The Emerging Role of HE4 in the Evaluation of Epithelial Ovarian and Endometrial Carcinomas

HE4 (human epididymis protein 4) is overexpressed in both ovarian and endometrial cancers. Levels of the shed HE4 protein are elevated in sera from ovarian and endometrial cancer patients. HE4 is less frequently elevated than cancer antigen 125 (CA 125) in benign gynecologic conditions and is found in a fraction of endometrial and ovarian cancers that lack CA 125 expression. Consequently, HE4 has emerged as an important biomarker that complements CA 125 in discriminating between benign and malignant pelvic masses, monitoring response to treatment, and detecting recurrences of both ovarian and endometrial cancer. The “risk of ovarian malignancy algorithm” (ROMA) incorporates CA 125, HE4, and menopausal status to distinguish benign from malignant adnexal masses, and has been approved by the US Food and Drug Administration to aid in referring patients who are likely to have ovarian cancer to specially trained gynecologic oncologists for surgery. HE4 also promises to augment the sensitivity of CA 125 for detecting early-stage ovarian cancer. In this review, we discuss the discovery and biologic significance of HE4 and evaluate available evidence regarding the utility of HE4 as a biomarker for ovarian and endometrial cancer.

Introduction

Some 22,280 women were diagnosed with ovarian cancer in the United States in 2012, and 15,500 died from the disease.[1] While the cure rate for all stages of ovarian cancer has remained approximately 30%, 5-year survival rates have improved significantly (P < .05)-from 37% in the 1970s to 45% at the turn of the century-as a result of improved surgery and combination chemotherapy with carboplatin and paclitaxel. In contrast to surgical management of cancers at some other sites, gynecologic oncologists attempt to remove as much of an ovarian cancer as possible, even when all macroscopic disease cannot be resected. Prognosis relates to the maximum size of tumor nodules that remain after surgery. Gynecologic oncologists are specially trained to perform “surgical cytoreduction” prior to chemotherapy. Outcomes are improved when primary operations are performed by gynecologic oncologists, but less than half of women are referred to these subspecialists. To facilitate appropriate referral of patients who are likely to have cancer, a “risk of malignancy index” (RMI) was developed in 1990 to identify patients with malignant pelvic masses; it used menopausal status, appearance of the pelvic mass during ultrasonography, and levels of the biomarker cancer antigen 125 (CA 125).[2] In different studies, the sensitivity of RMI for detecting malignant masses ranged from 71% to 88%, and specificity from 74% to 97%,[3] leaving room for improvement in sensitivity, while retaining specificity.

CA 125 is overexpressed in 80% of ovarian cancers. Serum CA 125 levels have been elevated in 50% to 60% of patients with stage I ovarian cancer and in 90% of patients with stage III/IV disease, related to release of CA 125 not only from cancer cells, but also from the inflamed peritoneum. In patients with elevated CA 125 levels, changes in biomarker levels have tracked tumor burden with greater than 90% accuracy. Persistent elevation of CA 125 following chemotherapy has correlated with residual ovarian cancer in 90% of cases, leading to approval of CA 125 by the US Food and Drug Administration (FDA) for detection of disease that has survived primary chemotherapy. Monitoring CA 125 levels in patients with a complete clinical response can detect recurrence of cancer in up to 70% of patients, with an average lead time of 3 to 4.8 months. The clinical value of early detection of disease recurrence has been questioned based on a single study with significant limitations, but it does provide time for patients to receive multiple conventional drugs and to participate in clinical trials with novel agents.[4]

Biomarkers could have the greatest impact on survival as part of an effective screening strategy. When ovarian cancer is detected at an early stage, where the disease is still contained within the ovaries (stage I), 5-year survival rates can approach 90% with optimal surgery and currently available combination chemotherapy. By contrast, ovarian cancer that has spread throughout the peritoneal cavity or outside the abdomen (stages III and IV) is associated with 5-year survival of less than 30%.[1] At present, less than 25% of ovarian cancers are detected in an early stage.[5] Although promising trials are underway in the United States and the United Kingdom, there is no established screening strategy for early detection of ovarian cancer in women at conventional risk for the disease. In women with a strong family history, CA 125 levels and transvaginal sonography (TVS) are recommended for early detection, although there is not yet evidence that this improves prognosis.[5] Prophylactic surgery is recommended for women from BRCA1, BRCA2, or Lynch syndrome kindreds when they have completed their families. Surveillance with CA 125 in premenopausal women is complicated by the fact that antigen levels can be elevated by several physiological and benign conditions, including endometriosis, adenomyosis, uterine fibroids, and normal menstruation, compromising specificity. To achieve a positive predictive value of at least 10% (ie, 10 operations for each case of ovarian cancer detected), a screening strategy must have high sensitivity (> 75%) for preclinical early-stage disease, and an extraordinarily high specificity (> 99.6%).[6] While neither CA 125 nor TVS alone can meet these standards, a two-stage strategy in which a rising CA 125 level triggers TVS in a small fraction of patients has achieved sufficient specificity that only three operations are required for each case of ovarian cancer detected.[7,8] With this strategy, early-stage disease has been detected, but there is still a need to improve sensitivity, since CA 125 testing will miss at least 20% of cases. Use of multiple biomarkers, including human epididymis protein 4 (HE4), could fill this need.

In 2012, an estimated 47,130 women were diagnosed with endometrial cancer and 8,010 women died from this disease.[1] In contrast to the lack of specific symptoms in ovarian cancer, endometrial cancer is typically associated with postmenopausal vaginal bleeding, leading to early-stage (stage I) detection in almost 70% of cases-which makes for an excellent prognosis.[9] While opportunities to improve early detection are not as great as in ovarian cancer, biomarkers for endometrial cancer might aid in prognostication, in the detection of recurrence, and in the monitoring of therapy for metastatic disease. Women with high-grade tumors, deep myometrial invasion, and lymphovascular space invasion have recurrence rates of 20% to 30%.[10] Current methodologies for detecting recurrence rely on imaging and symptom presentation for the diagnosis of recurrent disease.[9] CA 125, the FDA-approved biomarker for ovarian cancer, is elevated in only a small fraction (25%) of recurrent disease.[11]There is a clear need for other biomarkers that might complement CA 125 in the identification of women who are at a higher risk for recurrent endometrial cancer.

