CA 125 is a better marker to differentiate endometrial cancer and abnormal uterine bleeding

Background Incidence of endometrial cancer in India is increasing due to lifestyle changes and obesity. As 5 year survival rate of cancer confined to uterus is good, there is need for serum tumor marker for early diagnosis. This study was designed to identify a tumor marker which differentiate endometrial carcinoma and abnormal uterine bleeding (AUB) because common presentation of endometrial carcinoma is AUB. Objectives To estimate and compare serum prolactin, Cancer Antigen 125 (CA-125), Cancer Antigen 15-3 (CA15-3), and Carcino embryonic antigen (CEA) levels in patients with endometrial cancer and abnormal uterine bleeding; To evaluate the role of these markers in diagnosing endometrial cancer. Methodology Thirty eight patients with endometrial cancer and 40 patients with AUB were recruited in this study. Serum prolactin, CA 125, CEA, and CA 15-3 levels were estimated in both groups. Results The levels of CA 15-3, CA 125, CEA, and prolactin were increased in endometrial carcinoma patients, on comparison with AUB patients. CA 125 alone was found to be a better marker to detect endometrial cancer with 52.63% sensitivity, 80.00% specificity. Conclusion As individual tumor marker, serum CA 125 has the ability to detect endometrial cancer in patients with abnormal uterine bleeding.


Introduction
Endometrial cancer is a common gynecological malignancy in women and its prevalence in India is increasing in last 5 years. According to GLOBOCAN 2012 statistics, approximately 12,300 new cases are diagnosed yearly, of which about 4700 women die from the disease each year in India 1 . Recently published data showed that the incidence of Endometrial cancer 4.3 per 100000 in India 2 . The overall 5-year survival rate is 86% when all stages combined and for the disease confined to the uterus is 96% 3,4 . Most common presentation of endometrial cancer is abnormal uterine bleeding. Among them, around 75% of the women were diagnosed to have early stage of endometrial cancer 5 . AUB being most common presentation in many gynecological diseases and there is a need for markers there is a need for markers to diagnose endometrial carcinoma, in patients with similar symptoms such as AUB. endometrial biopsy specimen or by endometrial cytological examination of endometrial brush. These methods are invasive and, less sensitive in diagnosing endometrial cancer and chances of false negative rates are high 6 . This warrants the use of non-invasive serum markers to detect endometrial cancer.
Earlier studies have estimated serum concentrations of Cancer Antigen -125 (CA 125) and Cancer Antigen 15-3 (CA 15 -3) in endometrial cancer. These studies have demonstrated an association between preoperative serum CA 125 and CA 15-3 levels with tumor stage and showed that these markers can be used to predict of extra uterine spread of cancer and can be used to monitor the response of chemotherapy as well 7,8 . Elevated CA 15-3 indicate poor prognosis in these patients 9  As Prolactin is subjected to physiological variation, measuring this hormone will have limited use in diagnosing endometrial cancers at initial stage. Hence, it is difficult to use either Prolactin or CA-125 as a single marker to screen endometrial cancer. The same study also showed that these two markers should be included together as a part of biochemical screening panel in future 12 .
The objective of this study includes comparison of tumour markers in two different conditions have same presentation. So the patients with AUB were included as comparison group. The tumour markers level were compared between these two groups to find out whether any of the included tumour markers can be able to differentiate endometrial carcinoma and AUB though there is common clinical presentation.
The present study is undertaken to estimate and compare the concentrations of serum prolactin, CA 125, CA 15-3, and CEA in patients with endometrial cancer and abnormal uterine bleeding and also to evaluate whether the above parameters can be used to diagnose endometrial cancer.

Methodology
This study was conducted in Department of Biochemistry along with Department of Obstetrics and Gynaecology, JIPMER, Puducherry, India. The study was approved by JIPMER institute ethics sub-committee-human studies, and was funded by Intramural grant from JIPMER.

Selection of study participants
The study subjects were enrolled in the study based on inclusion and exclusion criteria. All perimenopausal women with endometrial carcinoma diagnosed by histopathological examination were taken into group 1. Similarly weight matched peri-menopausal women with abnormal uterine bleeding other than endometrial carcinomas were considered into group 2. All endometrial cancer patients on treatment and those who were taking hormone replacement therapy were excluded from the study. Patients with abdominal tuberculosis were also excluded as elevated CA 125 levels are associated with this condition 13 .
All patients attending Gynaecology Department, JIP-MER Hospital with symptoms of peri-menopausal bleeding during reference period of January 2011 to April 2012 were recruited into study groups as per inclusion and exclusion criteria after obtaining a written informed consent. Thirty eight patients with endometrial carcinoma were included in Group 1, whereas forty weight matched patients with abnormal uterine bleeding other than endometrial cancers were considered into group 2.

