HE4 Tissue Expression and Serum HE4 Levels in Healthy Individuals and Patients with Benign or Malignant Tumors: A Systematic Review

The rapid development in bioinformatics and molecular techniques has accelerated the intense search for tumor markers detectable in tissues and/or bodily fluids to correctly diagnose cancer or identifying subgroups of patients for special treatment. For this purpose, the glucoprotein human epididymis protein 4 (HE4) encoded by the Whey-Acidic Four-Disulfide Core domain protein 2 (WFDC2) gene has received attention (1). HE4 is a secretory protein originally identified in the distal human epididymis (2). The function of HE4 has not been definitely demonstrated; however, HE4 shows significant structural similarity to proteinase inhibitors and is proposed to have a function in sperm maturation (1–4). Widespread expression of HE4 has since been demonstrated in several normal tissues, especially in the epithelia of the respiratory and reproductive tracts of both genders except from the ovaries, where no expression is seen (5–8).

Increased HE4 tissue expression has been demonstrated in a range of malignant neoplasms, especially of gynecologic and pulmonary origin (5, 6, 9, 10). Corresponding, significantly elevated HE4 serum levels have been widely investigated in patients with ovarian cancer (5, 7, 11–13), and large studies have reported serum HE4 as a putative tumor marker for differentiating between benign gynecologic tumors and ovarian cancer, claiming HE4 to be as good as the clinically approved serum cancer antigen 125 (CA125) used in ovarian cancer risk management worldwide (14–16). In 2008, the serum HE4 EIA analysis was cleared by the Food and Drug Administration (FDA) in the United States as a diagnostic tool to aid in the diagnosing process of ovarian cancer (17). So far, no comprehensive overview of HE4-expressing tissues and conditions influencing serum levels of HE4 exist. The aim of this review is to provide an outline of published studies investigating HE4 tissue expression in normal, benign, and malignant tissues, and to report the different clinical conditions in which serum HE4 levels are influenced. Possible limitations or benefits of HE4 in a clinical setting will be addressed.

This systematic review was performed with guidance from the general principles of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The PubMed and Scopus databases were used in search for literature. Two reviewers (N.S. Karlsen and M.A. Karlsen) were primarily responsible for literature search, and each reviewer double-checked the relevancy of the literature found. The keywords “Human Epididymis protein 4,” “HE4,” or “WFDC2” were combined with the terms “gene/tissue/protein expression,” “serum/blood level/concentration,” “bio/tumour marker,” and “cancer/malignancies.” When HE4-expressing tissues or conditions influencing serum HE4 concentration were revealed, the respective terms were used in further literature search (Fig. 1). We also performed a screening for literature in reference sections of relevant studies. We aimed to cover a wide spectrum of published studies investigating RNA/protein expression and serum concentration of HE4, yet large and recent studies published in English were favored, whereas articles with no clear definition of methods to analyze expression or concentration levels in tissue or serum were excluded. A total of 42 studies published from 2000 to 2014 were included. Because of discrepancy in expression quantification among the studies included, a consistent definition of tissue expression levels was used in this review described as low, moderate, or high, based on the study results of the individual article as well as assessed in relation to other studies included in the review.

HE4 tissue expression and serum levels are analyzed by a variety of methods. HE4 RNA expression was for the majority of the referred studies measured as mRNA assessed by either the use of microarray technology or quantitative real-time PCR (qRT-PCR; Table 1). HE4 protein expression was measured by immunohistochemistry using either tissue microarray (TMA) or whole-tissue slides. Different HE4 antibodies were used in the reported studies (Table 1). High concordance has been demonstrated between the results obtained using microarray and qRT-PCR (>87%; P < 0.05; ref. 18); however, so far no study compared the concordance between protein expression levels using different HE4 antibodies. Serum HE4 is determined by HE4 immunoassays, and several standardized assays have been developed (19, 20). Different assays for measuring HE4 serum levels may not result in identical results. Discrepancy in HE4 levels has been demonstrated when comparing two different manufacturers' HE4 assays: Fujirebio HE4 assay measuring 14% (11.3%–16.9%) higher serum HE4 levels compared with the Abbott Architect assay (21). The influence of storage temperature on HE4 stability has only been investigated by one study, recommending HE4 serum and HE4 EDTA plasma to be stored at −80°C for longer-term storage to maintain stability (22). For the majority of the studies included, serum samples have been stored at either −80°C or −70°C (Table 2).

