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Mais, V.;  Fais, M.L.;  Peiretti, M.;  Fanni, D.;  Massa, E.;  Carboni, G.;  Fais, G.;  Deo, G.;  Angioni, S. Serum Human Epididymis Protein 4 and Endometrial Cancer. Encyclopedia. Available online: (accessed on 19 June 2024).
Mais V,  Fais ML,  Peiretti M,  Fanni D,  Massa E,  Carboni G, et al. Serum Human Epididymis Protein 4 and Endometrial Cancer. Encyclopedia. Available at: Accessed June 19, 2024.
Mais, Valerio, Maria Luisa Fais, Michele Peiretti, Daniela Fanni, Elena Massa, Giulia Carboni, Giuseppina Fais, Giuseppe Deo, Stefano Angioni. "Serum Human Epididymis Protein 4 and Endometrial Cancer" Encyclopedia, (accessed June 19, 2024).
Mais, V.,  Fais, M.L.,  Peiretti, M.,  Fanni, D.,  Massa, E.,  Carboni, G.,  Fais, G.,  Deo, G., & Angioni, S. (2022, November 23). Serum Human Epididymis Protein 4 and Endometrial Cancer. In Encyclopedia.
Mais, Valerio, et al. "Serum Human Epididymis Protein 4 and Endometrial Cancer." Encyclopedia. Web. 23 November, 2022.
Serum Human Epididymis Protein 4 and Endometrial Cancer

Human epididymis protein 4 (HE4), also known as epididymal secretory protein 4, is a glycoprotein encoded by the Whey-Acidic Four-Disulfide Core domain protein 2 (WFDC2) gene.  There may be a correlation between the tissue expression of HE4 and the molecular classification of endometrial cancer.

endometrial cancer endometrial hyperplasia cancer genome atlas human epididymis protein 4 HE4