In the past decade, HE4 has emerged as a promising biomarker to address the unmet clinical needs in ovarian and endometrial cancer. In this review, we discuss the discovery and biologic significance of the biomarker, and we explore the promise of HE4 for addressing the various clinical needs in these cancers.

HE4: Discovery and Biologic Significance

HE4 was discovered by Kirchhoff et al in 1991 and belongs to the “four-disulfide core” family of proteins, which typically function as proteinase inhibitors.[12]In initial studies, HE4 messenger RNA (mRNA) was localized to the distal regions of the epithelial cells of the epididymal duct, indicating a possible role for HE4 in sperm maturation.[12] Its role as a potential biomarker for ovarian cancer emerged after cDNA comparative hybridization experiments found increased primary expression of HE4 in some ovarian cancers, relative to normal tissues.[13] Further studies using serial analysis of gene expression (SAGE) to analyze ovarian cell lines, tissues, and primary cancers corroborated HE4 as a potential marker that is upregulated in ovarian cancers.[14]

HE4 promotes migration and adhesion of ovarian cancer cells. In in vitro studies, HE4 knockdown resulted in tumor growth inhibition.[15] HE4 overexpression in endometrial cancer cell lines induced cancer cell proliferation in vivo and in vitro, supporting a function for HE4 in tumor progression.[16] In recent studies by LeBleu et al, HE4 was found to serve as a protease inhibitor, decreasing the activity of serine proteases Prss35 and Prss23, which degrade the type I collagen that accumulates in kidney fibrosis.[17] Fibrosis was inhibited in three mice models when HE4-neutralizing antibodies were administered, implicating HE4 as a therapeutic target in renal fibrosis.[17] HE4 may have an additional role in maintaining the innate immunity of the respiratory tract and the oral cavity.[18]

Tissue Expression

As with most biomarkers, HE4 is expressed in a number of normal and malignant tissues. Galgano et al completed a comprehensive study of HE4 mRNA and protein expression in normal and malignant tissues.[19] Normal glandular epithelium of the breast, female genital tract, epididymis, vas deferens, distal renal tubules, respiratory epithelium, colonic mucosa, and salivary glands all show HE4 immunoreactivity.[19] Among normal tissues, the highest levels of HE4 were found in the trachea and salivary gland. Lower expression was found in lung, prostate, pituitary gland, thyroid, and kidney. Positive HE4 immunoreactivity was prominent and consistent in ovarian cancer, while some positivity was observed in other cancers, including mesothelioma, lung, endometrial, breast, gastrointestinal, renal, and transitional cell carcinomas.[19] Among the different tumor sites, the highest expression levels were found in ovarian cancer; moderate levels in lung adenocarcinoma; and lower levels in breast, transitional cell, gastric, and pancreatic carcinomas.[19-21] Among malignant conditions, the highest levels of HE4 have been noted in ovarian cancer for women and in lung cancer for men.[22] Recently, LeBleu et al identified HE4 as a significantly-and the most frequently-upregulated gene in fibrosis-associated myofibroblasts in patients with kidney fibrosis.[17]

FIGURE

Normalized Expression Values of the HE4 Gene Across Different Cancers

Given the high expression levels noted for HE4 in ovarian cancer, Drapkin et al utilized immunohistochemistry to compare HE4 expression between malignant ovarian, benign, and nonovarian malignant tissues.[23] They found that a majority of nonovarian carcinomas did not express HE4 in their studies, with expression in normal tissues confined to the reproductive tracts and respiratory epithelium.[23] Interestingly, HE4 expression varied between histologic subtypes of ovarian cancer: HE4 was positive in 93% of serous tumors, in 100% of endometrioid tumors, and in 50% of clear-cell tumors, but in no mucinous tumors.[23] Expression has been found in primary tubal carcinomas and the normal fallopian tube epithelium, which may contribute to decreased specificity.[24] Similarly, in endometrial cancer, tissues from endometrial cancer patients had significantly upregulated HE4 in comparison with normal endometrium, implicating HE4 as a biomarker for endometrial cancer.[25]

From the cancer genome atlas (TCGA) database, we have compared the expression of HE4 across various cancer types utilizing the RNA-seq data (Figure). The results summarized in the Figure clearly reinforce the overexpression of HE4 in ovarian and uterine cancer. Lower expression is also noted for cervical cancer, lung adenocarcinoma, and chromophobe renal cell cancer. Sporadic extreme elevation was noted in head and neck cancers, and little or no expression was noted for all other cancers.

Secreted HE4

Since HE4 is overexpressed in ovarian cancers relative to normal tissues, Hellstrom et al examined the potential of HE4 as a secreted biomarker for ovarian cancer.[26] Murine monoclonal antibodies 2H5 and 3D8 were prepared against HE4 protein produced in mammalian cells from an HE4-IgG2a Fc fusion construct.[26] A heterologous double determinant immunoassay was established with the two antibodies that bound to distinct domains on the HE4 protein. A blinded study demonstrated comparable specificity and sensitivity for HE4 and CA 125, but serum HE4 was less frequently elevated in patients with benign gynecologic conditions.[26]

HE4 has also emerged as a serum biomarker for lung cancer,[27] pulmonary adenocarcinoma,[21] chronic kidney disease,[28] renal failure,[22] and kidney fibrosis.[17] Each of these conditions must be considered when interpreting HE4 levels in ovarian cancer. With a molecular weight of 25 kD, which is below the glomerular filtration cutoff, HE4 levels have also been found to be elevated in the urine of ovarian cancer patients compared with urine from healthy individuals or controls with benign disease.[29] Consequently, urinary HE4 has the potential to provide a noninvasive method for monitoring and diagnosing ovarian cancer.

