The dire need to develop a clinically validated screening method for the detection of early-stage ovarian cancer

For almost half a century, the WHO has urged the medical community to create screening methods with the primary purpose of detecting disease at an early, treatable stage [1]. This is of particular importance in epithelial ovarian cancer (EOC) given that there are no clinically validated biomarkers or screening protocols available for the accurate detection of early-stage disease. This year, approximately 22,000 women will be newly diagnosed with ovarian cancer, while an additional 15,000 deaths will be attributed to this disease [2]. These statistics make EOC the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy, with more women dying from this disease than all other gynecologic malignancies combined [2]. In addition, during the past 40 years, the overall survival for women with advanced-stage (III/IV) EOC has remained relatively unchanged, at less than 30%, despite surgical advancements and improvements in chemotherapeutics [2]. However, women diagnosed with early-stage EOC (stage I) require less morbid surgical procedures, may not require adjuvant chemotherapy, have an improved quality of life and, most importantly, have an overall survival approaching 93% [2]. Unfortunately, the majority of women (75%) continue to be diagnosed with advanced-stage EOC owing to the rather stealth nature of early-stage disease and the lack of a clinically validated screening method. Therefore, increasing the proportion of women detected with early-stage EOC, particularly stage I, would significantly improve women’s healthcare.

The standard screening methods currently in use to detect ovarian cancer include pelvic exams, transvaginal or transabdominal ultrasonography and the serum cancer antigen (CA)125 level. Quantitative CA125 levels have long been the ‘gold standard’ biomarker test for the surveillance of women with EOC; however, it is only elevated in 47% of women with early-stage disease. Moreover, CA125 is nonspecific and is elevated in many disease states, such as other abdominal malignancies, benign diseases and physiological conditions. Therefore, its effectiveness as an independent screening test is compromised owing to its lack of sensitivity and specificity. Although many other tumor biomarkers have been identified in ovarian cancer during the past decade, few of them have been widely tested for screening purposes. Furthermore, traditional ultrasonography is not an effective population screening test owing to its rather low sensitivity and specificity, both alone and combined with CA125.

Given ovarian cancer’s relatively low general population incidence rate (1.8%) [2], a valid screening test must have both a high sensitivity and a high specificity to decrease the number of false-positive test results and the subsequent invasive surgical procedures undoubtedly associated with this result. The ideal screening test for the postmenopausal population (with a prevalence of one in 2500) would require a specificity of 99.6% to yield an approximate positive predictive value of 10%, where a surgeon performs ten operations for each case of ovarian cancer detected. The positive predictive value of most screening tests is determined by specificity and disease incidence – given ovarian cancer’s low frequency, achieving an acceptable positive predictive value requires an extremely high specificity, especially in average-risk populations.

Multiple initiatives have been undertaken to discover strategies that detect and diagnose early-stage EOC, including the search for novel serum biomarkers and the development of new technologies, such as contrast-enhanced ultrasonography, with a number of them demonstrating hopeful results. The ideal screening test for ovarian cancer would be a simple measurement of biomolecules in bodily fluids, such as blood, serum or urine, whose absolute concentrations could differentiate cancer from noncancer and be organ specific. In the last decade, insights into the EOC microenvironment, as well as technological advances, such as microarrays and proteomics, have triggered the discovery of hundreds of potential clinically valuable biomarkers:

• ▪ Lysophosphatidic acid (LPA) is a phospholipid derivative consisting of a glycerol backbone, a single fatty acid chain and a free phosphate group. LPA has a variety of isoforms depending on fatty acid-chain variability at the sn-1 position. LPA was found to be elevated in the serum, plasma and malignant effusions of women with ovarian cancer and has known functions in cell proliferation, invasion and angiogenesis [3]. This molecule initially became of interest in 1998 for its reported high sensitivity and specificity to detect early-stage ovarian cancer [3]; however, its utility as a screening biomarker has been limited owing to the difficulty of isolating and detecting the different isoforms in patients’ serum and its specificity for ovarian cancer;

• ▪ Human epididymal protein (HE)4 is a relatively new biomarker used to monitor disease recurrence and disease progression in patients with ovarian cancer. It is the product of the WFDC2 (HE4) gene, which is overexpressed in ovarian cancer. HE4 has exhibited increased sensitivity to detect stage I disease [4] and has demonstrated promise as a sensitive and specific biomarker when combined with CA125 and other molecules [5]; although, more studies remain to be done to warrant its use as a biomarker for the detection of early-stage EOC;

