The use of HE4, CA125 and CA72-4 biomarkers for differential diagnosis between ovarian endometrioma and epithelial ovarian cancer

Endometriosis is a common chronic disease, present in 5-10% of women in reproductive age [1]. The disease, characterized by the presence and growth of endometrial tissue outside the uterine cavity, is often associated with infertility and pelvic pain and it tends to recur [2, 5]. Endometriosis can be diagnosed by clinical and ultrasound examinations (US) but the most accurate procedure to confirm the diagnosis is laparoscopy that allows visualization of lesions and histological confirmation [6] .

Endometriosis is a benign disease but it shares several characteristics with invasive cancer. Cancer antigen 125 (CA125) measurement is an important component in the work-up of a woman with an adnexal mass [7]. However, CA125 is characterized by a low diagnostic specificity, as abnormally high concentrations can be found in malignancies of different origin including non-ovarian gynecological cancer, such as endometrial, pancreatic, lung, breast and colorectal cancer [8]. In patients with endometriosis CA125 levels can be high. In fact, CA125 is the most extensively investigated and used peripheral biomarker of endometriosis [9]. In addition, elevated serum levels of CA125 are associated with non-gynecological diseases such as tuberculosis, liver cirrhosis and also in physiological conditions such as pregnancy or different phases of the menstrual cycle [10, 11]. Thus, CA125 has a limited role in the differential diagnosis between endometriosis and ovarian cancer due to the lack of specificity [12]. Recently, the role of surgery for the treatment of ovarian endometriosis in women with pregnancy desire has been criticized because of the fear of ovarian health tissue damage [6, 13, 14]. In selected cases, particularly in women undergoing assisted reproductive techniques, it is mandatory rule out an ovarian malignancy before ovarian stimulation and embryo-transfer [15]. Misdiagnosed ovarian cancer has been found in women with suspected ovarian endometriosis [16, 17].

Therefore identification of non-invasive and accessible markers of epithelial ovarian carcinoma (EOC) is valuable. For this reason serum tumor markers are being increasingly used for the differential diagnosis of adnexal masses.

Recently, the human epididymis protein 4 (HE4) has proved to be a promising marker for epithelial ovarian cancer with higher specificity and sensitivity than CA125 in distinguishing malignant from benign pelvic masses [18, 19]. Particularly, measuring both HE4 and CA125 serum concentrations increases the accuracy of ovarian cancer diagnosis and provides valuable information for discriminating ovarian tumors from ovarian endometriotic cysts [20, 21] or other gynecologic conditions [8]. However, an increased HE4 levels have been observed also in other types of cancer, i.e. lung adenocarcinoma [22] and in patients with impaired renal function [23].

Other biomarkers have been studied for clinical application in EOC. Among these, cancer antigen 72–4 (CA72-4), a glycoprotein, which increases in gastric, colon, breast and ovarian adenocarcinomas, may be employed alone or in combination with CA125 and HE4. CA72-4 is less sensitive than CA125 for EOC, but it is not influenced by pregnancy or the phase of menstrual cycle [24, 25].

The aim of this study was to evaluate the role of those biomarkers, which are usually elevated in patients with EOC, that could be useful to confirm the nature of ovarian cystic endometriosis or other benign ovarian masses.

From June 2012 to February 2013, 115 consecutive Italian women (mean age: 35 years, range: 22–82) referred to the Department of Gynecology, Obstetrics and Urology at the University of Rome ”Sapienza” for the presence of an adnexal mass detected at clinical and ultrasound examinations were enrolled in the study. Control group consisted of 50 healthy women (mean age: 30 years, range: 21–57) with clinical and US outcome negative for ovarian masses.

Exclusion criteria included current hormonal therapy, pregnancy, chronic diseases or other types of cancer. Two patients were excluded from the study because they were at the beginning of pregnancy. The study was part of a study protocol approved by local Ethics Committees. All patients signed written informed consent to the study. At enrolment, medical history was collected and peripheral blood samples were drawn from all women and immediately sent to the laboratory for analysis of tumor markers. All groups underwent complete physical and gynecological examination and transvaginal ultrasound (TVUS) with color Doppler imaging.

Women diagnosed with a pelvic mass subsequently underwent surgery. Disease was confirmed by histopathological examination. The women were divided into the following 4 Groups:

Group 3: 57 patients with ovarian endometrioma (mean age: 36 years, range: 23–48). Diagnosis of endometriosis was achieved on the basis of medical history, clinical and pelvic transabdominal and/or transvaginal US examinations. Patients with indeterminate findings underwent pelvic magnetic resonance imaging (MRI) to confirm suspected endometriosis using the previously described technique [26, 27]. At laparoscopy, the disease was staged according to the rASRM classification [28]. Mean diameter of endometriomas was 33 ± 18.9 mm (range 10–80). One patient had both endometriosis and an ovarian dermoid cyst.

