Ovarian tumors grow in combined cystic and solid formations. There is no reliable diagnostic test or imaging technique available to distinguish benign from malignant cysts [1]. The decision to operate is based on clinical findings, transvaginal sonography, computed tomography and/or magnetic resonance imaging, and cancer antigen 125 (CA-125) levels. Approximately 7 to 10 benign lesions are operated on for each case of ovarian cancer found [2]. Consequently, several patients undergo extensive surgical staging, including oophorectomy, without an assured diagnosis of a malignant tumor, resulting in increased morbidity. Thus, there would be both medical and socio-economic benefits if we could preoperatively identify patients with benign cysts and safely be able to recommend conservative laparoscopic staging operations [3].

The tumor marker CA-125 is used to predict the presence of malignancy in patients with a pelvic mass [4]. CA-125 is elevated (>35 U/mL) in 80% of cases of advanced cancer, but in fewer than 50% of cases in early stages. Moreover, CA-125 is often elevated in different benign gynecologic and non-gynecologic conditions and in premenopausal women [5]. Human epididymis protein 4 (HE4) is a prominent new candidate for the detection of ovarian cancer [6,7]. The marker was discovered in RNA expression arrays comparing normal epithelial cells and malignant tumors [8,9]. Hellstrom and colleagues [10] constructed HE4 antibodies and reported that in a rather small study population HE4 and CA-125 in serum samples detected ovarian cancer equally, but HE4 had a better capacity to distinguish healthy women and women with benign disease from those with malignant tumors. The findings indicated that HE4 used in combination with CA-125 might be a promising tool for the detection of ovarian cancer. Moore and colleagues [11] have created an algorithm for risk scoring called the risk of ovarian malignancy algorithm (ROMA) based on menopausal status and preoperative measurement of the dual markers. ROMA classifies women with ovarian cyst or pelvic mass scheduled to undergo surgery into low- and high-risk groups. Today, the question still remains whether HE4, ROMA, or both add sufficient information to CA-125 to be implemented in clinical practice [12,13].

It is evident that an early diagnosis is the most important factor for substantial improvement in the survival rate of patients with ovarian cancer, as shown by 5-year survival rates of over 80% for women in early stages to under 30% for those in stages III-IV [14]. The first published studies indicated that HE4 has higher sensitivity in identifying early stage disease than does CA-125 [7,15]. Conversely, later studies have shown CA-125 to be the best marker for detecting early stage disease [6,16].

We first hypothesized that known ovarian cysts in a study population might be better classified with HE4 alone or in combination with CA-125 than with CA-125 alone. Second, we intended to estimate an optimal cut-off value for HE4 in our study population [11,17]. Third, we intended to independently validate ROMA. We prospectively collected blood and tumor tissue from patients presenting with a suspicious ovarian cystic pelvic mass from 2001 to 2010. The patients had already been assigned to operation at our center of excellence for ovarian cancer surgery.

We used consecutively collected blood samples from 374 patients scheduled for surgery for a suspicious cystic ovarian or pelvic mass and analyzed the levels of HE4 and CA-125. We found that HE4 did not perform better than CA-125 results in the total material, but may be more useful in premenopausal women. Negative predictive value was high (92%) if CA-125, HE4, or both were elevated, and all false negatives were found in early stage EOC. This finding suggests that HE4 adds information in the preoperative workup.

Women presenting with a suspicious ovarian cyst or cystic pelvic mass are presently commonly exposed to open laparotomy and an extensive staging operation. A patient with this diagnosis should always be operated on at a unit where the gynecologic surgeons are trained in optimal debulking surgery [18]. On the other hand, approximately 7 to 10 benign cysts are removed for each single discovery of ovarian cancer [2]. The lack of preoperative screening modalities to predict diagnostic outcome increases unnecessary extensive abdominal ovarian staging operations. The risk of malignity index (RMI), an algorithm based on CA-125, menopausal status, and transvaginal sonography, was the first to be used for referral of ovarian cancer patients [19]. Another algorithm, ROMA, based on HE4, CA-125, and menopausal status, was shown to stratify patients into high- and low-risk groups, enabling more accurate triage of patients to centers of excellence for ovarian cancer than RMI [20], while McDonald et al. [21] concluded that patients with solid or complex ovarian tumors and elevated CA-125 were those at high risk of ovarian malignancy. The results from our investigation suggest that adding measurements of HE4 to CA-125 in the preoperative evaluation of a suspicious ovarian cyst or cystic pelvic mass may help to choose the correct type and setting for the operation. If we had evaluated HE4 and CA-125 in addition to TVS and CT preoperatively, the majority of the patients in our study population could have had conservative laparoscopic staging, without the risk of an incorrect diagnosis [3] since every one of the 13 false negatives were in the early stage. With the incorporation of this measure preoperatively, not only will the patient have fewer preoperative complications and less co-morbidity due to unnecessary abdominal procedures, but she will also have a shorter stay at the hospital, be back to her ordinary life more quickly, and be left with one intact ovary.