Tables 1 and 2 demonstrate general study conditions, expression quantification, case numbers, and sensitivities and specificities of all studies included. In Table 1, it is further specified which studies had published microarray data of HE4 expression according to the Minimum Information About a Microarray Experiment (MIAME) guidelines (Table 1; refs. 23, 24).

The highest expression of HE4 RNA and protein are reported in the glandular epithelium of the female and male genital tracts and the respiratory tract (5, 6, 8). In the male genital tract, HE4 is highly expressed in the epithelial cells of the epididymal and spermatic ducts, whereas no expression is found in the surrounding stroma of the testes (5, 6). Low focal expression of HE4 is inconsistently found within the glandular epithelium of the prostate (5, 6) High HE4 expression in the female genital tract is seen in the fallopian tubes, endometrium, and cervical glands. Low HE4 expression is detected in the Bartholin glands, and no expression is seen in the myometrium, vulva, and ovaries (5, 6, 9). In the respiratory tract, HE4 is highly expressed in the epithelium of the oral cavity, the excretory ducts of the salivary glands, nasopharynx, and especially the trachea (5, 6, 8), whereas results of HE4 expression in the lungs are diverging. Three lung studies have detected HE4 RNA and protein expression in the lung tissue (5, 6, 8); however, two other studies could not confirm these findings (25, 26).

Focal and low HE4 expression is detected in the distal convoluted tubules of the kidney, and no expression is found in the rest of the kidneys, ureteres, bladder, and urethra (5, 6, 9). Breast epithelium demonstrated variably HE4 expression with higher staining in the ducts compared with the lobules (5, 6). Low and focal HE4 expression is seen in scattered cells in the anterior pituitary, thyroid, lacrimal, and eccrine sweat glands, whereas no expression is seen the adrenal cortex and medulla (5, 6). Expression is absent in the gastrointestinal canal (5, 6, 10, 27), as well as in hematolymphoid tissue (bone marrow, lymph nodes, spleen, thymus, and tonsils), musculoskeletal tissue (cartilage, fat, skeletal muscle, and synovium), neural tissue, skin, and vascular tissue (aorta, heart, and lymphatics; ref. 5).

The amount of studies investigating HE4 tissue expression in benign tumors is sparse. To our knowledge, HE4 tissue expression has only been demonstrated in various benign ovarian tumors, endometriosis, preneoplastic metaplasias of the upper gastrointestinal canal, and oncocytomas of the kidney. One study investigated HE4 expression in benign ovarian tumors, including benign serous (N = 12) and mucinous (N = 12) cystadenomas, ovarian endometriosis (N = 12), and benign ovarian surface inclusion cysts (N = 12; ref. 9). Moderate-to-high HE4 tissue expression was detected in 11 of 12 mucinous cystadenomas and 11 of 12 ovarian surface inclusion cysts, whereas 5 of 11 serous cystadenomas demonstrated low expression. Moderate-to-high expression was detected in all samples of ovarian endometriosis analyzed (9). Furthermore, Drapkin and colleagues (6) found that HE4 expression in 11 ovarian surface inclusion cysts was mainly present in cysts lined by the Mullerian epithelium, compared with no HE4 expression in the cysts that kept the flat morphology of the ovarian surface epithelium. In the upper gastrointestinal canal, HE4 expression has been investigated in preneoplastic metaplasias of the esophagus, stomach, and pancreas. In 5 of 12 samples of Barret esophagus scattered HE4 expression was demonstrated (10). In the stomach, intestinal metaplasia and spasmolytic polypeptide–expressing metaplasia showed high HE4 expression (27). Furthermore, elevated HE4 tissue expression was demonstrated in gastric epithelium from stomachs infected with Helicobacter pylori, associated to gastric cancer (28). In the pancreas, pancreatic intraepithelial neoplasia has been investigated and no HE4 expression was seen in the tissues analyzed (N = 55; ref. 10). Finally, low to high expression was demonstrated in eight of 12 oncocytomas from the kidney (5).

In the female genital tract, high HE4 tissue expression is consistently found in epithelial ovarian adenocarcinomas of serous histology, whereas a lower and more inconsistent HE4 expression is found in endometrioid and clear cell histology. No HE4 expression is detected in epithelial ovarian cancer of mucinous histology (5, 6, 9), as well as no expression is present in nonepithelial ovarian cancer, including sex cord stromal tumors (nine granulosa cell tumors) and germ cell tumors (four dysgerminomas and a single yolk sac tumor; ref. 5). Furthermore, HE4 expression is reported in serous borderline tumors of the ovary, yet, with weaker expression compared with HE4 expression in epithelial ovarian cancer, and diverging results exist regarding the mucinous borderline ovarian tumors (5, 9). High HE4 expression has been demonstrated in primary tubal carcinomas (N = 12; ref. 9) and endometrial carcinomas (5, 6, 9, 29, 30). Bignotti and colleagues (29) found that HE4 tissue expression was significantly higher in endometrial carcinoma tissues compared with surrounding normal endometrium (P < 0.0001), and HE4 expression in the endometrioid subtype was significantly higher compared with nonendometrioid subtype (P = 0.016).

