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Abstract

Ovarian cancer is the most lethal gynecological malignancy. However, effective screening strategies have not been established and continue to be elusive. A good screening test must adequately address validity, reliability, yield, cost, acceptance and follow-up services. An ideal screening test for ovarian cancer must have a high sensitivity in order to correctly diagnose all women with the disease and a high specificity to avoid false-positive results. The current screening modalities of bimanual examination, CA-125 and transvaginal ultrasonography together allow us to detect only 30–45% of women with early-stage disease. Recent developments in proteomic and genomic research have identified a number of potential biomarkers. Although panels of tumor markers and proteomic-based technologies may improve the positive predictive value, all markers require validation and interfacing with newly developed diagnostic imaging technologies. While a large amount of information on miRNAs has been promising, much remains to be elucidated. This review will examine the current status of biomarkers and technologies of interest in the effort of early detection of ovarian cancer.

Keywords

biomarker early detection miRNA ovarian cancer proteomics

Medical Education Online

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Release date: 12th March 2013; Expiration date: 12th March 2014

Learning objectives

Upon completion of this activity, participants should be able to:

Describe properties of ideal screening tests for ovarian cancer and diagnostic performance of current screening modalities

Describe the potential role of biomarkers in detecting early-stage ovarian cancer

Describe the potential role of new technologies in detecting early-stage ovarian cancer

Current challenges of early ovarian cancer detection

Owing to the low prevalence of ovarian cancer, an effective screening method for detection of early-stage disease requires a specificity of at least 99.6%, sensitivity of at least 75% and a positive predictive value of at least 10% to be considered an effective tool [5]. Despite intensive research, presently no such test exists. Currently, two large clinical trials are evaluating ovarian cancer detection using CA-125 and transvaginal ultrasonography. The PLCO screening trial including 39,105 women aged between 55 and 74 years with a median follow-up of 12.4 years, showed that screening simultaneously with CA-125 and transvaginal ultrasonography did not reduce ovarian cancer mortality in women from the general population. There were 118 deaths caused by ovarian cancer in the screened group compared with 100 deaths among the controls. In addition, of the 3285 women with a false-positive result, 1080 underwent surgery and 163 (15%) experienced at least one serious complication [6]. The UKCTOCS trial involves 202,638 low-risk postmenopausal women. The screening differs from the PLCO screening trial in the use of algorithms to interpret CA-125 (risk of ovarian cancer algorithm) [7]. Although initial results in 2009 yielded an encouraging sensitivity and specificity [8], the outcome of the UKCTOCS trial will not be available by 2015, until then there will be continuing uncertainty about the efficacy of ovarian cancer screening in the general population.

Currently, recommendations from professional societies remain unchanged for ovarian cancer screening. The US Preventive Services Task Force guideline, released in April 2012 incorporating the results from the PLCO screening trial, has continued recommending against screening for ovarian cancer in the general population and reaffirmed the US Preventive Services Task Force position that the potential harm of a false-positive result outweighs any potential benefits. The US Preventive Services Task Force recommendations are consistent with guidelines from other professional societies in the USA including the American Congress of Obstetricians and Gynecologists [9] and the Society of Gynecologic Oncology [10]. The recommendations from professional societies have not changed over time mostly due to the lack of strong evidence of clinical benefit. The challenge continues to find a better tool or strategy with a high sensitivity and specificity for malignancies to detect early-stage ovarian cancer and to minimize the false-positive number in such a low incidence disease.

Risk factors of ovarian cancer

Until an effective screening algorithm is available, analysis of family history and identification of women at high risk for ovarian cancer may help to select those women who would most benefit from current screening strategies. These risks include a personal or family history of breast or ovarian cancer, genetic predisposition based in inherited genes or family pedigree of malignancies associated with increased risk, age, nutrition, parity and reproductive factors. The most important risk factor is a family history of breast or ovarian cancer. In contrast to the 1.8% general population risk for ovarian cancer, a family history of ovarian cancer in a first-degree relative triples a woman's lifetime risk of developing ovarian cancer [10]. Women who have inherited high-penetrance cancer susceptibility genes, such as mutated BRCA1 or BRCA2 genes or those who inherited Lynch syndrome-associated mutations, are at a greatly increased risk of developing ovarian cancer. A number of familial ovarian cancer syndromes have also been identified such as Cowden disease, also known as multiple hamartoma syndrome, and Li–Fraumeni syndrome.

Although inheritance of a genetic mutation accounts for 10% of cancers, 90% of epithelial ovarian cancers (EOCs) occur sporadically [5]. A woman with a BRCA1 mutation (located on chromosome 17q) has a 39–45% lifetime risk of developing ovarian cancer and for BRCA2 mutation carriers (located on chromosome 13q) a risk of 11–25% [11]. Women of eastern European or Ashkenazi Jewish descent, are at higher risks of being positive carriers of BRCA1 or BRCA2 mutations. Of patients diagnosed with ovarian cancer, 36–41% of Ashkenazi women have a BRCA1 or BRCA2 mutation compared with the 10–15% of the general population [202]. The phenotype of BRCA-positive ovarian cancers is more likely to be of the serous subtype, the next most common histologic type is endometrioid, followed by mucinous and then borderline. BRCA-positive tumors are also more likely to be high grade and found in advance stages [12]. Women who have endometriosis are linked to a more than threefold chance of developing clear cell ovarian cancer and double the risk of developing endometrioid carcinoma and low-grade serous ovarian cancers [13].

