Journal of Medical Sciences and Health

Introduction

Ovarian cancer is the third most common cancer of the genital tract in females globally and the fourth among Indian females. ^1, 2 It most often originates from the epithelial cells but may also originate from germ cells and stromal cells. ³ In a study published by Chaturvedi M. et al. ⁴ on the Indian population, roughly 89% of the ovarian cancers identified were epithelial in origin. Due to non-specific symptoms and lack of specific biomarkers, ovarian carcinoma is often diagnosed in advanced stages when prognosis is poor. ⁵

Currently, CA125 is the most commonly used biomarker for the diagnosis of Ovarian Carcinoma. A value <35 units/mL is taken as normal, while >35 units/mL is suspicious. However, raised CA125 has also been associated with benign conditions of ovaries ⁶ as well as in conditions apart from ovarian masses such as abdominal tuberculosis ⁵ , thus making CA125 a non-specific marker for ovarian carcinoma. In a meta-analysis by Dodge JE. et al., the sensitivity and specificity of CA125 were found to be 78% each. ⁷ Other non-specific markers associated with ovarian cancers are Human epididymis protein 4 (HE4), Carcinoembryonic Antigen (CEA), Cancer antigen 19-9 (CA 19-9), etc.

 Osteopontin (OPN) is a calcium-binding protein expressed in bones, kidneys, and the brain, among ^8, 9 It is also known as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), and secreted phosphoprotein 1 (SPP1). Although the precise functions of OPN are unknown, studies have suggested its role in cell attachment and signalling. ⁹ It has been found to be increased in various cancers such as colorectal, breast and small lung cell carcinoma. ¹⁰ Studies have also evaluated its role as a diagnostic marker of ovarian cancer. ¹¹

Folate, being a part of one-carbon metabolism, is important for the synthesis of various molecules, including purines and pyrimidines and hence DNA synthesis and cell multiplication. ¹² Folate is transported across the cellular membrane in three ways- through the reduced folate carrier (RFC), through the proton-coupled folate transporter (PCFT), and through folate receptors, of which there are four glycopolypeptide members (FRα, FRβ, FRγ and FRδ). ¹³ FRα has been reported to be overexpressed in solid tumours such as ovarian, lung and breast carcinomas.14 Given the important role of folate in cancer, aside from methotrexate and other drugs targeting folate, targeted therapy against the Folate receptor Alpha is also a subject of further investigation under the context of precision medicine.15, 16

In this study, we evaluated OPN and FRα as diagnostic marker for ovarian carcinoma.

Material and Methods

This study was conducted in a tertiary care hospital by the Department of Biochemistry in collaboration with the Departments of Obstetrics and Gynaecology, and Pathology. The study was approved by the Institutional Ethical Committee (F.No.17/IEC/MAMC/2018/02, dated 26.10.2018). It was a hospital-based case-control study. Written informed consent was obtained before sample collection prior to surgery. Three groups of participants were recruited as follows:

Group I (Epithelial Ovarian Cancer Patients): The study included newly diagnosed treatment-naive patients with epithelial ovarian carcinoma aged between 20 and 70 in this group. Blood samples were collected before and after surgery (sample collection mentioned below), and patients who were confirmed to have cancer histopathologically were included in group I. In contrast, those with benign histopathology were considered for group II.

Subjects were recruited consecutively on admission in the wards till the sample size was met.

Group II (Benign Ovarian Tumor Patients): Patients in the similar age group as group I diagnosed with benign ovarian tumors as confirmed by histopathological examination of biopsy samples.

Group III (Control subjects): Clinically normal healthy women belonging to similar age group, without any ovarian abnormality as seen on ultrasound were included along with group II as controls.   

Subjects with concurrent cancer at any other site, with present or past history of any bone diseases or with any other major systemic illness were excluded from the study.

Results

Level of serum biomarkers

(a) Pre-surgery:

Estimation of baseline levels (Pre-surgery) of CA125, OPN, FRα and Folate are presented in Table 2.

Table 2: Median and interquartile range of Basal serum Folate, CA125, Osteopontin (OPN) and Folate Receptor Alpha (FRα) levels in healthy controls, patients with benign ovarian tumour and cases with ovarian cancer

Parameter	Healthy Controls (n=25)	Benign ovarian disease (n=25)	Ovarian Cancer (n=25)
Serum CA125 (U/mL)	12.90(8.85-41.2)	39.2(16.25-52.50)*	295.3(59.95-799.3)*#
Serum OPN (pg/mL)	2483(1379-3680)	2216(889.2-4663)	4095(1421-5044)
Serum FRα (pg/mL)	54(0.1-199.1)	212.2(22.33-337.8)*	397.1(153.4-1794)*#
Serum Folate (ng/mL)	9.0(5.66-12.25)	4.32(3.47-8.0)*	5.15(4.3-6.0)*#
*p < 0.05 compared to healthy controls, # p < 0.05 compared to Benign ovarian disease by Kruskal Wallis test followed by Bonn Ferroni corrected post-hoc Mann Whitney U-Test.