In the respiratory tract, one study has demonstrated moderate-to-high HE4 expression in various malignant neoplasms of the salivary gland (N = 24; ref. 5). In addition, HE4 expression has been demonstrated in several lung cancer cell lines (7, 31). Compared with small cell, squamous, and large cell lung cancer, adenocarcinomas of the lung demonstrate the highest HE4 tissue expression (5, 8, 25, 26). Finally, Galgano and colleagues (5) demonstrated low to high HE4 expression in 28 of 47 mesotheliomas investigated.

Two studies have investigated HE4 tissue expression in carcinomas of the breast, and found a moderate increase in HE4 expression of ductal carcinomas (5, 32).

In the gastrointestinal canal, HE4 tissue overexpression is seen in gastric cancer (27) and pancreatic cancer as well as occasionally in colon and hepatocellular cancer (5, 10, 33). In a study cohort comprising more than 550 tissue samples from all stages of gastric cancer, high HE4 tissue expression was demonstrated in 70% of intestinal type, in more than 90% of diffuse type and in 75% of mixed type (10). In 103 of 220 pancreatic ductal adenocarcinoma samples (172 individual patients), low HE4 expression was seen in more than 5% of the epithelial cancer cells (10), a finding supported by two other studies (5, 33). Low HE4 RNA expression was demonstrated in 18 of 23 colon cancer samples and all seven hepatocellular carcinomas investigated. However, when performing immunohistochemical staining for HE4 protein expression, merely 6 of 27 colon cancer samples demonstrated low HE4 protein expression, whereas no HE4 expression was detected in the hepatocellular carcinomas investigated (5).

In the urologic tract, low to high HE4 expression is seen in clear cell carcinomas of the kidney, papillary renal cell carcinomas, and chromophobe carcinomas of the kidney (5, 7, 11). Also transitional cell carcinomas have demonstrated high or moderate RNA expression in only two of eight samples investigated, whereas prostatic adenocarcinomas in the male genital tract show no expression of HE4 (5). Finally, Galgano and colleagues (5) demonstrated no HE4 expression in basal cell carcinomas of the skin.

In healthy individuals, serum HE4 significantly varies between sexes with a pronounced age-related increase (13, 21, 34–36). In a multivariate analysis with 1,591 samples comprising 801 women and 790 men of ages 18 to 86 years, age was shown to be the main determinant of serum HE4 level, and age-dependent reference limits were determined for men and women excluding all smokers. The lowest limit at the age of 18 years was 43.4 pmol/L for men and 51.3 pmol/L for women, whereas the highest limit at the age of 82 years was 78.4 pmol/L for men and 69.7 pmol/L for women (21). Significantly increased HE4 levels are also measured in smokers (29% increase) compared with nonsmokers (P = 0.007; refs. 21, 34, 37). Furthermore, significantly higher median HE4 levels in serum are measured in postmenopausal women compared with premenopausal (P < 0.001; refs. 36, 38), and serum HE4 varies within the different phases of the menstrual cycle (39, 40).

Finally, patients with renal failure have been demonstrated to have significantly increased serum HE4 concentration correlating to elevated serum creatinine levels (13, 21, 35, 41, 42). One study measured serum HE4 in a group of 113 female patients at different stages of chronic kidney disease with no history of ovarian cancer or lung cancer. The HE4 cutoff values were 70 pmol/L in premenopausal women and 140 pmol/L in postmenopausal women. Compared with 68 healthy females, a significant positive correlation between increasing severity of renal failure and elevated serum HE4 levels was found in both the pre- and postmenopausal group (P = 0.003–0.0001 and P = 0.001–0.0001, respectively; ref. 41). Liver disease has been proved to induce only small, yet, significant increases in serum HE4 levels (P = 0.001) in one study (13).

