T lymphocytes, macrophages, and leukocytes, and found in the extracellular matrix, sites of inflammation and body fluids [75] Osteopontin is not only expressed in ovarian cancer but also in endometrial, cervical, breast, colorectal, nonsmall cell lung, prostate, hepatocellular and gastric cancer. OPN is associated with tumour progression, invasion and metastasis. In 2001, OPN was identified with a cDNA microarray system using RNA isolated from several ovarian cancer cell lines, with surface epithelial cells as controls [50,76]. The levels of OPN were also significantly higher (p < 0.001) in the plasma samples of 51 women with EOC (486.5 ng/mL, n = 51) compared with the healthy controls (147.1 ng/mL, n = 107), benign ovarian disease (254.4 ng/mL, n = 46), and other gynaecological cancers (260.9 ng/mL, n = 47) [77].

In addition, OPN has been utilised to predict the progression of disease in advanced EOC, as the prognosis of patients with peritoneal spread is poor. The levels of osteopontin in 32 out of 40 peritoneal metastatic biopsies were found to be significantly elevated compared to the levels found in primary ovarian tumour tissues among women with Stage III EOC [78]. In addition, the elevated OPN levels were independently correlated with extremely poor prognosis among these women (n = 32), whereas 75% of the women found with no increase in OPN levels had a 36-month survival rate (n = 8). Furthermore, the high levels of osteopontin could be measured in the urine samples of patients with high-grade ovarian cancer, so this test could potentially be used clinically as a noninvasive tool for the early diagnosis of ovarian cancer [79].

Kallikreins are a subgroup of serine proteases with different physiological roles. The human kallikrein gene family has now been entirely defined to include 15 members on chromosome 19q. They are expressed in epithelial and endocrine tissues regulated by hormones in cancer and they are shed and detected in human body fluids [34]. Therefore, many studies have been carried out to find their role in cancer diagnosis and prognosis. A total of 12 out 15 KLKs are upregulated in ovarian cancer, with some KLKs correlating to poor prognosis and late-stage disease (4–7, 10 and 15), as well as chemoresistance (KLK 4 and 7) to a first line paclitaxel agent [80].

In a study by Luo et al., the preoperative serum level of human kallikrein (hk10) in 146 patients with ovarian cancer was significantly elevated compared to 97 healthy women and 141 women with benign gynaecological diseases [81].

Bikunin is a multifunctional glycoprotein, which mediates the suppression of tumour cell invasion and metastasis. The measurement of bikunin levels in the tissue of patients with malignant diseases has been introduced as a simple diagnostic tool for the evaluation of the prognosis. High preoperative bikunin levels have been reported to be a strong favourable prognostic marker for ovarian cancer [13]. Matsuzaki et al. found, in an extensive study, that bikunin protein in the plasma of women with ovarian cancer (N = 327), compared to those with benign ovarian mass (N = 200) and healthy controls (N = 200), may be useful in evaluating the prognosis of the disease.

A low bikunin level (≤11.5 ug/mL) was found to be associated with the late-stage (Stage III/IV) disease, the presence of large residual tumours (>2 cm) and poor response to chemotherapy. The median survival time was also shorter, at 26 months compared to 60 months, in those with high levels of bikunin (p = 0.002), thus corresponding to a 2.2-fold higher risk of dying (hazard ratio, 0.45; p = 0.023) [13]. Measuring levels of bikunin in plasma is easy and relatively inexpensive; therefore, it has the potential to be included as a prognostic biomarker for ovarian cancer. However, there is a significant overlap in bikunin levels across cancer, benign and healthy controls, which needs to be further investigated before it can be of clinical use.

