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Clin Chem Lab Med 2011;49(3):443445 2011 by Walter de Gruyter Berlin New York. DOI 10.1515/CCLM.2011.073

Editorial

ROMA or death: advances in epithelial ovarian cancer diagnosis

Mario Plebani and Bohuslav Melichar

Epithelial ovarian cancer (EOC) is the fourth most common cause of cancer mortality in women in the Western world, and the leading cause of death from gynecological malignancy. The high death rate from EOC is mostly attributable to its late detection at an advanced stage and the high tendency for diffuse metastasis and recurrence. In fact, while early EOC detection results in a 92% 5-year survival rate, the overall 5-year survival rate is -50%. This is because only 19% of EOC cases are diagnosed prior to extra-ovarian spread owing to the lack of obvious symptoms prior to progression (1). Besides the obvious importance of early detection of cancer, EOC diagnosis represents a specific challenge. While for most malignant tumors, e.g., breast cancer, gastrointestinal malignancies or carcinoma of the uterine cervix, the diagnosis can be easily histologically confirmed before surgery, histological verification of EOC before surgery is usually not possible and the diagnosis is obtained from the tumor specimen obtained at surgery. The surgical approach for benign and malignant tumors differs fundamentally, and pre-operative diagnosis allows for careful planning of the surgical procedure, including referral to high volume centers, composition of the surgical team and post-operative care. As with cancers in other primary locations, the quality of primary surgery remains a principal factor determining the long-term prognosis of EOC patient. Thus, obtaining the diagnosis before histological verification is of fundamental importance for patient management in EOC, and considerable efforts have been devoted in the past decades for identifying imaging techniques and laboratory tests that would diagnose EOC prior to surgery. The serum tumor marker CA 125 is commonly used to predict the presence of a malignancy in women with a pelvic mass. The discovery of this cancer antigen by Bast et al. (2) in 1981 represented a milestone in the pathway for the development of a non-invasive and biochemically-driven approach to the diagnosis of EOC. However, measurement of CA 125 has several limitations. CA 125 is increased in less than half of early-stage EOC cases, and in approximately 80% of all EOC patients, potentially leaving 20% of ovarian cancer patients without a useful biomarker for the management of their disease. In addition, many pre-menopausal women with common benign gynecologic disorders will have increased serum CA 125 concentrations, and many medical conditions affecting post-menopausal women can also increase serum concentrations, thus resulting in a reduction of the diagnostic sensitivity and specificity of this biomarker (3). In fact, it is

well-known that CA 125, in addition to marked overexpression in neoplastic ovarian lesions, is frequently increased in association with irritation of the peritoneum and mesothelium caused by benign conditions, such as menstruation, pregnancy and the post-partum state. In 2002, patterns of mass proteomic peaks generated by mass spectroscopy (surfaceenhanced laser desorption and ionizations, SELDI) were reported to diagnose ovarian cancer, also at an early stage, with 100% sensitivity and 95% specificity (4). Because such a high degree of diagnostic discrimination for cancer had never been achieved by a blood test and would cause high expectations by both physicians and patients, interest predictably followed among investigators, funding agencies, industry and the public. However, the results of this study as well as of other studies performed with the same technique have never been reproduced, stressing the need not only of standardization in pre-, intra-, and post-analytical procedures but, even more important, study design that would avoid confounding due to bias and chance (5). The human epididymal secretory protein E4 (HE4) was first identified and characterized by Kirchhoff et al. upon differential cDNA screening of human epididymal tissue (6). Subsequent studies revealed the expression of HE4 in a number of tissues outside the male reproductive system, and it was observed that HE4 is among the most upregulated genes in epithelial ovarian carcinomas based on gene expression profiles (7). Hellstrom and colleagues first (8), and Scholler et al. (9) later on, developed specific antibodies to recognize and measure the HE4 antigen in human sera through conventional immunoassays. A fundamental further step was the availability of the first diagnostic assay for HEA commercialized by Fujirebbio Diagnostics Inc. (Malvern, PA, USA). Several publications have demonstrated superiority of HE4 over CA 125 as a biomarker for EOC (10). Regarding sensitivity, HE4 is increased in more than 50% of women whose tumors do not express CA 125. Additionally, in patients with early stage disease, HE4 has been shown to have greater sensitivity than CA 125 alone (11). When analysed as a single marker along with other serum biomarkers including CA 125, CA 72-4, mesothelin-related peptide, activin, inhibin, osteopontin, and EGF receptor, HE4 had the highest sensitivity, at 72.9%, when specificity was set at 95% (12). In addition, and even of greater interest, the ability of HE4 to distinguish benign diseases from malignancies, due to its specificity, is significantly increased in comparison to CA 125. For example, it was demonstrated that diamiderelated erythrocyte band 3 P-Tyr concentrations and the decrease in total glutathione content are significantly higher