Significantly elevated levels of serum HE4 are detected in patients with epithelial ovarian cancer, endometrial cancer, lung adenocarcinomas, and transitional cell carcinomas when compared with serum HE4 levels in benign and healthy individuals. The most pronounced increase of serum HE4 is measured in patients with epithelial ovarian cancer, especially in serous and endometrioid epithelial ovarian cancer (13, 35). Using the serum HE4 concentration 70 pmol/L as cutoff value (14), serum HE4 has been demonstrated by several to be significantly increased in women with epithelial ovarian cancer compared with healthy controls and various benign gynecologic diseases (13, 14, 16, 30, 42–44), where a modest serum HE4 increase can be detected (14, 30, 44, 45). HE4 has been demonstrated to yield a higher sensitivity and specificity than that of CA125 serum marker when differentiating epithelial ovarian cancer from benign gynecologic disease, and when serum HE4 is combined with serum CA125, the diagnostic accuracy increases even further (13–16, 42–44, 46). Serum HE4 is significantly elevated in women with endometrial cancer regardless of stage compared with healthy, postmenopausal women (P < 0.001; refs. 29, 30, 47, 48). For all stages of endometrial cancer (N = 171), Moore and colleagues (47) found that serum HE4 had a sensitivity of 45.5% at a predefined specificity of 95% compared with a sensitivity of 24.6% for CA125 when differentiating from healthy individuals, whereas Zanotti and colleagues (47) found a sensitivity at 66% for HE4 at a set specificity at 95%. Combined with CA125, the sensitivity increased to 50.1% (47). When distinguishing patients with endometrial cancer from women with benign disease of the uterus, Angioli and colleagues (50) found a sensitivity of 59.4% and a specificity of 100% at a HE4 cutoff level of 70 pmol/L.

Significantly elevated level of serum HE4 has been detected in patients with primary lung adenocarcinoma compared with levels in healthy control subjects and patients with benign lung tumor (P = 0.0001; refs. 13, 25, 26, 35, 37, 51, 52). One recent study investigated the diagnostic eligibility of serum HE4, and found a sensitivity of 67.9% at a specificity of 93.4% using an optimal cutoff value of 77.48 pmol/L when differentiating patients with lung cancer (N = 190) from healthy controls (N = 106; ref. 53). Nagy and colleagues (37, 52) found a sensitivity of 64.3% and specificity of 95.9% using a cutoff value of 97.6 pmol/L; however, HE4 was not shown to be superior to other tumor markers investigated: CEA, TPA, Cyfra 21-1, and CA125. Only if serum HE4 was combined with CEA and CA125, the diagnostic efficacy was enhanced yielding a sensitivity of 91.8% and a specificity of 92.8% (37).

In a study comprising 222 serum samples, HE4 serum levels were significantly higher in patients with transitional cell carcinomas (N = 102; median, 66.7 pmol/L; mean, 42.1 ± 108.8), compared with HE4 levels in patients with benign urinary diseases (N = 60; median, 58.3 pmol/L; range, 51.8–63.6; P < 0.001) and healthy controls (N = 60; median, 37.3 pmol/L; range, 28.9–44.4; P < 0.01). The sensitivity and specificity of HE4 at a cutoff value of 45.7 pmol/L were 67.6% and 88.3%, respectively (54).

Park and colleagues (42) found that serum HE4 level was significantly increased in patients with pancreatic cancer measuring a median of 213 pmol/L. Yet three other studies investigating serum HE4 levels in patients with various malignancies of the gastrointestinal canal, including pancreatic cancer, found only a nonsignificant elevation compared with levels found in healthy patients (13, 26, 43).

Escudero and colleagues (13) found that serum HE4 was significantly related to the presence of liver metastases from any cancer origin (P = 0.001). Furthermore, Elsammak and colleagues found that in 88 patients with different types of pleural effusions, both serum HE4 levels and pleural effusion HE4 levels were significantly higher in patients with malignant effusions than in patients with transudative or nonmalignant exudative effusions (P < 0.001 and P = 0.002, respectively). In pleural fluid a cutoff value of 1.675 pmol/L was found to predict malignant pleural effusions with a diagnostic sensitivity of 85.3% and specificity of 90.7% (53).

The purpose of this review was to provide a comprehensive outline of HE4 expression in normal, benign, and malignant human tissues and HE4 serum levels to evaluate the clinical eligibility of HE4 as a potential tumor marker. The vision of HE4 as a potential tumor marker has expanded through the past decades due to an increase of studies demonstrating an upregulation of HE4 in various meta- and neoplasias occasionally followed by elevated levels of the substance in blood.