No significant difference was found in the median values of serum OPN in the three groups.

A significant difference in median values of FRα and CA125 was found in the ovarian cancer group when compared to the benign ovarian tumor group and control group. Serum folate level was significantly lower in both ovarian cancer and benign ovarian tumor group in comparison to controls, however, it was within normal reference range (5-20 ng/mL).

(b) Post Surgery:

Follow-up of Group I & II patients: Levels of serum biomarkers were compared in the subjects before and after surgery (Figure 1) using 2-tailed Wilcoxon Signed Ranks test in cancer subjects and benign subjects. There was a significant (p<0.05) decrease in serum levels of Folate receptor Alpha (FRα) and CA125 post-surgery in cancer subjects; however, the decrease in serum Osteopontin (OPN) levels was insignificant (p>0.05).

In benign subjects, there was no significant difference in serum levels of OPN and CA125 before and after surgery, however, FRα decreased significantly post-surgery.

Spearman correlation showed a significant positive correlation between baseline levels of FRα and CA125 (r= 0.506, p=0.01) in cancer subjects, however, no significant correlation was found between OPN and CA125 (r=0.043, p=0.838).

Serum levels of FRα and CA125 were significantly increased in high-grade serous adenocarcinoma in comparison to mucinous adenocarcinoma, with p values of 0.019 and 0.02, respectively. However, OPN did not show any significant difference among different histopathological grading. None of the markers showed any significant difference with FIGO surgical staging.

Discussion

The demographic profile of cancer participants is presented in Table 1. As seen in the table, age distribution in the 3 groups was not different, hence, age is not a confounder while interpreting biomarkers in ovarian cancer. None of the subjects gave a history of use of oral contraceptives, which has been reported as a protective factor against ovarian cancer by several studies. ¹⁷ There was no significant family history of breast or ovarian cancer in these cases.

However, menopausal status and parity were different in the cancer group when compared with healthy controls and benign ovarian tumors. Most females in the cancer group were multiparous, despite the fact that increased parity ^17, 18 is protective in ovarian cancer, which suggests that parity was not a confounding factor in the study. Most subjects (68%) of the ovarian carcinoma group were postmenopausal with an average of 37 years of reproductive years before menopause, compared to 36% of benign subjects and 28% of healthy controls. Menarche at an early age and menopause at a late age increases lifetime ovulatory years, which is a risk factor for ovarian carcinoma. ¹⁷

As shown in Table 2, basal serum levels of CA125 and Folate Receptor Alpha (FRα) were significantly higher in patients with benign ovarian tumors and patients with ovarian cancer when compared to healthy control; similarly, the difference was also significant between benign and ovarian cases. CA125 is a known biomarker for ovarian cancer; however, due to its limitations, there is a need for a different biomarker. With this study, we infer that FRα has the potential to be a biomarker in diagnosing ovarian cancer as its AUC was close to that of CA125; few studies have found that such potential exists. ^19, 20 FRα has been studied with HE4 by Leung F. et al. and was found to be a strong predictor of ovarian cancer diagnosis. 20 It can distinguish between histological subtypes; a few studies have also demonstrated it being raised in high-grade serous ovarian histology. ^21, 22 Studies have shown the therapeutic role of FRα ^23, 24 and the role of intraoperative imaging of cells expressing it. ^25, 26

Although FRα levels were high in benign and cancer groups, the serum folate levels in all the groups were within the reference range. Hence, the difference in FRα levels is probably not attributed to serum folate levels.

There was a significant decline in levels of CA125 and FRα after surgery in the cancer group; the decrease in OPN levels was, however, not significant (Figure 1). This again proves that CA125 and FRα are potential biomarkers of ovarian cancer. An increase in their levels during follow-up would be suggestive of a possible recurrence of cancer.

In the benign group, only serum levels of FRα showed a significant decline post-surgery.

There was a positive correlation between basal levels of CA125 and FRα.

Our ROC analysis shows that CA125 and FRα can be used to differentiate cancer from healthy controls and even from benign cases. For benign cases, AUC for FRα doesn’t improve much, but it increases significantly for cancer cases; thus, its discriminatory power increases (Table 3). There was a marginal increase in the Area under the curve when CA125 and FRα were combined to diagnose cancer from healthy and benign groups. So, we conclude that FRα in serum may act as an independent marker; in combination with CA125, its distinguishing ability increases.

Conclusion and Recommendation

Based on our study, CA125 is superior to FRα in sensitivity and specificity for the diagnosis of Ovarian Carcinoma. However, a combined study of the two markers with a bigger sample size is recommended to further evaluate its role in diagnosis.

With the surgical removal of cancer, there was a significant reduction in the levels of CA125 and FRα, suggesting a significant secretion of these biomarkers in serum by cancer cells. This finding can be explored further for monitoring cases of disease recurrence. In addition, increased expression of folate receptor alpha may reflect an increased demand for folate by the rapidly multiplying cancer cells.

Disclosure

Funding: None

Conflict of Interest: None