DIAGNOSTIC SIGNIFICANCE OF CHANGES IN SERUM HUMAN EPIDIDYMIS EPITHELIAL SECRETORY PROTEIN 4 AND CARBOHYDRATE ANTIGEN 125 IN ENDOMETRIAL CARCINOMA

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1 Acta Medica Mediterranea, 2018, 34: 785 DIAGNOSTIC SIGNIFICANCE OF CHANGES IN SERUM HUMAN EPIDIDYMIS EPITHELIAL SECRETORY PROTEIN 4 AND CARBOHYDRATE ANTIGEN 125 IN ENDOMETRIAL CARCINOMA PATIENTS JING-LI LIU, XIAO-LIN GAO, CONG-ZHE HOU, LIN ZHU, HUI XU* Department of Obstetrics and Gynecology, The Second Hospital of Shandong University, Jinan City, Shandong Province, , China ABSTRACT Objective: To study changes in serum human epididymis epithelial secretory protein 4(HE4 and carbohydrate antigen 125(CA125 in endometrial carcinoma (EC patients, and the diagnostic significance of these changes. Methods: A total of 40 cases of EC patients were assigned to the EC group, 20 patients with endometrial hyperplasia were in the endometrial hyperplasia group, while 30 healthy subjects were in the control group. Changes in HE4 and CA125 levels in the three groups were analyzed before and using ELISA. Positive expressions and sensitivity indicators were used to determine efficiency and relationship between the changes in serum HE4 and CA125, and histological grades and surgical staging. Results: Before operation, serum HE4 and CA125 levels in the EC group were significantly higher than those in the endometrial hyperplasia and control groups (p < 0.05). Serum HE4 and CA125 levels of endometrial hyperplasia group were significantly higher than those of the control group (p < 0.05), and the sensitivity of the combination of HE4 and CA125 in the diagnosis of EC was significantly higher than that of either marker alone (p < 0.05). Serum HE4 and CA125 levels in EC 7 days were significantly lower than those before operation (p < 0.05). Analyses of histological types showed that the highest positive expression of HE4 was pathological grade 3 (G3) while the lowest positive expression of HE4 was G1 (p < 0.05); G3 was significantly higher than G2 (p < 0.05). In analyses of surgical stages, the highest positive expressions of HE4 and CA125 were stage Ⅲ, and the lowest were stage I (p < 0.05); stage Ⅲ was significantly higher than stageⅡ(p < 0.05). Serum HE4 and CA125 levels in the recurrence group at 18 and 24 months were significantly higher than those in non-recurrence group (p < 0.05). Conclusion: Combined determination of serum HE4 and CA125 in the diagnosis of EC has high sensitivity. This has important clinical value for early diagnosis, and is useful for guiding operation and prognosis. Keywords: Endometrial carcinoma, Serum human epididymal epithelial secretory protein 4, Carbohydrate antigen 125, Histological types DOI: / _2018_3_120 Received November 30, 2017; Accepted January 20, 2018 Introduction Endometrial carcinoma (EC is one of the three major malignant tumors of the female genital tract. Early treatment is one of the important means for improving the prognosis of EC patients (1). Most EC patients are diagnosed through abnormal symptoms such as vaginal bleeding or fluid discharge, but nearly 30% of the patients are diagnosed in already advanced stage. The traditional examination of EC is by hysteroscopy combined with curettage, but curettage is an invasive treatment with great potential for injury/damage. Clinically, tumor markers are preferred for use in screening EC. However, there is still lack of high-sensitivity and specific tumor markers for early diagnosis, for guiding surgical treatment and evaluating prognosis (2). Serum carbohydrate antigen-125 (CA125 is used to detect epithelial ovarian cancer, and it is a tumor marker for the early screening of EC, but its clinical value is limited (3). In recent years, it has been reported that serum human epididymal secre-