Although EOC could originate either from normal ovarian surface epithelium itself or from the crypts or inclusion cysts arising from this surface epithelium [14], recent evidence suggests that high-grade serous ovarian cancer can arise from serous tubal intraepithelial carcinoma. This is based on the observation that the most common premalignant pathologic findings in risk-reducing salpingo-oophorectomy specimens from BRCA mutation carriers are in the distal fallopian tube [15]. Tumors classified as primary ovarian serous carcinomas often involve the endosalpinx and 48% of these contain both serous tubal intraepithelial carcinoma and p53 signature [16]. Recently, an algorithm has been suggested to standardize the classification of serous tubal intraepithelial carcinoma by incorporating morphologic and immunohistochemistry markers for p53 and Ki-67 [17].

In terms of factors that may lower the risk of ovarian cancer, there is strong evidence that the use of oral contraceptives for 5 or more years is associated with an approximate 50% reduction in ovarian cancer risk. A meta-analysis of 18 studies including 13,627 BRCA-mutation carriers reported a significantly reduced risk of ovarian cancer (significantly reduced risk: 0.50) associated with oral contraceptive use [201]. In general, the lifetime risk of developing ovarian cancer decreases among women who breastfeed their children [18], the risk is estimated to decrease by 15% with each live birth [19]. Gynecologic procedures such as hysterectomies and tubal ligations also decrease risk [20].

Currently, for women with identified hereditary ovarian cancer syndromes, the National Comprehensive Cancer Network and the Society of Gynecologic Oncology recommend screening every 6 months with CA-125 and transvaginal ultrasonography beginning between the ages of 30 and 35 years or 5–10 years earlier than the earliest age of first diagnosis of ovarian cancer in the family [10,203]. The American Congress of Obstetricians and Gynecologists committee opinion in March 2011 is consistent with the National Comprehensive Cancer Network guideline [9].

Ovarian cancer presentation

Ovarian cancer has been dubbed a silent killer because it has been relatively asymptomatic until late stages. Patients may report abdominal pain, bloating, early satiety, changes in urination and increased abdominal size. As the disease progresses patients may present with abnormal uterine bleeding, shortness of breath, ascites and palpable inguinal lymph nodes. Approximately 95% of women reported symptoms 3 months before presenting to their physicians. There are two types of ovarian cancer with different presentation [21]. Type I ovarian cancers, including well-differentiated serous, mucinous, malignant Brenner, clear cell and endometrioid tumors, exhibit slow progression. The pathogenesis of this first type includes microsatellite instability and mutations in KRAS, BRAF, β-catenin and PTEN. Type II ovarian cancers, including malignant mixed mesodermal tumors, carcinosarcomas, undifferentiated carcinomas, serous carcinomas of the moderately or poorly differentiated variety, have a rapid onset, early metastasis and mitigate the possibility of detection of early-stage disease. The pathogenesis of this second group frequently involves mutations in the p53 tumor suppressor gene [16].

Since 2007 an ovarian symptom index has been developed for screening purposes [22]. A positive symptom index was the result of persistent symptoms for more than 12 days in a month but lasting less than 1 year. Rossing et al. reported sensitivity of 60% for early-stage disease and 79.1% for advanced-stage disease, but unfortunately yielded a very low positive predictive value (0.6–1.1%) [21].

Available tools for ovarian cancer detection

Pelvic examination

Currently, the pelvic examination, transvaginal ultrasonography and serum CA-125 levels are the standard modalities in detecting ovarian cancer. However, pelvic examinations are not efficient in distinguishing an early or premalignant lesion from a normal ovary [23]. Evidence demonstrates that the sensitivity and specificity of detecting a pelvic mass based solely on a pelvic examination are approximately 40 and 90%, respectively [24], which are below the required criteria for an effective screening test.

Diagnostic imaging

Transvaginal ultrasonography is the initial diagnostic modality of choice for the evaluation of the adnexa. However, the sensitivity and specificity of transvaginal ultrasonography for the definitive diagnosis of ovarian cancer is limited. In a screening study from the National Ovarian Cancer Early Detection Program, 4526 women at high risk for ovarian cancer were screened with transvaginal ultrasonography, which demonstrated the limited value of this modality when all ovarian, primary peritoneal and fallopian tube cancers detected in asymptomatic women were stage III [25]. Recently, a prospective study from the University of Kentucky (KY, USA) evaluated 37,293 asymptomatic women aged 50 years or older, and asymptomatic women aged 25 years or older with a documented family history of ovarian cancer, who received annual ultrasonographic screening, with a mean of follow-up of 5.8 years. A total of 47 invasive EOC and 15 epithelial ovarian tumors of low malignant potential were detected. Stage distribution for invasive epithelial cancers was: stage I, 47%; stage II, 23%; stage III, 30%; and stage IV, 0%. The authors achieved a specificity of 98.5% and a positive predictive value of 8.9% with 11.1 operations per case of primary invasive EOC. The 5-year survival rate for all women with invasive EOC detected by screening was 74.8% compared with 53.7% for unscreened women with ovarian cancer [26]. However, whether the results reflect a difference or biases related to nonrandomized studies (healthy volunteer effects, lead time) have to be explained [27].