A prominent upregulation of HE4 expression was seen in epithelial ovarian cancer tissue, especially in serous and endometrioid adenocarnomas. No expression was detected in normal ovarian tissue, and a lower expression was observed in both benign and borderline ovarian tumors compared with protein expression levels in epithelial ovarian cancer. This finding is clinically relevant, because the morphologic distribution of HE4 protein expression in whole-tissue slides or TMA's could be a marker used in the diagnostic process of epithelial ovarian cancer. Furthermore, epithelial ovarian cancer can be differentiated from nonepithelial ovarian cancer, because no expression was demonstrated in nonepithelial ovarian cancer. A significantly higher level of HE4 in serum was also detected in women with epithelial ovarian cancer compared with the HE4 serum levels in healthy women and women with a benign disease. In combination with serum marker CA125, a significant increase in sensitivity and specificity was revealed in differentiating between benign gynecologic conditions and epithelial ovarian cancer. Differentiation between benign and malignant diseases is of great importance for correct referral of patients to a tertiary center and thereby optimal treatment and follow-up (55–59). When investigating tissue from the fallopian tube and endometrium, a positive HE4 expression should be more carefully interpreted because a high HE4 expression was present in normal tissue. However, we cannot decline the potential of a HE4 as a tumor marker in endometrial cancer, because HE4 measured in serum from patients with endometrial cancer was significantly elevated compared with serum levels in healthy individuals and women with benign uterine disease, yet, further investigation is needed. Furthermore, additional investigation of HE4 expression pattern in tissue from cervical, vaginal, and vulva cancer from tissue is needed to fully understand the relevance of HE4 in these cancers.

HE4 could potentially be a tumor marker in primary lung adenocarcinomas. A significant upregulation of HE4 was observed in lung cancer tissue compared with normal lung tissue and other primary lung cancers, where none or low expression was present. A significant elevated HE4 level in both serum and pleural fluid was detected in patients with primary lung adenocarcinomas compared with HE4 levels healthy control subjects and in patients with a benign lung tumor. However, to apply HE4 as a diagnostic tumor marker in a clinical setting, further clarification about tissue expression and serum levels in benign versus malignant lung disease as well as substudies of different lung cancers is needed.

Upregulation of HE4 was also demonstrated in malignancies of the gastrointestinal canal, urinary tract, bladder, and breast. However, these findings were demonstrated in few studies investigating relatively small tissue and serum materials. Therefore, we cannot make any conclusion about the clinical eligibility of HE4 as a tissue and serum tumor marker in these cancers, because larger studies are necessary before any statement can be taken. In addition, the inevitable heterogeneity in study conditions regarding specimen storage and analytic methods (Table 1 and 2) could have an impact on study results and also the influence of age, menstrual status, smoking habit, and renal function has on serum HE4 levels in healthy subjects were not considered in many study cohorts.

The widespread upregulation of HE4 seen in a range of malignancies implies that HE4 is neither organ nor tumor specific. Therefore, clinicians must be aware of the possibility for differential diagnoses in case of upregulated HE4 tissue expression or elevated levels of HE4 in serum. The implementation of the HE4 tumor marker test in the clinic would first of all require standardized preanalytic conditions, including specimen handling, storage, and analyzing method (60). Second, a set of well-estimated baseline concentrations should be prepared. So far, no optimal cutoff exist, as well as there is no consensus for correct parameters to include in the examination. Bolstad and colleagues (21) have suggested a set of baseline serum HE4 concentrations for men and women adjusted for age, and two recent studies (36, 38) have suggested reference intervals of serum HE4 in healthy women at 65.87 pmol/L for premenopausal and 90.76 pmol/L for postmenopausal measured in the study with the largest group included (N = 1,515 healthy women; ref. 38). In women with pelvic masses, Fujirebio Diagnostics (19) has defined the normal range below 150 pmol/L, whereas Abbott Diagnostics (20) defined normal ranges below 70 pmol/L for premenopausal women and 140 pmol/L for postmenopausal women. However, both the manufacturers recommend that reference intervals are determined for each population investigated yielding the highest sensitivity and specificity possible.

There is a valuable diagnostic eligibility of HE4 tissue expression and serum levels in patients with a risk of gynecologic malignancies, especially epithelial ovarian cancer. The diagnostic ability of HE4 in patients with risk of lung adenocarcinomas seems promising. More research is needed to correctly evaluate the diagnostic relevance of HE4 in other cancers; however, it is important to recognize that other malignancies can cause increased HE4 expression and HE4 serum levels. Finally, an awareness of the influence of age, menstrual status, smoking habits, and renal function may have on serum level is important for proper interpretation of a serum HE4 test result.

No potential conflicts of interest were disclosed.

The Danish Cancer Society kindly funded a scholarship for the first author (N.S. Karlsen) as part of a Research Year Project.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.