2 786 Jing-li Liu, Xiao-lin Gao et Al tory protein 4 (HE4 can also screen EC, and can become a new tumor marker of EC (4). The present study was aimed at determining changes in serum HE4 and CA125 levels in patients with EC, and analyzing their clinical significance for early diagnosis, surgical treatment and assessment of prognosis of EC so as to provide a basis for their clinical application. Materials and methods General information on subjects Forty cases of patients diagnosed with EC from June 2013 to December 2016 at the Department of Obstetrics and Gynecology, The Second Hospital of Shandong University, Jinan City, participated in the study. In addition, there were 20 cases of endometrial hyperplasia patients, while 30 normal healthy cases who underwent gynecological examination were in the control group. The study was approved by Ethical Committee of The Second Hospital of Shandong University, and all participants were informed and signed informed consent. The EC group were aged 48 to 82 years (mean age = ± years). According to the surgical-pathological staging criteria of the International Federation of Gynecology and Obstetrics (FIGO) (5), 13 cases were in stage I, 22 cases were in stage II, 4 cases were in stage III, while 1 case belonged to stage IV. Histological types showed that there were 7 cases of grade 1 (G1 i.e. highly differentiated carcinoma); 20 cases in G2 (moderately differentiated adenocarcinoma); and 13 cases in G3 (low differentiation adenocarcinoma). The endometrial hyperplasia group were aged 38 to 59 years (mean age = ± years). There were pure hyperplasia in 13 cases, 4 cases of complex hyperplasia, and atypical hyperplasia in 3 cases. The control group were aged 40 to 60 years (mean age = ± 7.86years). All patients were treated without hormone therapy, adjuvant radiotherapy, chemotherapy and immunotherapy before surgery. The exclusion criteria included all patients diagnosed with severe infection, congenital heart disease and congestive heart failure; patients treated with hormone drugs for more than 6 months, and patients with malignant diseases and severe complications. Determination of serum HE4 and CA125 levels Fasting venous blood samples (5 ml) were collected from the patients in the morning, and centrifuged at 3000 rpm for 3 min. The serum samples were kept at -20 prior to analyses. The specimens were collected and processed in the dark. The levels of serum CA125 and HE4 were measured by enzyme-linked immunosorbent assay (ELISA) kits (Beijing Jinhui Golden Medical Technology Co., Ltd, China) according to the manufacturer s protocol. Assessment of results CA125 assessment method: Normal CA125 levels are in the range 0 ~ 35U/mL. Thus, CA125 levels > 35 U/mL were deemed positive. Normal range of HE4 is ± 7.37pmol/L, and HE4 levels > pmol/l were taken as positive. Pathological biopsy was used as the gold standard to determine the true and false positive and negative expressions of HE4 and CA125, and the sensitivity and specificity were calculated. From a standard curve drawn using binomial fitting curve, if the result of the inspection of the control object was in the range of the marked values (0~150pmol/L), it was concluded that the result was referential and effective. Statistical analysis Data were analyzed by SPSS19.0. The enumeration data were analyzed between the groups with Chi-square test. Measurement data in line with the normality and homogeneity of variance were analyzed using t-test, but data not consistent with the normal distribution were analyzed with Wilcoxon rank sum analysis. Values of p < 0.05 were considered as indicative of statistical significance. Results Comparison of serum HE4 and CA125 levels in the three groups before operation The levels of serum HE4 and CA125 in the three groups before operation differed significantly, and were highest in the EC group and lowest in the control group (p < 0.05, Table 1). Comparison of positive scores of serum HE4 and CA125 in each group The sensitivity of HE4 combined with CA125 in the diagnosis of EC group was significantly higher than that of HE4 or CA125 alone (p < 0.05, Table 2).