In order to improve the efficacy of sonography, the technique that combines grayscale morphologic assessment with tumor vascularity in a diagnostic system is significantly better in ovarian lesion characterization than Doppler arterial resistance measurements, color Doppler flow imaging, or grayscale morphologic information [28]. There is evidence that the use of contrast agents with 3D power Doppler sonography is superior to that of nonenhanced sonography (95 vs 86.7%) [29]. Fleischer et al. has shown that using pulse inversion harmonic imaging with contrast enhanced ultrasonography is a more appropriate method to aid differentiation between benign and malignant ovarian tumors [30].

CA-125

To date, the CA-125 glycoprotein antigen is the most commonly measured tumor marker for epithelial ovarian tumors, which account for 85–90% of ovarian cancers. CA-125 was first detected using the OC125 murine monoclonal antibody [31]. CA-125 was originally developed to monitor patients previously diagnosed with ovarian cancer and not for screening. Alone, CA-125 is only elevated in 47% of women with early-stage ovarian cancer, while CA-125 levels are elevated in 80–90% of advanced-stage ovarian cancers [32]. As CA-125 levels are elevated in many benign conditions in premenopausal women, its utility as a tumor marker is more effective in postmenopausal women [21]. For detection of ovarian cancer in postmenopausal women, the CA-125 cut-point of 35 units/ml has been used. The 98th percentile in this population yielded a 2% false-positive rate, whereas the same cut-point in premenopausal women resulted in substantially higher false-positive rates. Baseline CA-125 values and clinical and demographic data from 3692 women participating in a screening study, which was conducted by the National Cancer Institute, recommended to achieve a 2% false-positive rate in ovarian cancer screening trials and in high-risk women, the cutoff point for initial CA-125 testing should be personalized primarily for menopausal status: 50 units/ml for premenopausal women, 40 units/ml for premenopausal on oral contraceptive, and 35 units/ml for postmenopausal women [33]. Clinically, CA-125 has been used to follow women diagnosed with ovarian cancer for prognosis, surveillance and optimization of care. However, as CA-125 has been the oldest and one of the best performing biomarkers, a biomarker panel used to detect ovarian cancer in its early stage will include CA-125 [34]. Together with the use of CA-125, the focus has incorporated other biomarkers with and without combined imaging techniques and simultaneous evaluation of multiple markers may achieve the required sensitivity–specificity.

A risk of malignancy index is a triaging tool obtained by multiplying serum CA-125 level by a score for specific pelvic ultrasound findings, and by a score for menopausal status. The risk of malignancy index yields a sensitivity of 90% and specificity of 89 % in determining malignancy in pelvic masses [35].

Serum biomarkers are under evaluation beside CA-125

Multiple publications have identified potential biomarkers for the detection of ovarian cancer beside CA-125.

HE4

This protein has a WAP-type four disulfidecore and is encoded by the WFDC2 gene found on chromosome 20q1213.1 [36]. It is elevated in ovarian cancer and there is increased HE4 mRNA expression in different types of EOC's [37,38]. HE4 is overexpressed in specific subtypes of ovarian cancer, 100% in endometrioid and 93% in serous ovarian cancer, possibly enabling one to distinguish among several tumor types [34]. Alone, the sensitivity of this marker has been reported to be 95% with a specificity of 72.9% [39]. Moore et al. reported a sensitivity of 76.5% and specificity of 95% when CA-125 and HE4 were combined to differentiate benign from malignant lesions in a retrospective study [39]. Recently, in a prospective trial, this panel yielded a sensitivity of 93.8%, a specificity of 74.9% and a negative predictive value of 99% [40]. Equally important, HE4 is less likely to be elevated falsely in the setting of benign neoplasms as compared with serum CA-125, and it can be used to differentiate endometriomas from malignant ovarian tumors, with sensitivity of 71% as compared with sensitivity of 64% of CA-125. HE4 is also noted as a potential marker for adenocarcinoma of the endometrium [41]. Recently, two meta-analysis studies independently published a similar conclusion that HE4 is a valuable marker in the diagnosis of ovarian cancer [42,43]. HE4 has recently obtained US FDA approval for monitoring of disease recurrence or progression but not for screening.

The Risk of Ovarian Malignancy Algorithm is a more effective tool that includes a patient's menopausal status, CA-125 and HE4 levels manipulated by a logarithmic formula. When HE4 and CA-125 are combined to detect malignancy, Moore et al. reported a sensitivity of 94.3% and specificity of 75% [44]. The ability to differentiate cancer from endometriosis by the Risk of Ovarian Malignancy Algorithm had a specificity of 80% and sensitivity of 85.4% [45]. To differentiate borderline tumors from healthy controls/benign disease HE4 alone was the better test in comparison with CA-125, with specificities of 81.8 and 85.9%, and sensitivities of 62.5 and 62.5%, respectively. HE4 is also a better test to differentiate a benign pelvic mass: in a study of 1042 women with benign disease HE4 levels were less often elevated than CA-125 (8 vs 29%) [46]. In summary, HE4 was superior to CA-125 in separating benign, borderline ovarian tumors, cancers of the fallopian tubes, as well as early-stage EOC.

