HE4 as a biomarker for ovarian and endometrial cancer management

Similar to OC, endometrial malignancies tend to carry favorable prognoses when early detection is realized. Diagnosis at a later stage, however, typically coincides with a poor prognosis and a relatively low 5-year survival rate. Fortunately, the presentation of signs and symptoms early on in the course of the malignancy, including postmenopausal bleeding, often precludes late diagnosis beyond the first stage. As a result, approximately 70% of ECs are diagnosed as stage I malignancies. Even so, the remaining 30%, along with certain high-risk groups, such as patients with human nonpolyposis colorectal cancer syndrome, phosphate and tensin homolog gene defects (Cowden syndrome), obesity, diabetes or breast cancer patients on tamoxifen, would certainly benefit from the advent of a reliable serum-marker panel to aid early diagnosis. Noninvasive means for detecting EC may improve detection, aid in the diagnosis of recurrence, and help monitor response to therapy. Some patients with stage I disease will carry intermediate- to high-risk factors such as depth of invasion and lymphovascular involvement, and may benefit from a reliable means of disease monitoring. Furthermore, an accurate biomarker for EC might provide preoperative prognostic value to help guide the extent of surgical staging, thus contributing to the goal of improving overall patient care [ 76 ]. Presently, recurrent EC is detected through presentation of clinical symptoms and imaging techniques, which generally only leads to advanced-stage detection. The use of CA-125 assays for recurrent EC detection is largely limited to later-stage detection since only 10–20% of women with stage I EC and 25% of those with asymptomatic recurrences will have elevated serum CA-125 levels [41,42,76–81]. Taken together, these data suggest that a more reliable EC biomarker is needed. Only a handful of research groups have begun investigating HE4's efficacy as a serum marker for endometrial malignancies. Data from these studies indicate a promising value for HE4 as a component of the biomarker panel in EC detection. Moore and colleagues measured preoperative serum samples from patients with endometrioid adenocarcinoma [ 76 ], along with healthy postmenopausal women as controls, for levels of HE4, soluble mesothelin-related peptide, CA72-4 and CA-125. The results suggested that, as a single marker for EC, HE4 was the most accurate of the group regardless of stage. The single marker ROC-AUC values for HE4 were higher than all other markers investigated for stage I, stages II–IV and all stages combined (ROC-AUC: 76.7, 83.6 and 78.7%, respectively). The sensitivity of the HE4 assay was also highest of all other single markers regardless of stage (sensitivity at 90% specificity for stage I, stages II–IV and all stages combined: 48.4, 71.4 and 55.0%, respectively). The addition of CA-125 to the HE4 assay considerably increased the sensitivity compared with that achievable by CA-125 alone (50.1 vs 24.6% at 95% specificity, respectively). The dual-marker combination assay of CA-125 plus HE4 was also advantageous according to ROC-AUC analysis compared with HE4 alone for stages II–IV (ROC-AUC: 86.6 vs 83.6%, respectively), but had no such advantage over HE4 alone in the analysis of stage I malignancies (ROC-AUC values for HE4 alone were identical to those for CA-125 plus HE4; 76.7%) or when all stages were combined (ROC-AUC for HE4 alone vs HE4 plus CA-125: 78.7 and 79.4%, respectively). These data suggest that HE4 is the most accurate and sensitive EC marker identified to date, and as such will likely prove beneficial alone or as a component of a biomarker panel for the detection of EC. Moreover, CA-125 is frequently used for monitoring response to treatment in EC patients, and HE4's considerable advantage over the others (including CA-125) in identifying stage I endometrioid adenocarcinomas indicates its potential prognostic value in the earlier detection of EC recurrence. In their analysis of serum HE4 and CA-125 levels in women with EC, OC, endometriosis and disease-free controls, Huhtinen et al. also reveal data suggestive of a role of HE4 in both OC and EC detection. HE4 mean serum levels were considerably elevated in both ovarian (1125.4 pM; p < 0.001) and endometrial (99.2 pM; p < 0.001) malignancies, but not in ovarian endometriotic cysts (46.0 pM) or other forms of endometriosis (40.5 pM), compared with the healthy control group. Serum CA-125 levels, on the other hand, were significantly elevated in OC (1117.1 U/ml; p < 0.001), aggressive endometriotic lesions with deep myometrial/peritoneal invasion (40.8 U/ml), and ovarian endometriotic cysts (44.3 U/ml), but not in the EC group (22.0 U/ml; p = 0.029), compared with healthy controls (8.9 U/ml) [ 73 ]. This study further corroborates HE4's superiority over CA-125 in discriminating between malignancies and benign diseases.