Several groups have investigated HE4 in a possible screening setting and report good ROC AUC separating women with either early or late stage epithelial ovarian cancer from healthy controls [10,16,22,23]. HE4 is a biomarker that increases progressively with disease and is markedly lower in healthy women than in women with benign ovarian cysts [12]. This fact makes comparisons between the above-mentioned studies, which include patients without disease of the ovaries, and studies like ours and others [6,24], which compare prospective data from known ovarian masses less relevant. Very promising results that separate benign from malignant disease using HE4 alone have presented ROC AUC of 95% [25] and 91% [7], while we and others reached an ROC AUC of 84%, 84% [6], and 86% [24]. Different cut-off points, more postmenopausal women in the benign cystic cohort, and the fact that we analyzed plasma instead of serum could have influenced our results. As in several other studies, CA-125 performed better than HE4 in our estimation, not only in the whole group but also when we divided the cohorts into benign versus borderline and stage I EOC.

The sample characteristics in our investigation differ from the normal distribution one could expect because the patients were already qualified for operation at our centre of excellence for ovarian cancer surgery. Compared to a population-based incidence of operated ovarian cysts our cohort included fewer benign samples (58%), more early stage EOC (50%), and more postmenopausal women (74%). The young, fertile women with simple cysts considered to have a low risk of malignancy were operated on at the benign gynecology unit and were never part of the investigation. It is important to be aware that the composition of study populations varies greatly between published studies and very few present data from a normal clinical setting without exclusions. Relatively high or low numbers of women with endometrioma or mucinous tumors also affect the overall results for both biomarkers in analyses [26,27]. A large older population in the benign cohort, as in ours, will affect analyses because HE4 increases every two years in healthy postmenopausal women [28] and CA-125 decreases after menopause [26,29]. Both Moore et al. [11] and Van Gorp et al. [24] had more premenopausal women (57% and 47% compared to our 26%) in the benign group than we did. Another difference is the large proportion of early stage EOC in our study. Aggressive EOC with large numbers of small peritoneal metastases and ascites (stage IIIIV) seldom present themselves with cystic formations. These stage III-IV tumors were excluded according to the study criteria and could account for a lower proportion of late stage tumors than expected.

In healthy premenopausal women CA-125 is more often elevated than HE4 in different benign gynecologic conditions [5,11,23]. This is also true for our data, suggesting that HE4 is a good complement to distinguish CA-125 false positives from true positives, especially in fertile women. A previous investigation of serum from healthy women and women with ovarian cancer showed that CA-125 is a better marker than HE4 for women age 50 and over, and that HE4 had better specificity among women under the age of 50 [23]. Further, an investigation comparing benign and malignant tumors showed that HE4 had greater sensitivity than CA-125 among premenopausal women, and that CA-125 and HE4 perform at the same level among postmenopausal women [7]. We can confirm that HE4 yields higher sensitivity and is a more stable marker in premenopausal women at a set specificity than CA-125. For postmenopausal women CA-125 (with a cut-off of 35 U/mL) yielded higher specificity than for premenopausal women (83% vs. 75%).

The cut-off value for HE4 at 75% specificity in our analysis was 85 pM but mean HE4 levels were lower in the premenopausal women. Estimated cut-off in this group was 71.8 pM, which accords with the cut-off of 70 pM used in other studies [11,24,27]. Most algorithms for the referral of patients are based on an evaluation of TVS alone or in combination with biomarkers and menopause status [1,19,21]. Indeed TVS was performed on all patients in our cohort, but not in a standardized way. Instead, we calculated ROMA in our cohort. Diagnostic accuracy of ROMA described by ROC AUC was estimated at 88.7% (postmenopaus) and 83.1% (premenopaus), which is well in line with other published data. If both markers are measured in the future, the clinician may look at the independent values for the two markers (highest negative predictive value but lower specificity) or be calculating ROMA. HE4 and CA-125 in each patient might also need to be evaluated according to other factors such as chronic or intermittent disease, BMI, and smoking [28]. In our benign group, the three highest values of HE4 belonged to two patients with chronic kidney failure, a disease known to have increased HE4 values, and one patient with breast cancer, suggesting that patient history should be accounted for more strictly when the results of new biomarkers are being evaluated.

CA-125 is known to have low sensitivity and specificity in women with early stage cancer. Previous findings of the performance of HE4 and CA-125 in early stage tumors are contradictory, favoring both CA-125 and HE4 as the best marker to detect early disease [6,7,10]. We found that the ROC AUC and sensitivity were slightly higher for CA-125 than for HE4. The number of early stage tumors in the studies range from 7 to 132 (with 47 in our study), and the menopausal status of the women differ between analyses. In our study CA-125 is again favored by the large cohort of postmenopausal women with very low levels. Dividing fertile and postmenopausal women with early stage cancer into two groups before the analysis could perhaps have changed the data, especially for the premenopausal group, but this was not done because of the small sample size.

Currently many women are diagnosed with an ovarian cyst during a visit to a gynecologist or during investigation of unknown abdominal pain by computer tomography. Women presenting with an ovarian cystic pelvic mass may be classified more correctly by measuring both CA-125 and HE4. We suggest, based on this study, that these biomarkers could be included in a prospective study of patients diagnosed with an ovarian cyst scheduled for surgery. According to their HE4 and CA-125 levels, patients would be assigned to either a conservative laparoscopic ovarian cancer staging operation or an explorative laparotomy with debulking.