Mesothelin

Mesothelin is a glycosylphosphatidylinositollinked cell surface molecule expressed by mesothelial cells. Mesothelin level can be measured in urine and elevated in mesothelioma, pancreatic and ovarian cancers. Elevated serum mesothelin was detected in 60% of ovarian cancers with a specificity of 98% [47]. In a study consisting of 44 ovarian tumor specimens, Obulhasim et al. found that mesothelin was expressed in 100% of serous cystadenocarcinoma and 100% of serous borderline tumors of the ovary. The average methylation of CpG sites in ovarian tumors ranged from 6 to 56% in mesothelin-positive, and from 13 to 79% in mesothelin-negative samples. The authors identified diverse levels of methylation/hypomethylation at CpG sites in the mesothelin promoter region in ovarian cancer [48]. Mesothelin may aid in the peritoneal implantation and metastasis of tumors through its interaction with mucin MUC16 (CA-125) [49]. Combination of mesothelin and CA-125 detected more ovarian cancers than each marker alone. Mesothelin was elevated in 42% of urine assays in comparison with 12% of serum assays of early-stage ovarian cancer patients at 95% specificity [50].

Transthyretin

Transthyretin (TTR) is prealbumin indicator of nutritional status, and an acute phase reactant involved in tumor development [51]. TTR is the major carrier for serum thyroxine and facilitates the transport of retinol via retinol binding protein. The serum level of full-length TTR was downregulated among patients with later stage ovarian cancer relative to that in healthy controls and patients with colorectal, breast or prostate cancer. It was identified that a truncated form of TTR showed a lack of the N-terminal ten amino acids. In addition to mutations on protein level, TTR exists in different isoforms [52]. It has been reported that TTR is decreased in EOC [53]. When combined with CA-125, ApoA1 and transferrin in a study of 358 serum samples, the panel yielded a sensitivity and specificity of 96% and 98%, respectively, for detection of early-stage ovarian cancer [54].

ApoA1

ApoA1 is constituent of high-density lipoproteins. Exogenous ApoA1 prevents tumor development in mice while lowered ApoA1 concentrations are associated with ovarian cancer. Decreased ApoA1 levels were previously reported in the serum of patients with ovarian cancer. The mechanism of this association remains unclear at this time; however, it has been proposed to be associated with free radical-mediated damage to cellular biomembranes resulting in lipid peroxidation [55]. ApoA1 can be a biomarker for the detection of early-stage ovarian cancer, and may also be a promising therapeutic agent for the treatment of ovarian cancer [56]. When ApoA1 was combined with CA-125 and TTR, the overall sensitivity and specificity were significantly improved in the receiver operating characteristic curve. For stage I and II detection, the sensitivity was 93.9% at 95% specificity [57].

VCAM

VCAM-1 is a cell surface receptor expressed on activated endothelial and mesothelial cells, which functions to regulate leukocyte attachment and extravasation at sites of inflammation. VCAM-1 protein was found to be preferentially expressed on the mesothelium of ovarian cancer patients compared with the mesothelium of women without cancer. Ovarian cancer cell invasion of the mesothelium was quantified using a coculture assay system. Inhibition of VCAM-1 function in the coculture system decreased ovarian cancer transmigration of the mesothelium [58]. When VCAM-1 was combined with CA-125 and other biomarkers, the panel yielded sensitivity of 86% for early-stage and 93% sensitivity for late-stage ovarian cancer at 98% specificity [59].

IL-6 & IL-8

IL-6 is an acute phase reactant and promotes inflammation. High levels of IL-6 were found in 50% of 114 patients with primary ovarian cancer. IL-6 sensitivity was lower than that of CA-125 [60]. IL-6 is also correlated with poor prognosis and clinical outcome [61]. A multimarker test which combines CA-125 with C-reactive protein, serum amyloid A (SAA), IL-6 and IL-8, was reported to have 94.1% sensitivity and 93.1% specificity for detection of ovarian cancer [62]. IL-8 is an acute phase reactant recruiting leukocytes. IL-6 and IL-8 are pleomorphic cytokines that have been implicated in aspects of tumor growth, disease progression and/or treatment [63]. Analysis of circulating concentrations of anti-IL-8 IgG allowed for prediction of early ovarian cancer (stages I and II) with 98% specificity and 65.5% sensitivity [61].

B7-H4

B7-H4 is a 282 amino acid protein, which is expressed on the surface of a variety of immune cells and functions as a negative regulator of T-cell responses. B7-H4 may promote malignant transformation. Tringler et al. found that B7-H4 expression was consistently higher in serous, endometrioid and clear cell ovarian carcinomas compared with mucinous subtypes or normal somatic tissues. These findings indicated that B7-H4 should be further investigated as a potential serum biomarker for ovarian cancer [64]. Using ELISA to analyze the level of B7-H4 protein in more than 2500 serum samples, ascites fluids and tissue lysates, Simon et al. found high levels of B7-H4 protein in ovarian cancer tissue lysates when compared with normal tissues. B7-H4 was present at low levels in all sera, but showed an elevated level in serum samples from ovarian cancer patients when compared with healthy controls or women with benign gynecologic diseases. In early-stage patients, the sensitivity at 97% specificity increased from 52% for CA-125 alone to 65% when used in combination with B7-H4 [65].