1. HE4

Human epididymis protein 4 (HE4), also known as epididymal secretory protein 4, is a glycoprotein encoded by the Whey-Acidic Four-Disulfide Core domain protein 2 (WFDC2) gene [1]. The genomic sequence coding for the WFDC family of proteins is present at the level of chromosome 20, in the 20q13 region, is 678 kb long and includes 14 coding genes [1]. HE4 is a secretory protein originally identified in the distal human epididymis and shows significant structural similarity to proteinase inhibitors [2][3]. Later, it was shown that the protein is also expressed by the vas deferens, the respiratory epithelium, the salivary glands, the distal tubule of the kidney, and the normal glandular epithelium of the female genital system but not by the superficial ovarian tissue [4]. The genomic sequence encoding the WFDC family of proteins was amplified in various pathological situations, such as carcinoma of the colon, stomach, pancreas, oral cavity, breast, endometrium, and ovary. Therefore, the role of the protein in carcinogenesis and tumor progression has been hypothesized, more precisely, in the migration and adhesion of ovarian cancer cells [4][5][6]. Increased tissue expression of HE4 was then demonstrated in a series of malignancies, particularly of gynecological and pulmonary origin [4][7][8].
Over the years, HE4 has acquired increasing clinical relevance thanks to its ability to differentiate benign adnexal masses from ovarian cancer. Moore’s group in 2012 showed that serum HE4 is less frequently elevated in benign pathologies than CA125 (8% vs. 29%), with better specificity, especially in premenopausal women [9]. In healthy women, serum HE4 levels vary up to 150 pmol/L. This wide range is related to the fact that HE4 values are influenced by age [10], BMI, smoking habits, and respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD) [11], as well as the presence of renal failure, which is the main cause of false-positives [12]. Serum levels of HE4 do not appear to be influenced by the phases of the ovarian cycle or by hormonal therapies, which is why HE4 can be measured at any stage of the menstrual cycle (unlike the CA125 marker) and during the use of oral contraceptives [13]. Finally, the concentrations of HE4 across the different trimesters of pregnancy are unchanged relative to the HE4 levels of nonpregnant individuals [10]. However, the serum levels of HE4 reported in the literature greatly depend on the dosage method used by the various authors. Differences between manual and automated HE4 assays have been reported [11]. Over time, several automated immunometric systems that have become available are not yet compatible with each other [14].
Regarding the use of HE4 as a serum biomarker, Moore et al. have developed an algorithm for the calculation of the “ROMA Risk Score” (risk of ovarian malignancy algorithm), which combines the levels of the two biomarkers HE4 and CA 125 and simultaneously takes into account the patient’s menopausal status [15]. The sensitivity of the ROMA Risk Score was 94% (with a specificity of 75%) in distinguishing benign from malignant ovarian pathologies [15][16]. A recent meta-analysis suggested that the ROMA Risk Score is probably more effective in diagnosing epithelial ovarian carcinoma than HE4 and CA125 taken individually in postmenopausal women. At the same time, HE4 is more effective than CA125 in diagnosing epithelial ovarian carcinoma in premenopausal women [17].
Immunohistochemical studies have shown overexpression of HE4 in 100% of ovarian carcinomas of the endometrioid histotype, 93% of ovarian carcinomas of the serous histotype, and 50% of clear cell carcinomas [7]. Lu et al. demonstrated in ovarian cancer cell cultures that the expression of HE4 is associated with cancer cell adhesion and migration [6]. Furthermore, HE4 suppression markedly inhibited cancer growth [6]. Overexpression of HE4 has also been correlated with the proliferation, invasion, and metastasis of ovarian cancer [18]. Finally, HE4 immunocytochemistry appears to be highly sensitive in identifying cells derived from high-grade serous ovarian carcinomas in malignant ascites [19].
However, different isoforms of HE4 protein exist, and different anti-HE4 antibodies recognize different HE4 epitopes [20][21]. In 2002, Bingle et al. [20] described the complex nature of the HE4 gene. The gene undergoes splicing, and HE4 protein exists as five isoforms. The full length HE4 protein contains two whey acidic protein (WAP) domains whereas the other protein isoforms contain only one WAP domain. Two isoforms (HE4-V1 and HE4-V4) contain a N-terminal WAP domain and two (HE4-V2 and HE4-V3) contain a C-terminal WAP domain [20]. The existence of five isoforms of HE4 protein suggested the need to use multiple different antibodies to identify these isoforms both in biological fluids and in healthy or tumor tissues. In 2019, Hellstrom et al. [21] published an interesting study describing the results obtained by using four different anti-HE4 mouse antibodies (mAbs) to characterize the different isoforms of the HE4 protein in ovarian cancer cells and in the blood of women affected by these neoplasms. Two mAbs recognized epitopes on the C terminal and two mAbs recognized epitopes on the N-terminal. The authors determined HE4 binding affinity to the four mAbs and performed sandwich assays for detection of HE4 epitopes. They also performed immunohistochemical evaluation of tumor sections and measured HE4 in sera from women with ovarian carcinoma and in culture supernatants after establishment of ovarian carcinoma cell lines [21]. They evaluated the binding of the four mAbs to tumor sections of 19 ovarian carcinomas and were able to show that one mAb bound to 18 of 19 sections and the other three mAbs bound to 14, 7, and 4 sections out of 19, respectively. Sera from only 12 of the 19 patients had HE4 levels higher than the standard 150 pM cutoff. Cultured cells from ovarian carcinoma lines which expressed high levels of HE4 at immunohistochemistry also secreted the HE4 molecule into the supernatant. Cells which expressed low levels of HE4 at immunohistochemistry did not secrete HE4 into the supernatant. The authors concluded that different anti-HE4 antibodies bind independently in formalin-fixed sections of ovarian carcinomas, identifying subpopulations of ovarian carcinoma cells [21]. Moreover, the overexpression of a specific epitope recognized by a specific anti-HE4 antibody was associated with platinum resistance of ovarian carcinoma cells [21].