Various factors aside from malignancy may influence serum HE4 levels and should be carefully considered in interpreting values of HE4. Unlike CA 125 levels, which decrease with age, HE4 levels increase significantly with age.[30]For example, the upper 95th percentile levels for HE4 were 128 pM in postmenopausal women, compared with 89 pM in premenopausal healthy women.[30] To account for the strong influence of age, Urban et al recommend age-specific thresholds for every decade based on HE4 levels in order to achieve 95% specificity for HE4: these ranged from 41.4 pM for women aged 30 years to 82.1 pM for women aged 80 years.[31] HE4 levels are also affected by pregnancy: pregnant women have significantly lowered levels of HE4 in comparison with age-matched nonpregnant premenopausal women.[30] Women with a later menarche and smokers also had significantly higher levels of HE4 compared with appropriate controls.[31,32] Menstrual cycle, endometriosis, and estrogen and progestin contraceptive usage do not alter serum levels of HE4.[33]

HE4 Biomarker Utility

Since the first reports by Hellstrom et al in 2003, HE4 has emerged as a valuable biomarker for both ovarian and endometrial cancer. HE4 has been evaluated for distinguishing malignant from benign pelvic masses, prognostication, monitoring, and screening.

Discriminating between malignant and benign pelvic masses

An estimated 289,000 women will be hospitalized each year for a pelvic mass or an ovarian cyst, and an estimated 10% of all women in the United States will undergo surgery for suspected neoplasms.[34] Given our knowledge that triaging women to centers of excellence that have significant experience in ovarian cancer management can lead to improved outcomes and significantly improved survival, biomarkers and algorithms that triage patients appropriately are needed to permit optimal care.[35,36]

CA 125 is frequently elevated in benign gynecologic conditions. HE4 is less frequently elevated than CA 125 in benign disease (8% vs 29%), improving specificity, particularly in premenopausal women.[37] In endometriosis, CA 125 was elevated in 67% of cases, compared with 3% for HE4.[37] Elevation of HE4 can occur in renal failure and in lung cancer.[38] HE4 is a superior biomarker for distinguishing benign from malignant gynecologic disease.[39] The overall diagnostic accuracy of HE4 in differentiating malignant ovarian tumors from benign gynecologic conditions was assessed by meta-analysis and found to have a pooled sensitivity of 0.74 and a pooled specificity of 0.87.[40] While one study, by Partheen et al, found that HE4 did not outperform CA 125, the majority of studies suggest otherwise.[41]

A combination of CA 125 and HE4 was found to be a better predictor of malignancy than either marker alone.[42] In a study assessing the performance of 65 ovarian cancer–related biomarkers for evaluation of adnexal masses, the CA 125–HE4 combination was superior, outperforming all other marker combinations.[43] For the evaluation of possible malignant disease in women presenting with pelvic masses, a combination of CA 125, HE4, and age provided a higher diagnostic value (area under the curve [AUC] of 0.797) than CA 125 alone (AUC of 0.677).[39] This combination was also found to have diagnostic relevance in the setting where there is a need to distinguish endometrial cancer from benign uterine disease, with a sensitivity of 60.4% and a specificity of 100%.[44] Although the studies are preliminary in endometrial cancer, the results are encouraging, and further research in this area is warranted to determine clinical utility.

ROMA

A “risk of malignancy algorithm” (ROMA) that combines the diagnostic power of the CA 125–HE4 marker panel with menopausal status has been approved by the FDA for distinguishing malignant from benign pelvic masses.[35] In a multicenter prospective study that included a total of 531 patients, preoperative levels of CA 125 and HE4 were measured, and separate logistic regression algorithms were used for premenopausal and postmenopausal women. The ROMA algorithm combines CA 125 and HE4 values along with the menopausal status into a predictive index, which in turn is used to calculate the predicted probability of ovarian cancer (from 0 to 100%). The regression formulae are as follows, where “exp” stands for “exponent” and LN is the natural logarithm[35]:

In premenopausal women,

Predictive Index (PI) = −12.0 + 2.38*LN(HE4) + 0.0626*LN(CA 125)

In postmenopausal women,

Predictive Index (PI) = −8.09 + 1.04*LN(HE4) + 0.732*LN(CA 125)

Predictive Probability (PP) = exp (PI) / [1/exp (PI)]

The ROMA algorithm performed better in the premenopausal population than in the postmenopausal cohort. In the premenopausal group, the algorithm had a sensitivity of 92.3% and a specificity of 75.0%; in the postmenopausal group, the sensitivity and specificity were 76.5% and 74.8%, respectively. Predicted probability (PP) values greater than 13.1% indicated high risk in the premenopausal population, and PP values higher than 27.7% indicated high risk in the postmenopausal population. Using this algorithm, 93.8% of epithelial ovarian cancers were correctly classified as high risk, prompting referral for treatment by a gynecologic oncologist.[35] When validated cutoff values were utilized to reexamine the diagnostic performance of ROMA, the sensitivity and specificity of the algorithm were 84.6% and 81.2%.[45] Similar results were achieved in an Asian population in a prospective multicenter study of patients from six Asian countries.[46] In comparing nonmalignant controls with early-stage ovarian and tubal cancers, Jacob et al found that ROMA provided a better diagnostic performance than CA 125 alone, with a sensitivity of 78.9% and a specificity of 85.9%.[47] HE4 was found to be better than CA 125 for detecting borderline tumors and early-stage ovarian and tubal cancers even when not combined with CA 125.[47]

A second trial was performed to evaluate ROMA in 472 community patients who had a total of 89 cancers.[48] Overall, ROMA achieved a sensitivity of 94% and a specificity of 75%. In premenopausal patients, a sensitivity of 100% was observed. Overall, the negative predictive value was 98%. After successful completion of this community-based trial, ROMA was approved by the FDA for distinguishing malignant from benign pelvic masses. Subsequent reports have provided conflicting results, with some confirming the predictive value of ROMA[49-53] and others finding that it does not improve upon algorithms which use CA 125 or HE4 alone.[53-55]