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444 Plebani and Melichar: ROMA or death: advances in epithelial ovarian cancer diagnosis

and associated with increased concentrations of CA 125 in patients with endometriosis, while none of these patients presented with abnormal HE4 concentrations. Therefore, this study confirmed that the HE4 assay should be useful in endometriosis to rule out ovarian cancer. Taken together, the data from recently available studies seem to demonstrate that HE4 is complimentary to CA 125 as it is not falsely increased in many of the benign gynecologic disorders that can cause an increases in CA 125, primarily in pre-menopausal women. In fact, the combination of CA 125 with HE4 achieved the highest sensitivity compared to all other single markers or dual-marker combinations (11, 12). Even more recently, Huhtinen et al. analyzed serum concentrations of HE4 and CA 125 in women with EOC, endometrial cancer, endometriosis and healthy controls. The combination of HE4 plus CA 125 showed much improved sensitivity of 92.9% at 95% specificity compared with either HE4 (78.6% sensitivity) or CA 125 (78.6% sensitivity) alone (13). The Risk of Ovarian Malignancy Algorithm (ROMA) is a predictive index developed and validated from two separate pilot studies which basically takes into account the serum concentrations of both biomarkers (CA 125 and HE4) and the pre- or post-menopausal status (11, 12). In a recently published paper, the ROMA was compared with another index (the so-called Risk of Malignancy Index, RMI), to predict EOC in women with a pelvic mass. The RMI is a widely used algorithm that employs ultrasound (US) findings and imaging features of a pelvic mass, CA 125 concentrations, and menopausal status allowing the stratification of patients into high- and low-risk groups (14). Examining benign and invasive EOC, the authors found that setting the specificity at 75%, RMI achieved a sensitivity of 84.6%, while the ROMA achieved a sensitivity of 94.3%, which was significantly higher. In particular, the ROMA achieved a higher sensitivity for patients with stage I and II invasive EOC, and for patients with tumor grossly confined to the pelvis or with -2 cm disease in the upper abdomen. In addition, when averaged over all specificities, the average sensitivity (or area under the curve, AUC) was higher in ROMA than RMI for comparison of many tumor subgroups with benign disease. In this issue of Clinical Chemistry and Laboratory Medicine, two papers deal with the value of HE4 in diagnosing EOC (15, 16). The paper published by Martina Montagnana et al. (15) confirms previously reported data on the diagnostic value of the HE4 assay. In fact, the performance of this test, evaluated by the mean of ROC curves, appears higher than that of CA 125 both in pre- (AUC: 0.77 vs. 0.64) and post-menopausal (AUC: 0.94 vs. 0.84) EOC patients. In these patients, a positive correlation between CA 125 and HE4 concentrations (rs0.70, p-0.0001) was observed. However, it was not the same in those affected by benign mass or control subjects, thus underlining the higher specificity of the more recently discovered marker. In addition, the authors provide some evidence on the possible higher diagnostic accuracy of the HE4 concentrations when compared with the ROMA index. In the pre-menopausal group, the AUC was 0.64 (ps0.12, 95% CI: 0.440.83) for CA125,

0.77 (ps0.003, 95% CI: 0.620.92) for HE4 and 0.77 (ps0.002, 95% CI: 0.630.92) for ROMA; in the postmenopausal group, the AUC was 0.84 (ps0.0003, 95% CI: 0.730.94) for CA 125, 0.94 (p-0.0001, 95% CI: 0.880.99) for HE4 and 0.92 (p-0.0001, 95% CI: 0.850.99) for ROMA. There are several limitations in the study, partly admitted by the authors. These limitations include the limited number of patients with EOC and subjects in the post-menopausal group. In addition, it should be underlined that the final sentence the measurement of CA 125 for the estimation of the ROMA predictive index might be unnecessary since it does not add significant information in this clinical setting, is debatable. Most studies on HE4 were performed on Caucasian population. The paper by Kim et al. published in this issue of the Journal (16) reports on comparison of diagnostic utility of CA 125 and HE4 in Korean women. In pre-menopausal women, HE4 was superior to CA 125 in distinguishing EOC from benign ovarian mass, while the opposite was true for post-menopausal patients. The diagnostic performance of the combination of HE4 and CA 125 was in most cases higher than CA 125 alone; The ROMA index allowed the patient classification into the high- or low-risk groups with 88% sensitivity and 94% specificity (16). EOC represents a paradigm of the utilization of tumor markers in the management of the patient, not only for the diagnosis, but also for the evaluation of the therapeutic response and the detection of recurrence. Most cases of advanced EOC respond, often repeatedly, to systemic chemotherapy. Because peritoneal metastases that represent the most common mode of metastatic spread in advanced EOC are usually difficult to detect and evaluate by the currently available imaging technologies, algorithms have been developed to define the response to therapy based on the changes in CA 125 concentrations (17). Thus, the measurement of CA 125 is currenly widely used for monitoring the response to therapy and the course of the disease course. From this perspective, the data by Montagnana et al. should be welcome because they underline the importance of further studies to provide evidence not only on the diagnostic value of the HE4 assay and the ROMA index, but possibly also on their potential prognostic value and as a biochemical tool for patients monitoring and follow-up.

Conflict of interest statement

Authors conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.

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Prof. Mario Plebani Leonardo Foundation, Abano Terme and Department of Laboratory Medicine University-Hospital of Padova Via Giustiniani 2 35128 Padova Italy Phone: q390498212792 Fax: q39049663240 E-mail: mario.plebani@unipd.it Prof. MUDr. Bohuslav Melichar, PhD Department of Oncology Palacky University Medical School and Teaching Hospital Fakultn nemocnice I. P. Pavlova 6 775 20 Olomouc Czech Republic Phone: q420 588 444 288 Fax.: q420 588 442 522 E-mail: bohuslav.melichar@fnol.cz; Web: www.fnol.cz