Serum amyloid A

SAA is an acute phase reactant, which is expressed primarily in liver as a modulator of inflammation and metabolism, and transport of cholesterol. Expression of SAA was increased as epithelial cells progressed through benign and borderline adenomas to primary and metastatic adenocarcinomas. Real-time PCR analysis confirmed the overexpression of the SAA1 and SAA4 genes in ovarian carcinomas compared with normal ovarian tissues [66]. Confirmation of proteomics data with immunoassays in some early-stage cases revealed a substantial increase of SAA levels in plasma in comparison with the values of healthy controls. When combined with CA-125 the panel yielded an accuracy rate of 95.2% for ovarian cancer screening [67].

Transferrin

Transferrin (79 kDa) is an iron-binding transport protein responsible for transporting iron from sites of iron absorption and heme degradation to areas of storage and utilization. Transferrin has been previously reported to be decreased in the serum of patients with ovarian cancer [68]. All of these molecules have been shown to play an important role in oxidative stress, for which there are extensive data linking it to carcinogenesis [69]. It functions as promoter of tumor development and survival via antiapoptotic effects [70]. Combination of CA-125, transferrin, TTR and ApoA1 using proteomic analysis yielded a sensitivity of 89% at specificity of 92% for early detection screening [71].

Osteopontin

Osteopontin (OPN) is an adhesive glycoprotein related to bone remodeling as well as immune function. It is synthesized by vascular endothelial cells and osteoblasts. OPN has the ability to inhibit apoptosis. Kim et al., in a study consisting of 107 plasma samples using cDNA array, found significantly higher levels of OPN expressed in invasive ovarian cancer and borderline ovarian tumors [72]. OPN is involved in metastasis and tumor progression, useful to monitor recurrence. OPN as a sole biomarker has a sensitivity of 81.3%, when combined with CA-125 the panel yielded a sensitivity of 93.8%, but a low specificity of 33.7% [73]. Using osteopontin in combination with leptin, prolactin and IGF, Mor et al. reported a sensitivity of 96% and a specificity of 94% [74]. Similar to mesothelin, a fragment of osteopontin can be detected in the urine of ovarian cancer patients [75].

Kallikreins

Kallikreins (KLK) are a family of serine proteases that regulate proteolytic cascades. KLK promote or inhibit cancer cell growth, angiogenesis, invasion and metastasis by proteolytic processing of growth factors, angiogenic factors and extracellular matrix components. Of the 15 family members that are encoded by a group of genes tandemly localized on chromosome 19q13.3–4, 12 KLK are overexpressed in ovarian cancer at the mRNA and/or protein level [76]. KLK6 and KLK10 were elevated in ovarian cancer tissues that had low levels of CA-125 [77], elevated KLK11 was found in 70% of ovarian cancer sera at a specificity of 95% [78].

OVX1

OVX1 is an epitope of high molecular weight mucin-like glycoprotein, also an ovarian or breast cancer related glycoprotein antigen. OVX1 is increased in 70% of ovarian cancers; also increased in 59% of ovarian cancers with normal CA-125 level. Although these results indicate improvement in sensitivity, preliminary data from different laboratories suggest that OVX1 may be unstable unless serum is rapidly separated, which could complicate its use in population screening if samples are sent by post [79]. In a study of 201 serum samples, Donach et al. found a sensitivity of 88% and specificity of 92.5% using the artificial neural network with a panel of OVX1, M-CSF, CA19–19 and CA 72–74 [80].

VEGF

VEGF is a glycosylated angiogenesis mediator with serum levels significatly higher in patients with ovarian or gastrointestinal carcinoma than in healthy individuals, and the VEGF concentrations in sera from patients with metastatic disease were higher than those in sera from patients with localized tumors [81]. VEGF levels were significantly elevated in the sera and cyst fluids of carcinoma patients compared with patients who had benign neoplasms. High VEGF levels in ascitic fluids appeared to be significantly associated with shorter disease-free survival and overall survival. The elevated VEGF levels in sera and tumor effusions of patients with Fédération Internationale de Gynécologie Obstétrique (FIGO) stages I/II indicated that angiogenesis promoted by VEGF is a continuous process, independent of clinical advancement of the disease [82]. When combined with CA-125 the sensitivity is 77% and specificity is 87% [83]. VEGF inhibition has been shown to inhibit tumor growth and ascites production, and to suppress tumor invasion and metastasis [84].

Other biomarkers

The following are other markers that have been evaluated as potential biomarkers for detecting EOC:

AGR-2 [85,86]

Inhibin [87 –89]

M-CSF [79,90,91]

uPAR [92 –94]

EGF receptor [95,96]

Matrix metalloproteinases [97,98]

Lysophosphatidyl acid [99 –101]

miRNAs

In parallel with the efforts to identify potential protein biomarkers, attention has been recently focused on miRNAs. miRNAs consist of approximately 22 nucleotides of noncoding RNAs that post-transcriptionally regulate mRNA translation into the protein of a large number of target genes [102]. miRNAs globally influence gene expression, which ultimately determines cellular behavior by targeting complementary gene transcripts for translational repression or degradation of the mRNA transcript [103]. Similar to other cancers, the initiation and development of ovarian cancer is characterized by disruption of oncogenes and tumor suppressor genes by both genetic and epigenetic mechanisms [104]. Previous miRNA-expression profiling studies of ovarian cancer have defined differentially expressed miRNAs in ovarian cancer relative to the corresponding normal control [105 –107], and various miRNAs may represent potential targets for detection, diagnosis, prognosis and therapy [108]. There is a variety of tumor miRNA-expression patterns including genetic alterations, epigenetic regulation or altered expression of transcription factors, which finally target the miRNA genes. In cancer cells, transcriptional gene silencing has frequently been associated with epigenetic defects [109].