Group 4: 39 patients with ovarian carcinoma (mean age: 64 years, range: 28–91). Histology confirmed the diagnosis and staging was made according to the International Federation of Gynecology and Obstetrics (FIGO)[29].

Population characteristics are summarized in Table 1.

Endometriosis is a known cause of CA125 elevation and represents a common gynecologic disorder in women of reproductive age [2]. Generally the diagnosis of ovarian endometriosis is made by clinical and imaging technique examinations [31] and confirmed by surgery with histological examination [6]. Recently surgical treatment of ovarian endometriosis in women desiring pregnancy has been criticized because of the risk of ovarian healthy tissue damage [13, 14]. Therefore, in selected cases with ovarian endometrioma treated by medical therapy or undergoing assisted reproductive techniques (ART) without prior surgery, a correct diagnosis is mandatory. In these cases, the use of tumor markers with high sensitivity and specificity could help to reduce the risk, even small, of undetected ovarian cancer. In fact, there is a recognized association between endometriosis and clear cell, low-grade serous and endometrioid ovarian cancer [32]. So far, very little is known about the underlying factors involved in the malignant progression of endometriosis. For more than two decades CA125 has been the only marker employed in the diagnosis of EOC, but, although overexpressed in more than 80% of ovarian cancers, it lacks of specificity [33].

In the present study, we investigated the role of serum CA125, HE4 and CA72-4 in the diagnostic evaluation of ovarian endometrioma and adnexal mass. In agreement with data reported in literature [19], more than 50% of women with endometriosis expressed high levels of CA125, confirming the low specificity of this marker.

It was recently observed that HE4 rarely increases in benign gynecologic conditions suggesting its complementary role to CA125 [18, 19, 25, 34, 35]. In women with endometriosis, Moore et al. observed a marked difference between HE4 levels, which was increased only in 3% of cases, compared to CA125, which was elevated in 67% of cases [19]. Hamed et al. showed that serum HE4 and CA125 concentrations were significantly higher in patients with ovarian cancer compared with levels observed in patients with benign disease or healthy controls. In their study CA125 and HE4 had high sensitivity (90% vs 83.3%) and combining the two markers EOC were correctly detected in 97% of cases [36]. Moreover HE4 measurement, in healthy premenopausal women as well as in women with endometriosis, can be carried out at any phase of the menstrual cycle, and irrespective of hormonal therapy, extending the benefits of HE4 use in clinical practice [37, 38]. However, HE4 overexpression has been also observed in non-oncologic conditions such as chronic kidney disease, which represents the most important known source of false-positive HE4 results [23]. In addition, recent studies have reported high HE4 levels also in some benign gynecological conditions such as uterine fibroma and pelvic inflammatory disease [8, 19]. Moreover, HE4 levels in healthy women are affected by age, BMI and smoking [39].

In our study CA125 and HE4 yielded a sensitivity of 90% and 87% and a specificity of 70% and 100% respectively in the diagnosis of epithelial ovarian cancer. HE4 never increased in women with endometriosis and it was able to correctly discriminate malignant from all benign ovarian masses. Therefore we agree with previous reports that HE4 is the most useful marker for the differential diagnosis between EOC and ovarian endometriosis [19]. However, since some benign conditions can be associated with high HE4 levels, in selected cases CA72-4 may be useful for the differential diagnosis. Nevertheless the role of CA72-4 in the differential diagnosis between benign form malignant ovarian mass is still controversial [18].

In our study a slight and not statistically significant increase of CA72-4 was found in a small number of patients with endometriosis, with the highest observed value of 6.2 U/mL, which is indeed a borderline value, found only in one woman. Our data confirm the results reported by Lenhard et al. who showed that CA125 but not CA72-4 tends to be increased in the presence of endometriosis [24]. A multimarker approach, consisting of HE4, CA125 and CA72-4, can provide a more accurate tool for a differential diagnosis of patients with ovarian endometriotic cysts, other benign ovarian masses and ovarian cancer.

In conclusion our results suggest that the use of serum HE4, CA125 and CA72-4 may be a valuable approach for distinguishing patients with ovarian endometrioma or other benign adnexal masses from those with ovarian malignancy. This approach could reduce medical costs related to more expensive diagnostic procedures and it may have a reassuring effect on the patient. Further studies are needed to confirm these results.