ROMA has performed reliably with both of the commercially available assays for HE4-namely, the Abbott ARCHITECT chemiluminescent microparticle immunoassay (CMIA) and the Fujirebio enzyme immunoassay (EIA). Although the algorithm is identical with either platform, cutoff values for ROMA may vary between the Abbott and Fujirebio assays, and manufacturer guidelines should be followed carefully to assure correct interpretation of results. It is evident from these studies that ROMA derives its sensitivity in detecting ovarian cancer from CA 125 and its specificity in discriminating ovarian cancer from benign gynecologic malignancies from HE4. The incorporation of premenopausal or postmenopausal status addresses the issue of marker fluctuations with age, and the resulting algorithm is now a powerful tool that is recommended for evaluation of patients with pelvic masses. Because optimal cytoreduction and comprehensive surgical staging in the hands of a gynecologic oncologist are cornerstones of ovarian cancer management, ROMA permits the best possible utilization of resources-and consequently has improved survival as a result of appropriate triage of patients to centers of excellence.

Another algorithm, OVA1, has also received FDA approval for distinguishing malignant from benign pelvic masses. OVA1 utilizes imaging, menopausal status, CA 125, and four other protein biomarkers-apolipoprotein A1, transthyretin, transferrin, and β2-microglobulin-to generate a multivariate index that identifies malignant pelvic masses with a sensitivity of 85% to 96%, a specificity of 28% to 40%, and a negative predictive value between 94% and 96%.[47] Of the available algorithms, only ROMA does not utilize imaging and offers a 92.3% sensitivity and a 76.0% specificity in the postmenopausal cohort, and 100% sensitivity and 74.2% specificity in the premenopausal cohort, with a combined negative predictive value of 99% in a large prospective, multicenter blinded clinical trial.[56] In a direct comparison between ROMA and RMI for prediction of malignancy in patients presenting with a pelvic mass, ROMA performed better than RMI, with a sensitivity of 94.3% (compared with 84.6% for RMI) at 75% specificity for both tests.[57] In early-stage cancers, ROMA performed with a significantly higher sensitivity of 85.3%, compared with RMI’s sensitivity of 64.7%.[57] Thus, ROMA appears superior to RMI. ROMA has not been directly compared with OVA1, but based on reported diagnostic performance to date, it is anticipated that both algorithms will perform at equivalent sensitivities, but that OVA1 will exhibit much lower specificity.

A comprehensive analysis of 259 candidate biomarkers was completed, and a panel of nine biomarkers (HE4, CA 125, interleukin 2 [IL-2] receptor α, α1-antitrypsin, C-reactive protein, YKL-40, cellular fibronectin, CA 72-4, prostasin) was identified that improved upon the performance of the OVA1 panel with a specificity of 88.9% (compared with 63.4% for the OVA1 panel), at 90% sensitivity for both the nine-marker panel and OVA1.[58] A blinded validation study must still be completed to confirm the specificity offered by this biomarker panel, but the study holds promise that additional and alternate marker combinations may improve upon current tests for discriminating between benign and malignant disease. It is notable that HE4 is included in the second generation of the OVA1 test (named OVA2), which benefits from the improved specificity provided by HE4 along with other biomarkers.

In studies by Van Gorp et al, triaging with subjective ultrasound interpretation was found to be superior at distinguishing between malignant and benign pelvic masses compared with RMI and ROMA.[59] However, as the name suggests, subjective assessment relies on the impression and experience of the ultrasound examiner in distinguishing benign from malignant pelvic masses. Recently, Kaijser et al developed an ultrasound-based predictive model (LR2) that was reported to perform better diagnostically than ROMA, suggesting that supervised higher-quality sonography is crucial to improving diagnostic performance.[60] These results suggest that combining the power of biomarker panels (ROMA) with an imaging algorithm may improve upon the current performance of each of the individual methods alone. In a recent study by Macuks et al, an ovarian cancer malignancy risk index that included levels of CA 125 and HE4, menopausal status, and ultrasound score provided an AUC of 0.939.[61]

Screening

As detailed above, two-stage strategies have improved the specificity and possibly the sensitivity of CA 125 and TVS for the detection of early-stage ovarian cancer. The “risk of ovarian cancer algorithm” (ROCA) utilizes each woman’s own baseline for annual evaluation of CA 125. Rising values of CA 125 analyzed by ROCA have prompted TVS as a second-line screen in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).[7] This multimodal screening format has achieved a positive predictive value of 43.3%, indicating that it shows strong promise as a screening strategy for ovarian cancer. Results regarding the impact of screening on mortality in this trial will be available in 2015.[55] Despite the promise offered by ROCA, 20% of ovarian cancers do not express CA 125, prompting the need for multiple biomarkers to improve sensitivity.[54]

HE4 complements CA 125 and is expressed in 32% of CA 125–deficient ovarian cancers, providing one candidate for a larger panel.[54] In a study of 96 biomarkers that were evaluated for the development of a multi-marker assay for early detection of ovarian cancer, the optimal biomarker panel that distinguished early-stage cancer from controls included CA 125, HE4, carcinoembryonic antigen (CEA), and vascular cell adhesion molecule 1 (VCAM-1)[62]; this panel had an 86% sensitivity for early-stage disease and 98% specificity. Several other biomarker panels exhibited similar diagnostic performance, and almost all such panels included HE4 and CA 125. Thus, HE4 will most likely be included in a first-line test comprised of multiple biomarker panels.