The Cancer Genome Atlas project is a large-scale collaborative effort that seeks to comprehensively catalog the molecular aberrations in various cancers [204]. A recent initial report from the Cancer Genome Atlas revealed 487 samples had corresponding miRNA data from 489 samples of high-grade serous ovarian adenocarcinomas [110]. Resnick et al. reported differences in serum miRNAs between normal women and patients with ovarian cancer; among the 21 miRNAs that the study identified, five miRNAs were found to be overexpressed (miR-21, miR-29a, miR-92, miR-93 and miR-126) and three miRNAs (miR-127 and miR155 and miR-99) were underexpressed in the sera of patients with ovarian cancer, suggesting a panel of miRNAs can be set as a screening tool for ovarian cancer [111]. Chang et al. found that miR-148b was overexpressed in 92.21% (71 out of 77 of the ovarian cancer samples) in asymptomatic women with ovarian cancer and may be involved in the early stage of ovarian carcinogenesis [112]. In addition, miRNA signatures of ovarian cancer could be helpful to distinguish the tumors based on their histological subtype, miR200b, miR-141, miR-21, miR-203 and miR-205 were upregulated in endometrioid and serous subtypes, whereas miR-145 was downregulated in both serous and clear cell carcinomas and miR-222 in both endometrioid and clear cell carcinomas [106].

Moreover, miRNAs are promising biomarkers as they are remarkably stable to allow isolation and analysis from tissues and from blood in which they can be found as free circulating nucleic acids and in mononuclear cells [113]. Recent efforts have been focused on establishing miRNA as novel molecular biomarkers for ovarian cancer and defining peripheral blood-derived miRNA as novel circulating biomarkers [114].

Current & future technologies of interest to identify unique ovarian cancer biomarkers

Mass spectrometry & quantitative proteome analysis

Mass spectrometry has played an important role in the identification of protein biomarkers and their post-translational modifications. However, the post-translational modifications generate large diverse and heterogeneous gene products, which are one of the challenges in proteomics research. To validate the significance of any candidate biomarker derived from pilot studies in appropriately designed prospective multicenter studies is mandatory; reproducibility of the clinical results must be shown over time and in different diagnostic settings [115].

Matrix-assisted laser desorption and ionization time-of-flight and its offshoot surface-enhanced laser desorption and ionization time-of-flight are two techniques commonly used with mass spectrometry. Using a mass spectrometer, multiple proteins within a very small sample can be identified. When compared with gel electrophoresis, mass spectrometry has better resolution with molecules of low molecular weight. Despite having been used for years, the contribution of proteomics in general to biomarkers in clinical use remains the main part of large efforts to compare different biomarker panels for the early detection of ovarian cancer [79].

Recently, quantitative proteomics has been used as a screening tool for identification of differentially expressed proteins. Using the high-throughput proteomic technology of isobaric tags for relative and absolute quantitation coupled with 2D liquid chromatography tandem mass spectrometry, many of the potentially differentially expressed proteins identified had previously not been linked to ovarian cancer such as KRT8, PPA1, IDH2 and S100A11 [116].

Microarrays

Microarray technology is one of the powerful tools to study genome-wide expression of genes. DNA microarray consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, each containing a specific DNA sequence. This can be a short section of a gene that is used to hybridize a cDNA sample [117]. Genome-wide expression profiling using DNA microarray technology has enhanced the understanding of the genes that influence ovarian cancer development, histopathologic subtype, progression, response to therapy and overall survival. Both diagnostic and prognostic information can be obtained by this method [117]. For example, osteopontin [72] and kallikrein 10 [118] are markers that have been identified by microarray analysis.

Microarray-based expression analysis is considered an ideal strategy for identifying candidate miRNAs. The small size of the mature miRNA is less susceptible to nuclease degradation. In addition, the small size makes it possible to extract miRNAs from paraffin-embedded formalin-fixed tissue blocks, which makes large archives of fixed tissue available for molecular analysis. The use of microarray can therefore generate molecular signatures of the disease stages. The expression data are archived in a standardized base, both National Center for Biotechnology Information – Gene Expression Omnibus and Array Express databases have been used for storage of miRNA microarray data [119].

Microvesicles & exosomes analysis

Microvesicles are generated by the outward budding and fission of membrane vesicles from the cell surface occurring in normal and pathologic cells, and more frequently, in tumor cells. Microvesicles can be widely detected from bodily fluids such as blood, urine, cerebrospinal fluid and ascites from which high-quality RNA, DNA and protein can be extracted and purified for analysis. In addition, by transferring these bioactive molecules, they are now thought to have vital roles in tumor invasion and metastases in cancer progression [120].