3 Diagnostic significance of changes in serum human epididymis epithelial secretory protein Comparison of serum HE4 and CA125 in patients with EC before and The serum levels of HE4 and CA125 were significantly lower in EC group at 7 days than that before operation (p < 0.05, Table 3). HE4 CA125 Items n % c 2 p n % c 2 p Pathological grade G Group n HE4 (pmol/l) CA125 (KU/L) EC ± ±3.81 Endometrial hyperplasia ±3.78* 23.65±3.65* Control ±4.48*# 15.28±5.28*# G G *# Surgical staging Stage I Stage II T p 0 0 Table 1: Comparison of serum HE4 and CA125 levels in the three groups before operation (χ ±s). * p < 0.05, compared with EC group; # p < 0.05, compared with the endometrial hyperplasia group Group n HE4 CA125 Combined EC % % % Endometrial hyperplasia % % % Control % % % x p Table 2: Comparison of positive scores of serum HE4 and CA125 in each group [n(%)]. Time HE4(pmol/L) CA125(KU/L) Before operation ± ±3.962 Stage III ab ab Table 4: Relationship between HE4 and CA125 levels and pathological grading and surgical staging of EC patients (n, %) * p < 0.05, compared with G1; # p < 0.05, compared with G2; ap < 0.05, compared with stage I; b p < 0.05, compared with stage II Post-operative recurrence and changes in serum HE4 and CA125 in EC patients Sub-group analysis showed that out of the 40 cases of patients with uterine membrane cancer who underwent surgical treatment, there were 8 cases of recurrence and 32 cases without recurrence at 12 months after surgery, which were divided into recurrence group and non-recurrence group. At 18 months and 24 months, the levels of HE4 and CA125 in the recurrence group were significantly higher than those in non-recurrence group (p < 0.05, Table 5). Group HE4(pmol/L) CA125(U/mL) 7 days 97.58± ±4.64 At 18 months At 24 months At 18 months At 24 months T p 0 0 Table 3: Comparison of serum HE4 and CA125 in patients with EC before and (χ ±s). Relationship between HE4 and CA125 levels and pathological grading and surgical staging of EC patients From analyses of pathological grading of 40 EC patients, the positive score of HE4 in G3 was the highest, while G1 was the lowest (p < 0.05), and G3 was significantly higher than G2 (p < 0.05). From surgical staging analysis, the positive scores of HE4 and CA125 in stage III were the highest, while stage I was the lowest (p < 0.05), with stage III being significantly higher than stage II (p < 0.05, Table 4). Recurrence group (n=8) Non-recurrence group (n=32) Discussion ± ± ± ± ± ± ± ±4.69 T p Table 5: Comparison of serum HE4 and CA125 levels between recurrence and non -recurrence sub-groups of EC patients (χ ±s). Elevated serum CA125 (a glycoprotein secreted by celomic epithelial cells) are related to the occurrence and development of EC (6). However, CA125 lacks sensitivity and specificity in the diagnosis of early EC, and has limited application value, which makes it suitable for use in the univer-

4 788 Jing-li Liu, Xiao-lin Gao et Al sal screening of malignant tumor markers. In this study, the sensitivity of CA125 alone in the diagnosis of EC was only 30%, which was similar to the results reported by researchers in China and abroad (7, 8). The HE4 gene was originally thought to be a protease inhibitor associated with sperm maturation, but studies have shown that HE4 is expressed in multiple normal epithelial tissues such as the respiratory tract and genital tract (9). It can be used to screen EC in normal populations, with a sensitivity of 45.5%. In this study, the levels of serum HE4 and the positive grade of HE4 in EC group were significantly higher than those in endometrial hyperplasia and control groups. The sensitivity of HE4 in the diagnosis of EC was better than that of CA125. This may be related to the limitation of tumor and the low level of serum CA125 caused by low CA125 in the systemic circulation. In this study, the combined sensitivity of serum HE4 and CA125 was 62.5 %, which was higher than the