Engrailed-2 (EN2) protein in cervical mucus: a novel biomarker for endometrial carcinoma
Authors: Wang Tong , Jia Ningyi , Gao Songkun , Liu Shengjie , Liu Ming , Zhang Hong 
Published: November 2024

Abstract
Objective
This study aims to demonstrate that the EN2 protein in cervical mucus may serve as a novel biomarker for screening endometrial cancer.

Materials and Methods
This study included 133 patients who were treated at Beijing Obstetrics and Gynecology Hospital. According to the pathological results of hysteroscopy endometrial biopsy, the patients were divided into endometrial cancer group (n = 55), endometrial atypical hyperplasia group (n = 16), benign lesion group (n = 28), and control group (n = 34). All patients collected cervical mucus before hysteroscopy, and the level of EN2 protein in cervical mucus was detected by ELISA in the four groups of patients. Nine patients who underwent surgical treatment for endometrial cancer were included, and endometrial cancer lesions and adjacent tissues without endometrial cancer infiltration were taken from the endometrial cancer lesions and cervical tissues. Fifteen patients who underwent hysterectomy for benign lesions were included, and endometrial and cervical tissues were taken from the endometrial cancer lesions and adjacent tissues without endometrial cancer infiltration. PT-PCR, immunohistochemistry, and WB were used to verify the expression differences of EN2 protein in cancerous lesions and endometrial tissues without endometrial infiltration, cervical tissues, and endometrial tissues without endometrial lesions. In addition, HEC1B, KLE, and HeLa cell lines were used to characterize EN2 overexpression in endometrial cancer cells. Immunohistochemistry, RT-PCR, and confocal analysis were used to detect the expression of EN2 protein in different cell lines.

Results
In different groups, the average concentration of EN2 protein in cervical mucus in the EC group was significantly higher than that in the benign group and the normal control group (573.9 ± 123.4 ng/mL vs 153.5 ± 106.2 ng/mL and 153.0 ± 107.5 ng/mL, P < 0.001). The concentration of EN2 protein in EIN3 case specimens was significantly higher than that in the normal control group (P < 0.001). ROC analysis showed that the optimal threshold for diagnosing endometrial cancer in cervical mucus was 321.1 ng/ml, with a sensitivity of 92.6% and specificity of 95.4% for distinguishing EINIII, EC, and non-cancerous cases. In clinical specimens, the expression level of EN2 protein in endometrial cancer tissues was significantly higher than that in endometrial tissues and cervical tissues without endometrial cancer infiltration. In addition, PT-PCR, immunohistochemistry, suggest that EN2 protein is overexpressed in endometrial cancer cell lines compared to cervical adenocarcinoma cells (P < 0.01).

Conclusion
The concentration of EN2 protein in cervical mucus can effectively identify endometrial cancer and precancerous lesions. The increased expression of EN2 protein in endometrial cancer lesions leads to an increase in the concentration of EN2 protein in the cervical mucus of endometrial cancer patients.This is a small single-center study, and larger studies are needed to determine the role of EN2 protein in the diagnosis of endometrial cancer.