Exosomes are one of many different subpopulations of microvesicles. Exosomes originate predominantly from preformed multivesicular bodies that are released upon fusion with the plasma membrane. Exosomes can also contain proteins, enzymes, miRNAs and mRNAs; and thus, exosomes served as bioactive shuttle vesicles that constitute a mode of selective transmitting of information between cells [121]. An inappropriate release of miRNAs via exosomes may cause significant alterations in biologic pathways that affect disease development. Exosomes play an important role in cell to cell communication. They transfer proteins, mRNA and miRNA into recipient cells. The interplay via the exchange of exosomes between cancer cells, and between cancer cells and the tumor stroma may promote the transfer of oncogenes (e.g., β-catenin, CEA, HER2, Melan-A/Mart-1 and LMP-1) and oncomi-RNAs (e.g., let7, miR-1, miR-15, miR-16 and miR-375) from one cell to another, leading to the modulation of the activity of cellular signaling pathways in the recipient cells [122]. Resnick et al. have used real-time PCR-based microarray to perform high-throughput investigation of miR-21, miR-92 and miR-93, which are known oncogenes with biomarker and therapeutic potential [111]. Recently, Gercel-Taylor et al. demonstrated that nanoparticle tracking analysis can measure size and concentration of exosomes, and if combined with flourescent-labeled antibodies against specific markers, the technique allows the determination of the ‘phenotype’ of the exosomes [123]. While the detection of miRNA profiling, one of best tumor markers, has been limited to tissue specimens, which are conventionally obtained by a biopsy procedure, recent study results suggest that miRNA profiling of circulating ovarian tumor exosomes could potentially be used as a noninvasive surrogate of diagnostic markers for biopsy profiling [124].

Some available results of recent multiple biomarker analysis

Recently a large number of combinations of biomarkers have been investigated to improve the sensitivity and specificity for the early detection of ovarian cancer. Gorelik et al. identified a panel of markers including CA-125, EGF, VEGF, IL-6 and IL-8. The study was able to reach a sensitivity of 84% and a specificity of 95% for a total of 126 serum specimens using multiplex analysis technology [125]. Lokshin et al. proposed a panel of biomarkers consisting of IL-8, anti-IL-8, and IL-8: anti-IL-8 complexes. This study of 211 serum specimens using a novel multiplex array system (serological analysis of recombinant tumor cDNA expression libraries [SEREX] technique) gave a prediction of early ovarian cancer with 98% specificity and 65.5% sensitivity [61]. Su et al. used a multiple logistic regression model, with values for CA-125, ApoA1, transferrin and TTR. This model provided a sensitivity of 89% and a specificity of 97% [71]. In a study of a total of 468 serum specimens using the artificial neural network, Zhang et al. proposed a combination of CA-125 II, CA 72–74, CA 15–13 and M-CSF which showed a sensitivity of 71% at 98% specificity for detecting early-stage ovarian cancer [126]. Anderson et al. proposed a panel of biomarkers including CA-125, HE4 and mesothelin. Serum specimens of 34 ovarian cancers and 70 matched control subjects provide evidence of an increase in three tumor markers 3 years before clinical diagnosis; however, the lead time was short [127]. Recently, Yurkovetsky et al. identified a panel of CA-125, HE4, CEA and VCAM-1 applied for sera from 139 patients with early-stage ovarian cancer, 149 patients with late-stage ovarian cancer and 1102 healthy women. The four-biomarker panel provided the highest diagnostic power of 86% sensitivity for early-stage and 93% sensitivity for late-stage ovarian cancer at 98% specificity [59].

The multivariate index assay, OVA1, has been FDA approved for triage of pelvic masses since 2009. The test consists of CA-125, β2-microglobulin, transferrin, ApoA1 and TTR. OVA1 scores range from 0 to 10, with cutoffs set as 5.0 for premenopausal women and 4.4 for postmenopausal women. Pelvic masses with scores higher than these thresholds indicate a higher likelihood of malignancy. The sensitivity of the test is initially reported to be greater than 90% with a negative predictive value of greater than 90% [128]. A recent study of 516 women demonstrated an improvement in sensitivity and negative predictive value while decreasing specificity and positive predictive value when replacing the CA-125 with the multivariate index assay [129].

In summary, these results, while requiring validation, suggest that combinations of biomarkers may provide improved detection as the first step in a multimodal screening protocol.

Conclusion

A good screening test must adequately address validity, reliability, yield, cost, acceptance and follow-up services. An ideal screening test for ovarian cancer will have a high sensitivity in order to correctly diagnose all women with the disease and a high specificity to avoid false-positive results. The current screening modalities of bimanual examination, CA-125 and transvaginal ultrasonography together allow us to detect only 30–45% of women with early-stage disease, the majority of women continue to be diagnosed with advanced-stage ovarian cancer. The outcome of the UKCTOCS trial will be known by 2015 and will provide answers as to whether the CA-125 algorithm followed by sonogram is sufficiently sensitive and specific to be considered as a screening tool for the general population. Globally, hundreds of clinically relevant biomarkers have been identified that are based on the molecular biology of ovarian cancer. Although panels of tumor markers and proteomic-based technologies may improve the positive predictive value, all markers require validation and interfacing with newly developed diagnostic imaging technologies. While a large amount of information regarding miRNAs has been promising, much remains to be elucidated.

It is anticipated that the detection of early-stage EOC will be achieved using a combination of serum-based biomarkers in conjunction with biologically-based imaging technologies to improve women's healthcare.

Future perspective

Ovarian cancer remains the most lethal gynecological malignancy due to the lack of highly sensitive and specific screening tools for detection of early-stage disease. Recent developments in identification of potential biomarkers and application of technologies may improve the positive predictive value. It is anticipated that the detection of early-stage EOC will be achieved using a combination of serum biomarkers in conjunction with imaging technologies to improve women's healthcare.

Executive summary

Current challenges of early detection of ovarian cancer:

– The prevalence of ovarian cancer is very low.