Also known as WAP 4-disulphide core domain 2 (WFDC2), HE4 was first introduced as an ovarian cancer biomarker in 1999 [36]. The expression of HE4 is associated with cancer cell adhesion, migration and tumour growth, which can be related to its effects on the EGFR-MAPK signalling pathway [82]. Many studies suggested that HE4 is absent in normal ovarian surface epithelium but is expressed specifically in 100% of human endometrioid epithelial ovarian cancers (n = 16) and 93% of serous ovarian carcinomas stained for HE4 (n = 60) [57]. An ELISA analysis of serum HE4 levels in 37 patients with ovarian cancer, compared with 65 healthy controls, showed that HE4 had the same specificity and sensitivity as CA125 and detected fewer false positives in patients without a malignant disease [74].

The marker HE4 is significantly increased in ovarian and endometrial cancer, but not in endometriosis. HE4 can be increased, although it is less frequently elevated than CA125 in patients with benign disease, especially in premenopausal patients. The alternate probability of a malignancy algorithm (ROMA) blends the values of CA125 and HE4 with menopausal status in the predictive index and has been shown to stratify patients into high and low risk categories, with differing outcomes across many trials [83].

VEGF is a vascular permeability factor that is a key regulator of physiological and pathological angiogenesis, and makes a major contribution to tumorigenesis [84]. VEGF levels are known to be elevated in patients with ovarian cancer and contribute to the accumulation of ascites [85]. An analysis associated with VEGF levels in the preoperative sera of 314 patients with ovarian cancer recorded that higher VEGF levels were separately correlated with shorter survival periods [86]. In addition, tumour samples from 18 patients with advanced stage serous epithelial ovarian cancer were evaluated for VEGF expression by a reverse-transcriptase polymerase chain reaction (RT-PCR) [87]. It was demonstrated that 12 samples were found to be strongly positive, whereas six samples had low/negative VEGF expression. The median survival was longer, at 60 months in the VEGF-low/negative group compared to 28 months in the VEGF-positive group (p = 0.058).

Bevacizumab, the first and most studied anti-VEGF agent, when used as maintenance therapy following surgical debulking and first-line chemotherapy, led to significant improvements in the progression-free survival of patients with ovarian cancer but did not have an impact on the survival. Bevacizumab, in addition to PARP inhibitors, is currently being studied in Phase III PAOLO-1/ ENGOT-ov25 trials and has shown promising results, with reduced risk of disease progression by 41% overall, and by 69% in the subset of women with BRCA-mutated disease [88].

PSN is a trypsin-like proteinase (40 KDa) found on chromosome 16p11.2. It plays a major role in the activation of epithelial sodium channels and in the reduction of invasive prostate and breast cancers in vitro [89]. Similarly, the epidermal tight junction forming and terminal differentiation are related to the matriptase-prostasin proteolytic pathway [90].

The potential use of prostasin as a novel biomarker for ovarian carcinoma was proposed by Mok et al. using microarray technologies to classify upregulated genes for secretive proteins [91]. The findings revealed an overexpression of PSN in malignant epithelial ovarian cells and stroma, relative to standard ovary tissue, with a sensitivity and specificity of 51.4% and 94%, respectively [92]. Gene expression analysis indicated that PSN was expressed in ovarian cancer at levels more than 100 times greater than those found in normal or benign ovarian lesions. This overexpression signature was found in the early stages of ovarian cancer and maintained in the higher stages and grades [93]. Costa et al., on the other hand, reported a slightly higher overexpression of mRNA prostasin in freshly frozen ovarian cancer tissues than in usual controls. Thus, it has the ability to be used clinically as a differential diagnostic marker for ovarian cancer [93]. In another study by Mok et al., the combination of CA125 and prostasin gave a sensitivity of 92% and a specificity of 94% for detecting ovarian cancer [91].