Cancer antigen 125 was first identified in 1981 by Bast et al. as a protein that is elevated in the serum of more than 80% of women diagnosed with epithelial ovarian cancer [ 20 – 22 ]. CA-125, a transmembrane protein with a large, glycosylated extracellular domain, resides on the luminal-cell interface and is actively secreted into the lumen where it is detected in the serum. Although it is currently the only serum tumor marker routinely used for detection of epithelial OC [ 19 , 20 ], CA-125 is expressed in a variety of coelomic-derived epithelial cells, including pleura, pericardium, peritoneum and Müllerian epithelia [ 21 , 23 ]. Its success as a molecular marker for OC detection stems from its low expression in normal ovarian epithelium coupled with a significant upregulation in serous and papillary tumors [ 21 , 24 ]. Moreover, the fact that CA-125 is luminaly secreted allows for relatively easy, noninvasive detection in a patient's serum. In addition to its application for OC detection, several studies have suggested that CA-125 also has a promising potential as a prognostic marker. After total surgical removal of ovarian tumors, the CA-125 half-life is approximately 6 days, and results from a number of longitudinal studies indicated that women with a CA-125 half-life of greater than 20 days have a high potential for recurrence and a poor prognosis. Moreover, the initial level of CA-125 prior to therapeutic intervention has been shown to have some prognostic significance [ 19 , 25 – 30 ]. In fact, evidence shows that the serum half-life of CA-125 is an independent prognostic indicator for survival, rate of progression, time to progression, and the potential for total remission. A relatively high false-positivity (up to 20%) associated with this method has limited its usefulness for the assessment of individual patients [ 19 , 25 , 31 , 32 ]. Nevertheless, it is suggested that this system could serve as an effective surrogate for evaluating new therapeutic agents in clinical trials, thus serving an obvious benefit to both patients and clinicians [ 19 ]. The WHO and the Response Evaluation Criteria in Solid Tumor (RECIST) group have identified predictors to evaluate progression-free survival in clinical trials; however, the criteria neglects the use of CA-125 measurements in their definition of progression [ 21 ]. The Gynecologic Cancer Intergroup proposed a definition of disease progression that features both CA-125 concentrations and RECIST criteria [ 21 , 33 ]. For patients whose CA-125 levels have returned to normal following treatment, progression is defined as a twofold or more increase of CA-125 over the upper limit of normal on separate occasions at least 1 week apart. For those patients with elevated pretreatment CA-125 levels that fail to normalize, twofold or more increase of CA-125 over the nadir level is the criteria for progression. This definition was used retrospectively to compare the date of disease progression determined by CA-125 levels versus that determined by clinical and radiological criteria. Based on data from a trial of cisplatin and paclitaxel versus cisplatin and cyclophosphamide [ 34 , 35 ], the average time to determine progression was 54 days earlier when both criteria were employed, and the date of progression based on CA-125 measurement was up to 650 days earlier than the clinical diagnosis of progression [ 21 , 34 , 35 ]. Despite the many promising characteristics of CA-125 for OC diagnosis and monitoring, there are also several drawbacks. For example, in addition to marked overexpression in ovarian lesions, serum CA-125 is frequently elevated in association with irritation of the peritoneum and mesothelium caused by benign conditions such as menstruation, pregnancy and the postpartum state [ 21 , 36 ]. Likewise, elevated serum CA-125 concentrations are often seen in other diseases, such as liver disease (i.e., cirrhosis), congestive heart failure and primary liver cancer, especially if ascites is present. Furthermore, women with nonmalignant gynecological conditions, including benign ovarian cysts and endometriosis, tend to have serum CA-125 concentrations above the upper limit of normal, which is arbitrarily set at 35 U/ml. As a result, false-positives and low specificities are major concerns in regard to the use of CA-125 as an OC marker [ 19 – 21 , 37 ]. The low prevalence of OC (30–50 cases/100,000 women) further limits the achievable positive and negative predictive values of a population-wide, CA-125 screening test [ 38 ]. In fact, many specialists advise against CA-125 monitoring altogether because of the anxiety and stress it affords patients. Similarly, physicians struggle with the decision to start chemotherapy on asymptomatic patients with rising CA-125 levels. Consequently, several studies and clinical trials are