• ▪ Osteopontin (OPN) is a glycoprotein involved in cell adhesion, inflammation and tumorigenesis, with elevated levels seen in ovarian cancer [6]. Similar to HE4, OPN has been used in combination assays to identify ovarian cancer [7]; however, OPN is also elevated in other cancers and benign conditions, limiting its specificity to be used as an ovarian cancer biomarker;

• ▪ Kallikreins (KLKs) are serine proteases that function in cell growth, angiogenesis and invasion. KLKs are elevated in patient serum with ovarian cancer. KLK8 was reported to be associated with early disease, while KLK5, -6, -10 and -13 have been combined with CA125 to improve the accuracy of ovarian cancer detection [8,9];

• ▪ Claudins are components of tight junctions that create selective barriers and maintain cell polarity. Multiple claudins have been found to be elevated in ovarian cancer; however, their specificity has yet to be determined and verified [10].

In addition to the biomolecules mentioned previously, patient-derived tumor-reactive antibodies are also considered to be new diagnostic markers. A recent report suggests that the quantification of circulating tumor-reactive IgG can be used to identify the presence of ovarian cancer as well as distinguish early- and late-stage EOC. Tumor-reactive antibodies are stable and less sensitive to confounding factors, such as stress and sample manipulation. They are released in the circulation soon after tumor development and, thus, might represent a new class of markers for early detection of ovarian cancer [11].

Of particular note, traditional genetic pedigree analysis of ovarian cancer patients may provide information to help identify high-risk populations; for example, inherited BRCA1 and -2 mutations increase the risk for women to develop ovarian and/or breast cancer [12]. In addition, molecular profiling at the epigenetic level, such as miRNA profiling, may allow for the identification of novel biomarkers for early detection of ovarian cancer, given that these epigenetic changes might be detectable before the development of the physical tumor. Proteomic profiling allows for the identification of cancer-specific ‘fingerprints’ of proteins or peptides (proteomic patterns), the true effectors of genetic changes underpinning malignant transformation, from fluid samples without any prior knowledge of the protein’s characteristics. Unfortunately, proteomic patterns for detection are wrought with problems, especially the issue of reproducibility, as mass spectrometry is exquisitely sensitive to specimen handling. Nevertheless, these technologies, and others, are now continuing to identify and quantify innumerable potential markers for ovarian cancer. Separately, all these biomarkers, when used alone, do not appear to have the sensitivity and specificity required to accurately detect early-stage ovarian cancer owing to the heterogeneity of this disease. However, a biomarker panel or a multianalytes assay, which combines several biomarkers, may enhance the sensitivity and specificity of each individual marker and, subsequently, serve as an effective tool for the detection of early-stage disease. Nonetheless, vigorous clinical validation is required to verify their effectiveness in screening and early detection to optimize patient care.

Although many investigators are working to identify clinically relevant and validated biomarkers that can accurately detect EOC, a positive result will not immediately call for surgical intervention; clinical confirmation by imaging is warranted before such invasive procedures. Unfortunately, early-stage EOC has been notoriously resistant to detection by traditional ultrasonography; however, recent advances in ultrasound technology and the incorporation of intravenous contrast agents to enhance the visualization of the ovary may hold value. Intravenous contrast agents, akin to those used in CT and MRI, generate small, stabilized microbubbles of 1–10 µm in diameter that allow the visualization of the aberrant tumor neovascularization associated with early-stage disease by delineating the leaky vascular channels unique to malignancy [13]. This improved ability to quantify and distinguish vascular changes specific to early disease may help to detect early-stage cancer with greater accuracy. We have demonstrated that quantification of contrast-enhancement kinetics has 100% sensitivity and 96.2% specificity to differentiate benign from malignant tumors [13].

Despite these advances, at present, no clinically validated screening protocol for the detection of early-stage EOC exists. The discovery of novel biomarkers relies on obtaining a better understanding of the initiation and progression of EOC. Clinical validation and implementation of biomarkers will also benefit from advancements in new molecular and imaging technologies as patient care is optimized. Fortunately, hundreds of biomarkers have been identified; however, their clinical utility remains to be determined. In addition, the enhanced imaging capabilities of the ovary by ultrasound are providing practitioners with the ability to more accurately and precisely identify changes specific to the newly transformed ovary. The combination of these two modalities, biomarker panels and biologically based imaging may be the future. Therefore, we must forge ahead to develop a validated early-detection protocol that will not only decrease the number of advanced-stage diagnoses and deaths attributed to ovarian cancer but, most importantly, positively impact the future of women’s healthcare.