Data collection and Biochemical analyses
The details of age, height and weight of all participants were recorded and Body Mass Index (BMI) is calculated Body Mass Index BMI is calculated. The height was measured in centimeters using measuring tape and weight was measured in kilogram using Equinox weighing machine (New Delhi, India). Five ml of venous blood sample was collected from all study subjects. Serum was separated after centrifugation and stored at -80°C for assays of various parameters as per protocol. BMI was calculated by using Quetelet's index, [weight / height 2 ] The BMI was expressed as kg/m 2 . Serum concentrations of CA125, CEA and Prolactin were measured by a two-site sandwich immunoassay using direct chemiluminometric technology (ADVIA Centaur® CP Immunoassay System, Siemens, Switzerland). Whereas CA 15-3 was assayed using Enzyme Linked Immuno Sorbent Assay (ELISA) kit Syntron Bioresearch, California 17 . The reference ranges for serum CA-125, Prolactin, and CEA are less than 35 U/mL, 1.9-25 ng/mL and 0 -3µg/L respectively. The reference range for CA 15-3 is <33 U/ mL.

Statistical analyses
The normality of the continuous data was checked by Kolmogorov-Smirnov test. Data were reported as mean and standard deviation for normally distributed data and median with interquartile range was used for non-Gaussian data. The levels of tumor makers of two groups were compared either using independent t test or Mann Whitney U test according to their distribution. The possible relationship among the values of the tumor markers was assessed by correlation analysis. Sensitivity, specificity, positive predictive value and negative predictive value were calculated. Receiver operating Characteristics ROC curve was used to demonstrate the sensitivity of all these markers. The statistical analyses were done at 5% level of significance and p value < 0.05 was considered signifi-cant. The statistical analysis was carried out using Statistical Package for SOcial Sciences (SPSS), Version 20.0

Results
Thirty eight histo-pathologically confirmed endometrial cancer patients were included first group. Forty weight matched cases of abnormal uterine bleeding other than patients with endometrial cancer were included into group 2.
The demographic characteristics such as age, height, weight and BMI were compared between two groups. There parameters did not differ significantly between study groups. Table 1. The hormonal imbalance in post-menopausal stage can contribute to the change in the tumour markers levels. Therefore, menopausal status also matched between two groups. Details are given in Table 1. In study group 1, endometrioid adenocarcinoma In study group 1, endometrioid adenocarcinoma of uterus accounted for 36 (95%) of subjects on histopathological analysis. One case each of uterine papillary serous carcinoma and poorly differentiated carcinoma were included in the study.
Among the tumor markers, serum levels of CA15-3, CA 125, CEA and prolactin were significantly high in patients with carcinoma than AUB patients (Table 2). than AUB patients Table 2. We carried out correlation analysis be-tween/ among the different parameters in study group consisting of endometrial carcinoma cases. No significant correlation was observed between CA 125, CA 15-3, CEA, and Prolactin.   (Table 3).cut off value Table 3. All tumor markers in our study had a very good specificity, but very less sensitivity at the cut off when taken individually.  There is no significant difference in the level of other tumour markers including CA 125. In our study most of the women came to the hospital with the complaints of prolonged bleeding. Therefore phase of menstrual cycle cannot be predicted since they have prolonged menstrual phase 18 .
Even though several studies have reported the importance of CA 125 in the diagnosis of ovarian tumor, only limited studies have assessed its role in endometrial cancer. In our study the levels of CA 125 are higher in endometrial cancer patients when compared with abnormal uterine bleeding patients. These findings are in accordance with previous studies and the elevation of CA 125 is due to the increased synthesis of this cancer antigen by endometrial tumor cells 12,19,20 . Jiang T et al studied importance of preoperative CA 125 levels, age menopausal status and tumor histology in a large number of 995 endometrial cancer patients. The study showed that CA 125 levels had significant association with age and menopausal status, but it was not associated with tumour histology. They have suggested different cut-off values for prediction of lymph node metastasis and adnexal involvement and concluded that preoperative CA 125 levels has to be involved in the decision of management 21 . CA 125 levels are elevated in benign physiological and pathological conditions including menstrual cycle, pregnancy, endometriosis and also in malignant conditions such as endometrial carcinoma 22 . In order to differentiate those conditions from malignancy, patients with abnormal uterine bleeding due to non-malignant cause were included in this study for comparison.  25 .
Prolactin has been proposed as one of biomarkers in endometrial cancer and previous investigators have documented an increase in prolactin levels in endometrial carcinoma cases 11,12 . We observed significantly high serum levels of prolactin in cancer patients when compared to abnormal uterine bleeding group. The higher levels of prolactin may be explained by excessive prolactin release from the endometrial tumor cells 12 . Limitations of the study i. The study included small number of patients.
ii. Normal healthy controls were not included since the study was designed to differentiate or diagnose endometrial carcinoma in patients present with AUB and in future study design healthy control group as well as patients with benign uterine diseases will be included.

Conclusion
We found that serum concentrations of CA 125, CA 15-3, CEA, and Prolactin were significantly higher in endometrial cancer. In addition, we observed that as an individual tumor marker, serum CA 125 has the best diagnostic utility in differentiating endometrial cancer in patients presenting with abnormal uterine bleeding. With reasonable sensitivity and specificity observed in the study, we propose that CA 125 can be best, single and economical test in diagnosing endometrial cancer.