In the Carotene and Retinol Efficacy Trial, prediagnostic sera collected from women prior to the development of ovarian cancer and from corresponding controls were assessed for lead times for CA 125 and HE4.[63] Both CA 125 and HE4 started to increase at least 3 years prior to diagnosis, and detectable elevations were seen 1 year prior to diagnosis.[63] Longitudinal biomarker algorithms based on multiple biomarkers may not only offer additional specificity but also may improve lead times; our laboratory is currently focused on developing such an algorithm based on a panel that includes CA 125, HE4, matrix metalloproteinase 7 (MMP-7), and CA 72-4, choices that resulted from a study of 96 biomarkers.[62]

In a recent study, Urban et al compared various biomarkers as a second-line screen to augment the performance of TVS or to replace it.[64] HE4 outperformed TVS as a second-line screen in 39 cancers with rising CA 125 values, suggesting the development of a longitudinal screening algorithm that incorporates HE4.[64] Although the addition of HE4 improved lead time to a greater extent than CA 125 alone, using Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) biorepository specimens, it is likely that the use of a longitudinal algorithm, rather than evaluation at a specific time point, may result in better diagnostic performance.[65]

Currently, a phase I screening trial is underway to evaluate the performance of HE4 in a first/second-line multimodal screening strategy that combines CA 125, HE4, and TVS for women at high risk for ovarian cancer in a manner similar to the approach used in the UKCTOCS trial.[66] The goal of this ongoing trial is to evaluate the lead times, sensitivity, and specificity offered by the foregoing strategy.[66] In other efforts, a decision rule based on CA 125, HE4, and a symptom index has also been studied and was able to detect ovarian cancer with a sensitivity of 84% and a specificity of 98.5%; this may be of value in women who present with symptoms of ovarian cancer.[67]

Although most of the efforts in early detection with HE4 have been focused on ovarian cancer, HE4 has also been found to be a promising biomarker for distinguishing patients with early-stage endometrial cancer from healthy premenopausal women; HE4 was found to be elevated in all stages of endometrial cancer.[9] In a study comparing preoperative levels of various promising biomarkers, including HE4, soluble mesothelin-related protein (SMRP), CA 72-4, and CA 125, in surgically staged endometrioid endometrial adenocarcinoma, HE4 offered the highest sensitivity-45.5%, compared with 24.6% for CA 125 (at 95% specificity for both assays).[9] Thus, HE4 is a promising biomarker for early detection of endometrial cancer and warrants further exploration.

Prognostication

HE4 may serve as a useful prognostic biomarker for ovarian and endometrial cancer. Elevated levels of HE4 are associated with increases in International Federation of Gynecology and Obstetrics (FIGO) stage, grade, preoperative CA 125 levels, and residual tumor.[68] Elevated HE4 levels also correlate with the aggressiveness of ovarian cancers, poor prognosis, and overall survival.[68-70] Recent results from the multicenter European project “OVCAD” showed that higher HE4 levels corresponded with poor surgical outcome with respect to residual tumor mass and platinum resistance.[71] Advanced age and stage, lymph node involvement, presence of ascites, and suboptimal cytoreduction correlated with elevated ROMA, HE4, and CA 125.[45] Elevated HE4 and higher ROMA score were independent predictors of poor prognosis, defined in terms of shorter overall survival, progression-free survival, and disease-free survival.[46] In comparing CA 125 and HE4, HE4 was better at identifying patients with optimal cytoreduction. Using a cutoff value for HE4 of ≤ 262 pM and for ascites of < 500 mL, optimal cytoreduction was predicted with a sensitivity of 100% and a specificity of 89.5%.[72]

Much like in ovarian cancer, high levels of serum HE4 correlated with an aggressive phenotype of endometrial cancer, with higher levels associated with decreased disease-free survival, progression-free survival, and overall survival.[25] Higher HE4 values were found to be an independent prognostic factor for poorly differentiated endometrial cancers and may be useful in treating high-risk patients with aggressive adjuvant therapy regimens.[73] Higher HE4 levels also correlated with primary tumor diameter and increased myometrial invasion, supporting the biomarker’s utility in predicting a higher risk of metastatic disease preoperatively.[74] In a prospective, observational study of women with biopsy-confirmed endometrioid adenocarcinoma, preoperative levels of HE4 < 70 pM identified stage I disease with a sensitivity of 94% and identified stage IB disease with a sensitivity of 82%. Since staging determines myometrial invasion and influences corresponding clinical decisions based on lymph node dissection, HE4 may serve as a useful preoperative marker for guiding the clinical decision process.[75] Also, Kalogera et al noted that HE4 had a higher sensitivity than CA 125 in predicting the presence of advanced-stage endometrial cancer.[74] Much like in ovarian cancer, higher preoperative values of CA 125 and HE4 were significantly associated with aggressive tumor phenotypes, and the combination of both biomarkers was an indicator of higher hazard ratio for overall survival.[76]

Recurrence and Monitoring

Preliminary evidence suggests that HE4 may aid in detecting recurrent ovarian cancer. When four biomarkers-including CA 125, HE4, MMP-7, and mesothelin-were monitored in patients with advanced-stage ovarian cancer following surgery and chemotherapy, HE4 rose prior to recurrence with a lead time of 4.5 months.[77] In patients with rising CA 125 levels, HE4 was elevated prior to the rise in CA 125 in some patients. For a fraction of the patients in whom CA 125 and imaging were negative, HE4 levels stayed at or above cutoff.[77] In a recent prospective controlled study, HE4 was able to detect recurrent ovarian cancer with 74% sensitivity and 100% specificity at a cutoff of 70 pmol/L.[78] Using a combination of HE4 and CA 125 increased overall sensitivity to 76%. A combination of CA 125 and HE4 may offer better lead times and sensitivity for the detection of recurrent ovarian cancer.

HE4 levels may aid in monitoring response to therapy. Serum levels of HE4 obtained at the time of diagnosis of ovarian cancer differed significantly from levels following complete clinical remission (324.1 pM vs 23.3 pM), indicating a possible role in monitoring response to therapy.[79] In patients with peritoneal carcinomatosis, HE4 and CA 125 were both elevated in women with small implants and in women presenting with macronodular implants and omental thickening on imaging studies. A statistically significant difference was noted in HE4 levels, but not in CA 125 levels, in patients with small and macronodular implants. Elevated levels of HE4 also correlated with the severity of lymph node extension. A combination of sophisticated imaging techniques combined with HE4 marker levels may aid in following patients with peritoneal carcinomatosis.[80]

Conclusions

In the past decade, HE4 has emerged as a promising biomarker with many applications in ovarian cancer and endometrial cancer. One of the advantages of HE4 is its ability to complement CA 125. In healthy premenopausal women, HE4 increases the sensitivity of CA 125 without compromising its specificity, a phenomenon that made possible the development of the ROMA algorithm for distinguishing malignant from benign pelvic masses. HE4 levels have also been elevated in a fraction of cases where CA 125 cannot be detected, leading to its evaluation for monitoring response to therapy, for detecting recurrences, and for early detection. Preliminary results suggest that HE4 will also serve as a biomarker that can identify women with endometrial cancer who are at high risk for recurrence, as well as being used in monitoring and detecting recurrence.