sensitivity due to HE4 or CA125 alone. This suggests that sensitivity can be improved by combining the two markers. The occurrence and development of any kind of malignant tumor are possibly due to multiple gene interactions. Thus, combined use of multiple tumor markers is more consistent with the mechanism of occurrence and development of tumors than the use of a single marker. Therefore, compared with the use of a single biomarker, combined assay of serum HE4 and CA125 had a higher sensitivity for the early diagnosis of EC. The relationship between changes in serum levels of tumor markers and the histological grading and surgical staging can be used to guide clinical operations. CA125 has been detected in EC by experts, and serum CA125 level has been correlated with the grade, malignancy, depth of muscular invasion and extent of lymph node metastasis in EC (10, 11). In a study, it was shown that HE4 was expressed mainly in the cytoplasm of endometrial cells, while 90 % HE4 in EC was strongly positive, and had nothing to do with pathological types (12). On the other hand, it has been reported that the level of serum HE4 in patients with EC increased significantly with development of the disease, suggesting that HE4 may also be involved in the occurrence and development of tumors (13). However, another study found that serum HE4 levels were independent of pathological grading, but correlated with FIGO stage, depth of myometrial invasion, and tumor diameter (14). Through the combined assay of serum HE4 and CA125 levels, the present study found that the higher the histological grade, the higher the positive diagnostic value of HE4. This finding suggests that the levels of HE4 in serum before operation are significantly correlated with the pathological grade of EC, which may be associated with the higher degree of malignancy in patients with EC. These patients have higher expression of HE4, so that the higher level of serum HE4 entered into the systemic circulation, but the level of serum CA125 was not significantly correlated with the histological grading of EC. This study also showed that the higher the surgical stage, the higher the positive grade of preoperative HE4 diagnosis, suggesting that pre-operative serum levels of HE4 are significantly associated with FIGO surgical staging. With progression of the tumor, ulceration necrosis and degree of malignancy increased; vascular invasion, and lymphatic metastasis also increased, leading to increase in the patient s serum HE4. It is believed that advanced stage of FIGO, high-grade histological scores, and deep myometrial invasion are related to high concentration of HE4 (15, 16). Serum CA125 level was closely related to the surgical stage of EC, which is similar to previous findings (17), suggesting that combined assay of serum HE4 and CA125 levels in patients with EC can better guide clinical surgical treatment, and provide more comprehensive information on occurrence and development of EC. Serum CA125 levels have been associated with survival rate in patients with EC, and CA125 has been used as a prognostic factor for EC patients (18). The risk of post-operative death in patients with HE4 > 67.4 pmol/l was times than that of patients with HE4 <67.4 pmol/l, and the risk of post-operative recurrence was times of the latter, suggesting that serum HE4 is an independent prognostic factor for the overall survival and recurrence rate of patients with EC (19). In the present study, serum HE4 levels in the recurrence group and the non-recurrence group after surgery differed significantly, suggesting that serum HE4 levels may be one of the high risk factors for post-operative recurrence. The difference in CA125 level between the recurrence group and non-recurrence group was also statistically significant, indicating that CA125 and HE4 may have synergistic effects on the prognosis of EC, which is consistent with results from previous studies (20).