currently underway to develop a more reliable detection system, many of which employ a panel of two or more complementary markers. Considerably, fewer studies have been performed to evaluate the efficacy of CA-125 for EC detection and/or monitoring. This is partially due to the fact that timely EC detection (relative to OC) is often realized by virtue of early presentation of symptoms, such as vaginal bleeding. Nevertheless, EC's high incidence rate, together with the poor prognosis for advanced-stage patients justifies the need to identify diagnostic and prognostic biomarkers for EC. Although CA-125 is elevated in EC patients relative to healthy control subjects, this upregulation is slightly below the clinically defined cut-off point for OC diagnosis [ 39 , 40 ]. Moreover, serum concentrations of CA-125 are elevated in only 10–20% of women with early-stage EC, and only 25% of asymptomatic patients with recurrences will present with elevated CA-125 levels [ 18 , 41 , 42 ]. Thus, CA-125's application for EC detection is essentially restricted to advanced-stage diagnosis. Yurkovetsky and colleagues highlighted this idea in a recent publication confirming the presence of considerably elevated serum cancer antigen levels (CA-125, CA15–3 and carcinoembryonic antigen) in stage III EC patients compared with women with stage I disease. The authors suggested that this may be caused by the ‘shedding’ of cancer antigens during EC progression and/or disease aggressiveness, thereby depositing increasing amounts of these proteins into the lymphatic system as the malignancy advances in stage. Conversely, that same tumor aggressiveness and potential for metastasis may actually drive the upregulation of cancer antigens [ 40 ]. Of course, these are not mutually exclusive mechanisms as both can be at play simultaneously. Regardless of the culprit, the fact remains that no accurate biomarkers for EC detection are currently available. Recently, Yurkovetsky's group showed evidence of EC screening potential for prolactin, a single-chain peptide from the growth hormone family. The primary source of prolactin is the anterior pituitary gland, yet endometrial stroma also produce the protein during the secretory phase of the menstrual cycle. Studies have shown that prolactin's function is not limited to the regulation of breast development and lactation. Prolactin also acts as a cytokine with central roles in the immune and inflammatory processes [ 40 , 43 , 44 ]. Additionally, prolactin is able to serve as a paracrine/autocrine hormone, thereby influencing local angiogenic responses [ 40 , 45 , 46 ]. Given that blood vessel growth and remodeling is a key factor in cancer metastasis and lymphvascular invasion, prolactin could influence advancement of EC and other malignancies [ 40 ]. Accordingly, prolactin may not only serve as a marker for early detection of EC, but could also allow for reliable risk assessment for metastatic potential and/or probability for lymph node invasion. Early studies assessing prolactin's efficacy as an EC marker indicated its effectiveness in identifying recurrent diseases [ 40 , 47 ]. Recent data from Yurkovetsky and colleagues has also suggested that prolactin's diagnostic power in discriminating EC from healthy controls is superior to all other biomarkers examined to date. In their study involving 115 EC patients and 135 healthy control females, prolactin serum-marker assays were able to identify EC with a sensitivity of 98.3% and a specificity of 98%. No significant advantage was realized upon addition of other markers to the prolactin assay. Thus, prolactin measurement is sufficient to discriminate patients with cancer from healthy women. Since elevated levels of prolactin were detected in other cancers, including malignancies of the ovary, pancreas and lung, the authors concluded that the prolactin assay alone is not able to discern between the various cancer types and, consequently, cannot be used alone in the diagnosis of EC. The authors subsequently identified a panel of five markers, which included prolactin, eotaxin, growth hormone, E-selectin and thyroid-stimulating hormone. This combination has shown specificity for EC over OC and breast cancer, but it remains to be tested if it is effective in distinguishing EC from benign gynecologic diseases, such as endometriosis [ 40 ]. Future studies of prolactin's use as a biomarker for EC will need to address this issue prior to clinical trials. Besides CA-125 and prolactin, many potential markers have been investigated in the past decade. A comprehensive list of putative biomarkers for ovarian and ECs is provided in Table 1 . Crucial information on their specificity and sensitivity is also compiled. Owing to their in-depth coverage within the body of this review, HE4 and CA-125 have been excluded from the table.