Acknowledgements:These studies were supported in part by a grant from the National Cancer Institute R01 CA135354; the MD Anderson SPORE in Ovarian Cancer NCI P50 CA83639 grant; the Shared Resources of the MD Anderson CCSG NCI P30 CA16672 grant; the Ovarian Cancer Research Fund; the National Foundation for Cancer Research; and philanthropic support from the Zarrow Foundation and Stuart and Gaye Lynn Zarrow, Golfers Against Cancer, the Kaye Yow Foundation, and the Mossy Family Foundation.

Financial Disclosure:Dr. Bast receives royalties for CA 125 from Fujirebio Diagnostics, Inc, and serves on the scientific advisory board for Vermillion, Inc. Drs. Simmons and Baggerly have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.

References:

REFERENCES

1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10-29.

2. Jacobs I, Oram D, Fairbanks J, et al. A risk of malignancy index incorporating CA 125, ultrasound and menopausal status for the accurate preoperative diagnosis of ovarian cancer. Br J Obstet Gynaecol. 1990;97:922-9.

3. Bast RC Jr, Skates S, Lokshin A, Moore RG. Differential diagnosis of a pelvic mass: Improved algorithms and novel biomarkers. Int J Gynecol Cancer. 2012;10:S5-8.

4. Bast RC Jr. Commentary: CA 125 and the detection of recurrent ovarian cancer: A reasonably accurate biomarker for a difficult disease. Cancer. 2010;116:2850-3.

5. Das PM, Bast RC Jr. Early detection of ovarian cancer. Biomark Med. 2008;2:291-303.

6. Bast RC Jr, Brewer M, Zou C, et al. Prevention and early detection of ovarian cancer: mission impossible? Recent Results Cancer Res. 2007;174:91-100.

7. Menon U, Gentry-Maharaj A, Hallett R, et al. Sensitivity and specificity of multimodal and ultrasound screening for ovarian cancer, and stage distribution of detected cancers: results of the prevalence screen of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Lancet Oncol. 2009;10:327-40.

8. Lu KH, Skates S, Hernandez MA, et al. A two-stage ovarian cancer screening strategy using the risk of ovarian cancer algorithm (ROCA) identifies early stage incident cancers and demonstrates high positive predictive value. Cancer. In revision.

9. Moore RG, Brown AK, Miller MC, et al. Utility of a novel serum tumor biomarker HE4 in patients with endometrioid adenocarcinoma of the uterus. Gynecol Oncol. 2008;110:196-201.

10. Creutzberg CL, van Putten WL, Koper PC, et al. Survival after relapse in patients with endometrial cancer: results from a randomized trial. Gynecol Oncol. 2003;89:201-9.

11. Duk JM, Aalders JG, Fleuren GJ, de Bruijn HW. CA 125: a useful marker in endometrial carcinoma. Am J Obstet Gynecol. 1986;155:1097-102.

12. Kirchhoff C, Habben I, Ivell R, Krull N. A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors. Biol Reprod. 1991;45:350-7.

13. Schummer M, Ng WV, Bumgarner RE, et al. Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas. Gene. 1999;238:375-85.

14. Hough CD, Sherman-Baust CA, Pizer ES, et al. Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer. Cancer Res. 2000;60:6281-7.

15. Lu R, Sun X, Xiao R, et al. Human epididymis protein 4 (HE4) plays a key role in ovarian cancer cell adhesion and motility. Biochem Biophys Res Commun. 2012;419:274-80.

16. Li J, Chen H, Mariani A, et al. HE4 (WFDC2) promotes tumor growth in endometrial cancer cell lines. Int J Mol Sci. 2013;14:6026-43.

17. LeBleu VS, Teng Y, O'Connell JT, et al. Identification of human epididymis protein-4 as a fibroblast-derived mediator of fibrosis. Nat Med. 2013;19:227-31.

18. Bingle L, Cross SS, High AS, et al. WFDC2 (HE4): a potential role in the innate immunity of the oral cavity and respiratory tract and the development of adenocarcinomas of the lung. Respir Res. 2006;7:61.

19. Galgano MT, Hampton GM, Frierson HF Jr. Comprehensive analysis of HE4 expression in normal and malignant human tissues. Mod Pathol. 2006;19:847-53.

20. O'Neal RL, Nam KT, Lafleur BJ, et al. Human epididymis protein 4 is up-regulated in gastric and pancreatic adenocarcinomas. Hum Pathol. 2013;44:734-42.

21. Kamei M, Yamashita S, Tokuishi K, et al. HE4 expression can be associated with lymph node metastases and disease-free survival in breast cancer. Anticancer Res. 2010;30:4779-83.

22. Hertlein L, Stieber P, Kirschenhofer A, et al. Human epididymis protein 4 (HE4) in benign and malignant diseases. Clin Chem Lab Med. 2012;50:2181-8.

23. Drapkin R, von Horsten HH, Lin Y, et al. Human epididymis protein 4 (HE4) is a secreted glycoprotein that is overexpressed by serous and endometrioid ovarian carcinomas. Cancer Res. 2005;65:2162-9.

24. Georgakopoulos P, Mehmood S, Akalin A, Shroyer KR. Immunohistochemical localization of HE4 in benign, borderline, and malignant lesions of the ovary. Int J Gynecol Pathol. 2012;31:517-23.

25. Bignotti E, Ragnoli M, Zanotti L, et al. Diagnostic and prognostic impact of serum HE4 detection in endometrial carcinoma patients. Br J Cancer. 2011;104:1418-25.