5 Diagnostic significance of changes in serum human epididymis epithelial secretory protein Conclusion Changes in serum HE4 and CA125 levels in patients with EC are of great clinical significance. The sensitivity associated with the use of combined assay of serum HE4 and CA125 in diagnosing EC is much better than the sensitivity associated with the use of either marker alone. This finding has important application value in early diagnosis of EC, and it is useful for guidance of operation, monitoring curative effects, and evaluating prognosis. References 1) Soochit A. The significance of preoperative carbohydrate antigen (CA125 and human epididymis protein 4(HE4 serum level to predict pelvic or paraaortic lymph node metastasis in epithelial ovarian cancer patients. Gynecologic Oncology 2015; 137(1-4): ) Knific T, Osredkar J, Smrkolj Š, et al. Novel algorithm including CA-125, HE4 and body mass index in the diagnosis of endometrial cancer. Gynecol Oncol 2017; 147(1): ) Li X, Gao Y, Tan M, et al. Expression of HE4 in endometrial cancer and its clinical significance. Biomed Res Int 2015; 2015(13): ) Liu X, Zhao F, Hu L, et al. Value of detection of serum human epididymis secretory protein 4 and carbohydrate antigen 125 in diagnosis of early endometrial cancer of different pathological subtypes. Onco Targets Ther 2015; 1(8): ) Lewin S N, Ni H T M, Deutsch I, et al. Comparative performance of the 2009 international Federation of gynecology and obstetrics staging system for uterine corpus cancer. Obstet Gynecol 2010; 116(5): ) Modarresgilani M, Vaezi M, Shariat M, et al. The prognostic role of preoperative serum CA125 levels in patients with advanced endometrial carcinoma. Cancer Biomark 2017; 20(2): ) Zheng L, Qu J, He F. The diagnosis and pathological value of combined detection of HE4 and CA125 for patients with ovarian cancer. Open Med (Wars) 2016; 11(1): ) Bian J, Sun X X, Li B, et al. Clinical significance of serum HE4, CA125, CA724 and CA19-9 in patients with endometrial cancer. Technol Cancer Res Treat 2017; 16(4): ) Nagy B Jr, Nagy B, Fila L, et al. Human Epididymis Protein 4: A Novel Serum Inflammatory Biomarker in Cystic Fibrosis. Chest 2016; 150(3): ) Calis P, Yuce K, Basaran D, et al. Assessment of Cervicovaginal Cancer Antigen 125 Levels: A Preliminary Study for Endometrial Cancer Screening. Gynecologic & Obstetric Investigation 2016; 81(6): ) Jiang T, Huang L, Zhang S. Preoperative serum CA125: a useful marker for surgical management of endometrial cancer. Bmc Cancer 2015; 15(1): ) Caihong YANG, Yonghui XU, Xueyu ZHANG. Expression and significance of human epididymis gene product 4 in tissue and serum of patients with endometrial cancer. Chinese Journal of Clinical Oncology 2011; 38(9): ) Dongli Dong, Jinsong Gu, Shaojie Zhao. The Changes of Serum CA125 and HE4 In Endometrial Carcinoma and Clinical Significance. Shandong Medical Journal 2017; 57(3): ) Zamani N, Modares Gilani M, Zamani F, et al. Utility of Pelvic MRI and Tumor Markers HE4 and CA125 to Predict Depth of Myometrial Invasion and Cervical Involvement in Endometrial Cancer. J Family Reprod Health 2015; 9(4): ) Fang Yihua;Yan Shijie. The clinical value of serum human epididymis secretory protein 4 in diagnosis of endometrial cancer. Anhui Medical Journal 2015; 36(9): Kalogera E, Scholler N, Powless C, et al. Correlation of serum HE4 with tumor size and myometrial invasion in endometrial cancer. Gynecol Oncol 2012; 124(2): ) Stiekema A, Boldingh Q J, Korse C M, et al. Serum human epididymal protein 4(HE4 as biomarker for the differentiation between epithelial ovarian cancer and ovarian metastases of gastrointestinal origin. Gynecol Oncol 2015; 136(3): ) Nikolaou M, Kourea HP, Tzelepi V, et al. The prognostic role of preoperative serum CA 125 levels in patients with endometrial carcinoma. J BUON 2014; 19(1): ) Mutz-Dehbalaie I, Egle D, Fessler S, et al. HE4 is an independent prognostic marker in endometrial cancer patients. Gynecologic Oncology 2012; 126(2): ) Shu-ling LUO, Chun-yan ZHENG, Jin-hui CAO. Clinical Significance of CA125 and HE4 Assay In endometrial carcinoma. Chin J Lab Diagnosis 2016; 20(9): Corresponding author HUI XU Department of Obstetrics and Gynecology, The Second Hospital of Shandong University, Jinan City, Shandong Province, , China , xanhn6@163.com (China)