HE4 ( WFDC2 ) was first identified and characterized by Kirchhoff et al. upon differential cDNA screening of human epididymal tissue [ 48 , 49 ]. Subsequent studies revealed the expression of HE4 in a number of tissues outside of the male reproductive system. Using northern hybridization, Bingle et al. detected HE4 mRNA expression in lung, kidney and salivary gland [ 50 ]. Galgano et al. analyzed HE4 expression patterns in normal and malignant human tissues using a cDNA microarray. HE4 is expressed in relatively high levels in the trachea and salivary gland [ 51 ]. Applying quantitative realtime PCR, our laboratory has detected high levels of HE4 mRNA in the epididymis, trachea and lung, and intermediate levels in prostate, endometrium and breast. Little or no HE4 expression was detected in the colon, ovary, liver, placenta, peripheral blood cells and skeletal muscles [Jiang SW, Unpublished Data]. The deduced amino acid sequence of HE4 predicts a significantly large and odd-numbered cysteinyl content, suggesting that HE4 may participate in protein–protein interactions [ 48 , 49 ]. Further analysis showed that HE4 contains two whey acidic protein (WAP) domains. This conserved domain is common to whey proteins in the milk of several mammals and is characterized by a four-disulphide core arrangement of 50 amino acids, including eight cysteines. Generally, WAP domain-containing proteins are small, secretory molecules. Since the WAP domain proteins carry serine protease inhibitory activities and are secreted by pro-inflammatory cells, these proteins were thought to play a part in the natural defense against microorganisms [ 52 ]. Later in vitro studies using cloned WAP cDNA indicated varied functions that often include effects on cell growth and differentiation [ 50 , 53 , 54 ]. HE4 is one of several WAP proteins that are localized on human chromosome 20q12–13.1 [ 55 ]. Interestingly, results from several comparative genomic hybridization assays suggested that the 20q13 locus frequently exhibits chromosomal gains in various cancer types, including malignancies of the oral cavity, breast, ovary, colon, pancreas, stomach and uterus [ 56 – 59 ]. Indeed, this locus harbors several WAP proteins, including elafin and secretory leucocyte proteinase inhibitor (SLPI), that have been identified as candidate biomarkers for a number of cancers [ 60 ]. Although a significant proportion of those proteins containing WAP domains possess protease inhibitor function (e.g., SLPI and elafin), no such protease inhibitor activity has been assigned to HE4. While the high expression levels in epididymis suggest that HE4 may be involved in male fertility, the physiological role of HE4 has yet to be determined [ 61 , 62 ].