26. Hellstrom I, Raycraft J, Hayden-Ledbetter M, et al. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Res. 2003;63:3695-700.

27. Iwahori K, Suzuki H, Kishi Y, et al. Serum HE4 as a diagnostic and prognostic marker for lung cancer. Tumour Biol. 2012;33:1141-9.

28. Nagy B Jr, Krasznai ZT, Balla H, et al. Elevated human epididymis protein 4 concentrations in chronic kidney disease. Ann Clin Biochem. 2012;49(Pt 4):377-80.

29. Hellstrom I, Heagerty PJ, Swisher EM, et al. Detection of the HE4 protein in urine as a biomarker for ovarian neoplasms. Cancer Lett. 2010;296:43-8.

30. Moore RG, Miller MC, Eklund EE, et al. Serum levels of the ovarian cancer biomarker HE4 are decreased in pregnancy and increase with age. Am J Obstet Gynecol. 2012;206:349: e1-7.

31. Urban N, Thorpe J, Karlan BY, et al. Interpretation of single and serial measures of HE4 and CA 125 in asymptomatic women at high risk for ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2012;21:2087-94.

32. Lowe KA, Shah C, Wallace E, et al. Effects of personal characteristics on serum CA 125, mesothelin, and HE4 levels in healthy postmenopausal women at high-risk for ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2008;17:2480-7.

33. Hallamaa M, Suvitie P, Huhtinen K, et al. Serum HE4 concentration is not dependent on menstrual cycle or hormonal treatment among endometriosis patients and healthy premenopausal women. Gynecol Oncol. 2012;125:667-72.

34. Curtin JP. Management of the adnexal mass. Gynecol Oncol. 1994;55(3 Pt 2):S42-6.

35. Moore RG, McMeekin DS, Brown AK, et al. A novel multiple marker bioassay utilizing HE4 and CA 125 for the prediction of ovarian cancer in patients with a pelvic mass. Gynecol Oncol. 2009;112:40-6.


36. Carney ME, Lancaster JM, Ford C, et al. A population-based study of patterns of care for ovarian cancer: who is seen by a gynecologic oncologist and who is not? Gynecol Oncol. 2002;84:36-42.

37. Moore RG, Miller MC, Steinhoff MM, et al. Serum HE4 levels are less frequently elevated than CA 125 in women with benign gynecologic disorders. Am J Obstet Gynecol. 2012;206:351: e1-8.

38. Escudero JM, Auge JM, Filella X, et al. Comparison of serum human epididymis protein 4 with cancer antigen 125 as a tumor marker in patients with malignant and nonmalignant diseases. Clin Chem. 2011;57:1534-44.

39. Kondalsamy-Chennakesavan S, Hackethal A, Bowtell D, Obermair A. Differentiating stage 1 epithelial ovarian cancer from benign ovarian tumours using a combination of tumour markers HE4, CA 125, and CEA and patient's age. Gynecol Oncol. 2013;129:467-71.

40. Lin J, Qin J, Sangvatanakul V. Human epididymis protein 4 for differential diagnosis between benign gynecologic disease and ovarian cancer: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2013;167:81-5.

41. Partheen K, Kristjansdottir B, Sundfeldt K. Evaluation of ovarian cancer biomarkers HE4 and CA 125 in women presenting with a suspicious cystic ovarian mass. J Gynecol Oncol. 2011;22:244-52.

42. Moore RG, Brown AK, Miller MC, et al. The use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol Oncol. 2008;108:402-8.

43. Nolen B, Velikokhatnaya L, Marrangoni A, et al. Serum biomarker panels for the discrimination of benign from malignant cases in patients with an adnexal mass. Gynecol Oncol. 2010;117:440-5.

44. Angioli R, Plotti F, Capriglione S, et al. The role of novel biomarker HE4 in endometrial cancer: a case control prospective study. Tumour Biol. 2012;34:571-6.

45. Bandiera E, Romani C, Specchia C, et al. Serum human epididymis protein 4 and risk for ovarian malignancy algorithm as new diagnostic and prognostic tools for epithelial ovarian cancer management. Cancer Epidemiol Biomarkers Prev. 2011;20:2496-506.

46. Chan KK, Chen CA, Nam JH, et al. The use of HE4 in the prediction of ovarian cancer in Asian women with a pelvic mass. Gynecol Oncol. 2013;128:239-44.

47. Jacob F, Meier M, Caduff R, et al. No benefit from combining HE4 and CA 125 as ovarian tumor markers in a clinical setting. Gynecol Oncol. 2011;121:487-91.

48. Moore RG, Miller C, Disilvestro P, et al. Evaluation of the diagnostic accuracy of the risk of ovarian malignancy algorithm in women with a pelvic mass. Obstet Gynecol. 2011;118:280-8.

49. Bandiera E, Romani C, Specchia C, et al. Serum human epididymis protein 4 and risk for ovarian malignancy algorithm as new diagnostic and prognostic tools for epithelial ovarian cancer management. Cancer Epidemiol Biomarkers Prev. 2011;20:2496-506.

50. Kim YM, Whang DH, Park J, et al. Evaluation of the accuracy of serum human epididymis protein 4 in combination with CA 125 for detecting ovarian cancer: a prospective case-control study in a Korean population. Clin Chem Lab Med. 2011;49:527-34.

51. Lenhard M, Stieber P, Hertlein L, et al. The diagnostic accuracy of two human epididymis protein 4 (HE4) testing systems in combination with CA 125 in the differential diagnosis of ovarian masses. Clin Chem Lab Med. 2011;49:2081-8.

52. Molina R, Escudero JM, Augé JM, et al. HE4 a novel tumour marker for ovarian cancer: comparison with CA 125 and ROMA algorithm in patients with gynaecological diseases. Tumor Biol. 2011;32:1087-95.

53. Ruggeri G, Bandiera E, ZanottiL, et al. HE4 and epithelial ovarian cancer: Comparison and clinical evaluation of two immunoassays and a combination algorithm. Clinica Chimica Acta. 2011;412:1447-53.