– There is a lack of highly sensitive and specific screening tools.

– The PLCO screening trial showed that screening simultaneously with CA-125 and transvaginal ultrasonography did not reduce ovarian cancer mortality in women from the general population. The UKCTOCS trial involves 202,638 low-risk postmenopausal women. Although initial results in 2009 yielded encouraging sensitivity and specificity, the outcome of UKCTOCS will not be available until 2015; until then there will be continuing uncertainty about the efficacy of ovarian cancer screening in the general population.

– The current screening modalities of bimanual examination, CA-125 and transvaginal ultrasonography together allow us to detect only 30–45% of women with early-stage disease.

CA-125 and HE4 are the only two biomarkers US FDA approved for monitoring of disease recurrence or progression, but not for screening.

Combination of biomarkers has been investigated to improve the sensitivity and specificity for early detection of ovarian cancer.

The multivariate index assay, OVA1, has been FDA approved for triage of pelvic masses since 2009. The test yields an improvement of sensitivity and negative predictive value, while decreasing specificity and positive predictive value when replacing the CA-125 with the multivariate index assay.

miRNAs are promising biomarkers. Recent efforts have been focused on establishing miRNAs as novel molecular biomarkers for ovarian cancer and defining peripheral blood-derived miRNAs as novel circulating biomarkers.

miRNA profiling of circulating ovarian tumor exosomes could potentially be used as a noninvasive surrogate of diagnostic markers for biopsy profiling.

Footnotes

CME Author

Laurie Barclay, MD, Freelance writer and reviewer, Medscape, LLC Disclosure: Laurie Barclay has disclosed no relevant financial relationships.

Long Duc Nguyen, MD, Mount Sinai Medical Center, 11765th Avenue, 9th floor, New York, NY 10029, USA

Disclosure: Long Duc Nguyen has disclosed no relevant financial relationships.

Segundo Joel Cardenas-Goicoechea, MD, Mount Sinai Medical Center, 1176 5th Avenue, 9th floor, New York, NY 10029, USA

Disclosure: Segundo Joel Cardenas-Goicoechea has disclosed no relevant financial relationships.

Pierre Gordon, MD, Howard University Hospital, 2041 Georgia Avenue NW, Washington, DC 20060, USA

Disclosure: Pierre Gordon has disclosed no relevant financial relationships.

Christina Curtin, Mount Sinai Medical Center, 1176 5th Avenue, 9th floor, New York, NY 10029, USA

Disclosure: Christina Curtin has disclosed no relevant financial relationships.

Mazdak Momeni, MD, Mount Sinai Medical Center, 1176 5th Avenue, 9th floor, New York, NY 10029, USA

Disclosure: Mazdak Momeni has disclosed no relevant financial relationships.

Linus T Chuang, MD, Mount Sinai Medical Center, 1176 5th Avenue, 9th floor, New York, NY 10029, USA

Disclosure: Linus T Chuang has disclosed no relevant financial relationships.

David A Fishman, MD, Mount Sinai Medical Center, 1176 5th Avenue, 9th floor, New York, NY 10029, USA

Disclosure: David A Fishman has disclosed no relevant financial relationships.

Editor

Elisa Manzotti, Publisher, Future Science Group

Disclosure: Elisa Manzotti has disclosed no relevant financial relationships.

Ovarian cancer is a heterogeneous and rapidly progressive disease of low prevalence and poor survival. It is the fifth leading cause of cancer-related deaths in women, with 22,280 new cases and 15,500 deaths expected in the USA in 2012 [201]. The lifetime incidence for ovarian malignancies is one in 72 (1.39%) and the lifetime risk of death from ovarian cancer is one in 96 (1.04%) for women living in the USA [1]. Owing to symptoms associated with late-stage disease and suboptimal diagnostic modalities, women continue to be diagnosed with advanced-stage disease and the 5-year survival is less than 45% [2]. Although rare in comparison with other gynecologic malignancies, ovarian cancer is the most lethal gynecological malignancy accounting for more deaths than endometrial and cervical cancer combined [3]. This is mostly owing to the lack of highly sensitive or specific screening tools for early detection of early-stage disease. However, if one could detect early-stage disease, women require a less morbid operation, may not require adjuvant chemotherapy and have a 5-year survival approaching 90% []. Therefore, new tools are required to affect a paradigm shift from the detection of late- to early-stage ovarian cancer.

Biomarkers for early detection of ovarian cancer

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Biomarkers for Early Detection of Ovarian Cancer

Abstract

Keywords

Current challenges of early ovarian cancer detection

Risk factors of ovarian cancer

Ovarian cancer presentation

Available tools for ovarian cancer detection

Pelvic examination

Diagnostic imaging

CA-125

Serum biomarkers are under evaluation beside CA-125

HE4

Mesothelin

Transthyretin

ApoA1

VCAM

IL-6 & IL-8

B7-H4

Serum amyloid A

Transferrin

Osteopontin

Kallikreins

OVX1

VEGF

Other biomarkers

miRNAs

Current & future technologies of interest to identify unique ovarian cancer biomarkers

Mass spectrometry & quantitative proteome analysis

Microarrays

Microvesicles & exosomes analysis

Some available results of recent multiple biomarker analysis

Conclusion

Future perspective

Footnotes

Biomarkers for early detection of ovarian cancer

References