Although an inexpensive, sensitive and specific serum test would be the most attractive approach to screen women for ovarian and endometrial cancer, fundamental limitations of this method must be recognized. Measurement of CA-125 is very useful in the clinic setting for detecting recurrence in patients who have a known diagnosis of ovarian cancer in the past, and who displayed elevated CA-125 levels at diagnosis. In fact, over 95% of CA-125 elevations in this patient population are due to recurrent cancer. This is because of the high incidence of recurrence in this population. On the other hand, CA-125 is a very poor test to screen for ovarian cancer in the general population because of the low incidence. A screening test for ovarian cancer must have a very high specificity, over 99.9%, in order to be useful clinically and avoid unnecessary surgery in large numbers of patients. One of the strengths of HE4 is that it appears to correctly identify benign lesions in comparison to CA-125. But owing to the extreme heterogeneity of genetic abnormalities observed in ovarian cancer, such a screening test would require the use of multiple markers, of which CA-125 and HE4 may be a component. To achieve high specificity other modalities are likely to be required to be used in combination with serum markers, such as ultrasound and high-resolution MRI. Furthermore, patients at risk must be identified to improve the utility of such a test. For example, morbidly obese women have a risk of endometrial cancer that is ten times the risk of women of normal weight. Thus, a screen for endometrial cancer is likely to be most useful and most cost effective in obese women. Future efforts to diagnose ovarian and endometrial cancer in early stages will be dependent not only on improving screening methods, but also on continued epidemiologic investigation. The promise of effective, innovative and safe treatments for advanced endometrial and ovarian cancer has been slow to be realized over the past decade. With continued persistence and cooperation between disciplines, early diagnosis in targeted populations at high risk may become a reality and significantly reduce the impact of these diseases in women.

New technologies for diagnosis of EC and OC in early stages are urgently needed. This need is particularly apparent for women with ovarian cancer as the majority are diagnosed in advanced stages with low rates of cure. Many approaches have been tested, so far without success. Most recently, Petricoin et al. reported on the use of proteomic spectra generated by mass spectroscopy that completely discriminated serum from patients with and without ovarian cancer. However, that technique was ultimately discredited and now, 7 years later, we are still without an effective screen for ovarian cancer. Until now, CA-125 has been the most effective serum marker for ovarian cancer. In this review we discuss findings showing that HE4 may correlate even better than CA-125 with the presence of ovarian and endometrial cancer. Ultimately, a panel of markers, including CA-125 and HE4 may prove clinically useful. The most difficult challenge will be to identify the most effective markers to be used in that panel. This challenge is even more problematic with ovarian cancer, as the heterogeneity of gene expression between different tumors appears to be much more pronounced than in cancers of other organ systems.

Both ovarian and endometrial tissues develop from the Müllerian system [ 10 – 12 ] and are subject to tight control by reproductive hormones. As such, cancers arising from the two tissues share similarities in etiological factors, gene expression profiles, tumorigenic mechanisms, pathological changes and metastatic characteristics. For example, cyclin E and L1 adhesion molecule were independently found to be overexpressed in Müllerian-derived carcinomas [ 13 , 14 ]. Further, histological analysis of different cytological subtypes of EC and OC indicate a parallel spectrum of morphological changes [ 15 , 16 ]. Zorn and colleagues' analysis of gene expression profiles between subtypes of the two malignancies reveals a related expression profile in the clear-cell subtype across different organs. On the other hand, endometrioid and serous subtypes displayed expression profiles that were, for the most part, unique to their organ of origin. The authors were able to identify nine genes common to both diseases that were capable of distinguishing among the various histotypes [ 16 ]. Moreover, comparisons between uterine and ovarian carcinosarcomas, rare but characteristically aggressive gynecologic tumors, reveal no differences in patient demographics or overall survival for women presenting with these malignancies, perhaps lending justification for the use of similar therapeutic strategies in combating these diseases [ 17 ]. In addition, EC and OC share many of the same risk factors, including, nulliparity and frequency of ovulation [ 10 , 12 ]. The use of oral contraceptive hormones has been shown to reduce the risk of developing either condition [ 10 ]. In view of these parallels between EC and OC, it should be no surprise that a single marker or marker panel could be capable of recognizing both conditions. CA-125 has been found to be overexpressed in both OC and EC tissues, and increased CA-125 serum levels have been evaluated as a biomarker for the detection and monitoring of both malignancies. However, the unsatisfactory sensitivity and specificity of the CA-125 assay has prompted intensive efforts in the search for superior biomarkers [ 18 – 20 ].