54. Montagnana M, Danese E, Ruzzenente O, et al. The ROMA (Risk of Ovarian Malignancy Algorithm) for estimating the risk of epithelial ovarian cancer in women presenting with pelvic mass: is it really useful? Clin Chem Lab Med. 2011;49:521-5.

55. Van Gorp I, Cadron I, Despierre E, et al. HE4 and CA 125 as a diagnostic test in ovarian cancer: prospective evaluation of the Risk of Malignancy Algorithm. Br J Cancer. 2011; 104:863-70.

56. Moore RG, Miller MC, Disilvestro P, et al. Evaluation of the diagnostic accuracy of the risk of ovarian malignancy algorithm in women with a pelvic mass. Obstet Gynecol. 2011;118(2 Pt 1):280-8.

57. Moore RG, Jabre-Raughley M, Brown AK, et al. Comparison of a novel multiple marker assay vs the Risk of Malignancy Index for the prediction of epithelial ovarian cancer in patients with a pelvic mass. Am J Obstet Gynecol. 2010;203:228: e1-6.

58. Yip P, Chen TH, Seshaiah P, et al. Comprehensive serum profiling for the discovery of epithelial ovarian cancer biomarkers. PLoS One. 2011;6:e29533.

59. Van Gorp T, Veldman J, Van Calster B, et al. Subjective assessment by ultrasound is superior to the risk of malignancy index (RMI) or the risk of ovarian malignancy algorithm (ROMA) in discriminating benign from malignant adnexal masses. Eur J Cancer. 2012;48:1649-56.

60. Kaijser J, Van Gorp T, Van Hoorde K, et al. A comparison between an ultrasound based prediction model (LR2) and the Risk of Ovarian Malignancy Algorithm (ROMA) to assess the risk of malignancy in women with an adnexal mass. Gynecol Oncol. 2013;129:377-83.

61. Macuks R, Baidekalna I, Donina S. An ovarian cancer malignancy risk index composed of HE4, CA 125, ultrasonographic score, and menopausal status: use in differentiation of ovarian cancers and benign lesions. Tumour Biol. 2012;33:1811-7.

62. Yurkovetsky Z, Skates S, Lomakin A, et al. Development of a multimarker assay for early detection of ovarian cancer. J Clin Oncol. 2010;28:2159-66.

63. Anderson GL, McIntosh M, Wu L, et al. Assessing lead time of selected ovarian cancer biomarkers: a nested case-control study. J Natl Cancer Inst. 2010;102:26-38.

64. Urban N, Thorpe JD, Bergan LA, et al. Potential role of HE4 in multimodal screening for epithelial ovarian cancer. J Natl Cancer Inst. 2011;103:1630-4.

65. Cramer DW, Bast RC Jr, Berg CD, et al. Ovarian cancer biomarker performance in prostate, lung, colorectal, and ovarian cancer screening trial specimens. Cancer Prev Res (Phila). 2011;4:365-74.

66. Urban N. Designing early detection programs for ovarian cancer. Ann Oncol. 2011;22(Suppl 8):viii6-viii18.

67. Andersen MR, Goff BA, Lowe KA, et al. Use of a Symptom Index, CA 125, and HE4 to predict ovarian cancer. Gynecol Oncol. 2010;116:378-83.

68. Trudel D, Tetu B, Gregoire J, et al. Human epididymis protein 4 (HE4) and ovarian cancer prognosis. Gynecol Oncol. 2012;127:511-5.

69. Kalapotharakos G, Asciutto C, Henic E, et al. High preoperative blood levels of HE4 predicts poor prognosis in patients with ovarian cancer. J Ovarian Res. 2012;5:20.

70. Steffensen KD, Waldstrom M, Brandslund I, Jakobsen A. Prognostic impact of prechemotherapy serum levels of HER2, CA 125, and HE4 in ovarian cancer patients. Int J Gynecol Cancer. 2011;21:1040-7.

71. Braicu EI, Fotopoulou C, Van Gorp T, et al. Preoperative HE4 expression in plasma predicts surgical outcome in primary ovarian cancer patients: Results from the OVCAD study. Gynecol Oncol. 2012;128:245-51.

72. Angioli R, Plotti F, Capriglione S, et al. Can the preoperative HE4 level predict optimal cytoreduction in patients with advanced ovarian carcinoma? Gynecol Oncol. 2013;128:579-8.

73. Bandiera E, Franceschini R, Specchia C, et al. Prognostic significance of vascular endothelial growth factor serum determination in women with ovarian cancer. ISRN Obstet Gynecol. 2012;2012:
245756.

74. Kalogera E, Scholler N, Powless C, et al. Correlation of serum HE4 with tumor size and myometrial invasion in endometrial cancer. Gynecol Oncol. 2012;124:270-5.

75. Moore RG, Miller CM, Brown AK, et al. Utility of tumor marker HE4 to predict depth of myometrial invasion in endometrioid adenocarcinoma of the uterus. Int J Gynecol Cancer. 2011;21:1185-90.

76. Zanotti L, Bignotti E, Calza S, et al. Human epididymis protein 4 as a serum marker for diagnosis of endometrial carcinoma and prediction of clinical outcome. Clin Chem Lab Med. 2012;50:2189-98.

77. Schummer M, Drescher C, Forrest R, et al. Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to the established marker CA 125. Gynecol Oncol. 2012;125:65-9.

78. Plotti F, Capriglione S, Terranova C, et al. Does HE4 have a role as biomarker in the recurrence of ovarian cancer? Tumour Biol. 2012;33:2117-23.

79. Chudecka-Glaz A, Rzepka-Gorska I, Wojciechowska I. Human epididymal protein 4 (HE4) is a novel biomarker and a promising prognostic factor in ovarian cancer patients. Eur J Gynaecol Oncol. 2012;33:382-90.

80. Midulla C, Manganaro L, Longo F, et al. HE4 combined with MDCT imaging is a good marker in the evaluation of disease extension in advanced epithelial ovarian carcinoma. Tumour Biol. 2012;33:1291-8.