Creatine kinase plays a crucial function in the energy homeostasis of vertebral cells. CKB is a cytosolic isoform of creatine kinase that displays upregulated expression in a number of cancers. It has been reported that certain ovarian cancer tissues have improved protein CKB expression [94]. In addition, CKB decreased the intake of glucose and lactate, and improved the ROS output and consumption of oxygen. As a result, it was indicated that the suppression of CKB induced G2 arrest in the cell cycle through the PI3K/AKT and AMPK pathways. Clinically, this mechanism has helped clinicians to use this biomarker in cancer cell survival and tumour progression. CKB activity measured in preoperative serum samples was higher in women with ovarian cancer (N = 45), compared to those with benign ovarian mass (9.6 U/L, N = 49) and healthy controls (8.5 U/L, N = 37), p = 0.0096 [27]. CKB is highly expressed in early stage ovarian tumour tissues and is, therefore, a potential biomarker for the early detection of ovarian cancer; it should be further investigated [27].

Mesothelin, a tumour differentiation antigen found in mesothelial pleura, peritoneum and pericardium, was discovered in 1996 at the National Cancer Institute [95]. Mesothelin is widely expressed in many tumours, including 70% of ovarian cancers. Several mesothelin-directed treatments have been studied in clinical trials, including antimesothelin immunotoxins and antibody-drug conjugates (ADC) [96]. Quanz et al. showed the activity of anetumabravtansine in conjunction with conventional chemotherapy in ovarian cancer models. Anetumabravtansine is an ADC that produces a human antimesothelin antibody conjugated by a reducible disulphide linker to the DM4maytansinoid tubulin inhibitor. Both in vitro and in vivo experiments have indicated the selective activity of anetumabravtansine in injecting new expression cells and tumours, including low-sensitivity (68.2%) and high-specificity (80.5%) ovarian cancer [97]. In animal models with ovarian cancer, treatment with anetumabravtansine exhibits improved potency in combination with carboplatin, compared to either drug alone [97]. Similarly, Anetumabravtansine also demonstrates enhanced antitumour efficacy when combined with Bevacizumab, an anti-VEGF agent. A phase 1b study (NCT02751918) using anetumabravtansine in combination with pegylated liposomal doxorubicin in ovarian cancer patients is ongoing.

ApoA-I is a high-density lipoprotein (HDL) and apolipoprotein A-I in plasma. Apo A-I levels have been reported to decrease in the sera of patients with ovarian cancer [44]. A multiplexed magnetic nanoparticle-antibody conjugates (MNPs-Abs) based fluorescence spectroscopic system analysis combining CA125, β2-M and ApoA1 for the early detection of ovarian cancer performed by Pal et al. found that while CA125 detection only identifies 50–60% of early stage ovarian cancer, the combination of the three biomarkers achieved high sensitivity (94%) and high specificity (98%) in distinguishing early stage ovarian cancer patients from healthy individuals [98]. This proposed multiplexed panel assay is also cost-effective, and further clinical investigation should be conducted to develop a clinically beneficial test kit.

TTR is a natural serum protein synthesised mostly in the liver [99]. It attaches and transports the thyroid hormones and retinol protein binding to the retinal complex [100]. Low TTR serum levels were found in ovarian cancer and used with other biomarkers to detect ovarian cancer [29,101]. Using liquid chromatography with tandem mass spectrometry, Kozak et al. found that TTR, in combination with beta-haemoglobin, apolipoprotein AI, transferrin and CA125, significantly improved the detection of early stage ovarian cancer [101]. TTR was found to be an important marker for the detection of stage I–II ovarian cancer, with a sensitivity and specificity of 78.6% and 68.8%, respectively.

Transferrin is essentially synthesised in hepatocytes and responsible for delivering plasma iron to the cell. It plays a major role in cell division and proliferation [43]. Ahmed et al. documented the downregulation of transferrin in the sera of patients with ovarian cancer [102]. In another case-control study, the level of transferrin was measured using an immunological turbidimetric assay in the sera of 37 women with ovarian cancer and compared to those with benign ovarian diseases (N = 31) and age–matched healthy controls (N = 31). It was found that the use of the biomarker transferrin as a detection tool for ovarian cancer has only low sensitivity and specificity, at 72.9% and 74.1%, respectively [40]. Therefore, transferrin needs to be used in combination with other biomarkers to achieve clinical significance.