2. Serum HE4 and Endometrial Cancer

The first evidence suggesting that serum HE4 could be more useful as a tumor biomarker in women with endometrial endometrioid adenocarcinoma than serum CA125 dates back to 2008 [22]. Maintaining a specificity of 95%, the sensitivity of serum HE4 in differentiating women with endometrioid carcinoma of the endometrium at any stage from healthy women was 45.5%, significantly higher than that of serum CA125, which was 24.6% [22].
Since then, several authors have continued to verify the usefulness of serum HE4 as a marker to be used preoperatively in women with endometrial cancer to predict the risk of lymph node metastases [23][24][25]. In 2011, Moore et al. found a high specificity associated with a high sensitivity of serum HE4 in preoperatively distinguishing stage IA from stage IB, i.e., the invasion of less or more than 50% of the myometrium by the carcinoma in 96 women with carcinoma of the endometrium [23]. In 2012, Kalogera et al. verified the high specificity and sensitivity of serum HE4 in predicting myometrial carcinoma invasion and the maximum size of the carcinoma itself in another 75 women [24]. In 2014, Brennan et al. confirmed the previous observations by conducting a similar study in a group of 373 patients identified in a case–control study conducted in Australia between 2005 and 2007 [25]. Elevated serum HE4 levels were significantly associated with reduced recurrence-free survival. They reduced overall survival in the entire population of women with endometrial cancer and in the subpopulation of women with endometrioid cancer of the endometrium and ultimately in the subgroup of women with low-grade (G1/2) endometrioid endometrial cancer [25].
The large amount of data progressively produced by the various researchers was analyzed by various meta-analyses that took place over time from 2014 to 2020 [26][27][28][29]. A meta-analysis published in 2014 focused on the diagnostic accuracy of serum HE4, included six articles and identified high specificity as a strength of the marker [26]. A meta-analysis published in 2018 that included 23 articles also focused on the diagnostic accuracy of serum HE4. It concluded that diagnostic accuracy was higher when the control group consisted of patients with benign diseases than when the control group consisted of healthy women [27]. Different HE4 immunoassays were used in the different studies included in the meta-analysis, which makes the obtained results difficult to interpret [27]. Most of the studies used enzyme immunoassay (EIA), some studies used chemiluminescent microparticle immunoassay (CMIA), and few studies used electrochemiluminescence immunoassay (ECLIA) [27]. A meta-analysis published in 2019 compared the diagnostic accuracy of serum HE4 with that of CA125 in distinguishing women with endometrial cancer from women without cancer [28]. This meta-analysis included 12 studies involving 2586 women, of which 1106 had endometrial cancer, and concluded that the diagnostic accuracy of serum HE4 is better in the Caucasian population than in the Chinese population and is still better than that of CA125 [28]. A fourth meta-analysis was published in 2020 [29]. This meta-analysis included 17 studies with a total of 1807 women with endometrial cancer and 1260 women with healthy or benign uterine disease. It concluded that serum HE4 has good specificity in diagnosing endometrial cancer but relatively low sensitivity, with HE4 threshold values chosen by the various authors ranging from 45.5 to 141.5 mmol/L [29].
In 2021, two systematic reviews were published aiming to verify what advantages the serum HE4 dosage alone or in association with that of CA125 could offer in the diagnosis and in the prognosis and the forecast of survival endometrial carcinoma [30][31]. Degez et al. [30] reported that the sensitivity of HE4 in the diagnosis of endometrial cancer ranged from 44 to 91% for a specificity of 65 to 100%. Serum HE4 levels were related to prognostic factors such as the extent of invasion of myometrium, grade, and stage of carcinoma and presence of lymph node metastases. Behrouzi et al. [31] summarized the performance of serum HE4 as a diagnostic, prognostic, and predictive marker for relapse of endometrial cancer or response to progestogen therapy, especially in women not eligible for classic surgical management through hysterectomy. Serum HE4 appears to be a promising biomarker. However, its clinical use requires considering the limits of confounding variables like age, BMI, smoking habit, respiratory diseases, and the presence of renal failure, which may cause false positives [10][11][12][31]. Furthermore, the threshold levels of serum HE4 chosen by various authors to define the risk of the presence of carcinoma are at least disparate [31].


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