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Biomarkers for ovarian cancer detection and therapy

Biomarkers for ovarian cancer detection and therapy re VIew Cancer Biology & Therapy 9:9, 668-677; May 1, 2010; © 2010 Landes Bioscience Biomarkers for ovarian cancer detection and therapy Sonia Dutta, Feng-qiang w ang, Adrien Phalen and David A. Fishman* Department of Obstetrics, Gynecology and r eproductive Sciences; Mount Sinai School of Medicine; New York, NY USA Key words: epithelial ovarian cancer, early detection, serum biomarkers, signaling pathways, contrast enhanced ultrasound, proteomics novel technologic advances. Through the continuous efforts of Despite advances in surgical technologies and the develop- many investigators, hundreds of biomarkers that may potentially ment of more effective chemotherapeutics, epithelial ovarian serve to detect early stage EOC have been reported; however, cancer (eOC) remains the leading cause of death in women with to date, not a single biomarker has been proven to be clinically gynecologic malignancies. The high mortality and morbidity effective in detecting early stage EOC with adequate sensitivity associated with eOC is mostly attributed to the inability to and specificity. detect the disease before it is widely disseminated throughout In this article we will discuss current ovarian cancer biomark- the abdominal cavity. In the past decade, tremendous efforts have been taken to search for novel biomarkers that will detect ers that show promise to become potential screening markers, eOC at an early stage, which may also serve as new targets for as well as emerging technologies available to identify new EOC the prevention and control of metastasis. Here, we review biomarkers. In addition, we will discuss the signaling pathways recent developments in eOC early detection and targeted involved in ovarian cancer initiation and progression, in particu- therapy, as well as new technologies for the discovery of novel lar, those associated with EOC invasion and metastasis. biomarkers. Biomarkers for Ovarian Cancer Early Detection For the past several decades, the efforts to detect ovarian cancer Introduction at an early stage have been hampered by the lack of an effective screening tool. Ideally, this screening method would be a simple Ovarian cancer is deemed the “most lethal gynecologic malig- blood test with both a high sensitivity and a high specificity to nancy” in the United States given that more women die from decrease the number of false positive and false negative results. this disease than all other gynecologic malignancies combined. Unfortunately, an increase in the sensitivity of a test is often asso- The most common form of ovarian cancer (≥80%), thought to ciated with a reduction in its specificity and vice versa. Specificity derive from ovarian surface epithelium, is called epithelial ovar- of screening tools is of great importance in EOC because the ian carcinoma (EOC). Despite improvements in median survival majority of women with a positive result will require surgical through surgical advances and new chemotherapeutic regimens, intervention. For example, a screening test for postmenopausal the overall survival for women with late stage (III/IV) EOC has women would require a 99.6% specificity to yield an approximate remained relatively unchanged for the past 40 y, holding steady positive predictive value (PPV) of only 10%, where a surgeon at approximately 30% . In sheer contrast, women diagnosed with would perform ten operations for each case of EOC detected, EOC when the disease is confined to the ovary (stage I) not only given that the prevalence of EOC in this population is approxi- require less morbid surgical interventions and a decreased need for mately 1 in 2,500. However, lower specificities may be consid - adjuvant chemotherapy, but most importantly, have an overall 5-y ered acceptable in higher risk populations (such as those with a survival of approximately 93%, in addition to a much improved personal history of breast cancer or a recognized familial cancer quality of life. Unfortunately, the majority of women (70–75%) syndrome with an ovarian cancer predisposition) because as inci- continue to be diagnosed with advanced stage EOC because of dence increases, the specificity required to achieve a given PPV the rather asymptomatic nature of early stage disease and the lack decreases. of an adequate early detection screening method. To address this Currently physicians typically use a combination of pelvic problem, numerous researchers have focused on the identifica - exam, quantitative serum CA125 levels and transvaginal sonog- tion and validation of novel biomarkers for the early detection of raphy to monitor and screen women. Together, these techniques EOC with their increased knowledge of cancer biology and using are inadequate to detect early stage EOC due to their inability to delineate the subtle changes associated with the initial transfor- mation and their lack of specificity and sensitivity. Despite recent *Correspondence to: David A. Fishman; Email: david.s fi [email protected] Submitted: 02/19/10; Accepted: 02/24/10 advances in ovarian cancer biomarker research, no simple blood Previously published online: test with the required sensitivity and specificity has yet been clin - www.landesbioscience.com/journals/cbt/article/11610 ically validated. Fortunately, recent studies have identified and 668 Cancer Biology & Therapy Volume 9 Issue 9 re VIew re VIew validated a variety of new biomarkers or biomarker panels that disease and healthy controls. Their assay correctly identified have shown improved sensitivity and specificity over the current 47/48 patients with EOC, specifically 9/10 women with stage “gold standard”—CA125. Several biomarkers, in combination I disease, in comparison to only 2/9 early stage patients when with CA125, have enhanced the diagnostic accuracy of CA125 analyzed by CA125 alone. These results indicated that LPA had alone. Following, is a list of ovarian cancer biomarkers of recent a relatively high sensitivity and specificity of 95 and 89% , respec - interest. tively. However, follow-up studies by Meleh et al. reported no significant difference between LPA levels in benign and malig - Serum Protein Markers nant tumors. On the other hand, a later study by Sedlakova et al. corroborated with Xu’s in all respects and further added CA125 and the mucin family. To date, serum CA125 remains that LPA levels were associated with FIGO stage and histological the most useful tumor marker for the detection of recurrence after type. Although LPA appears to be a promising biomarker, as primary treatment. CA125, also known as MUC16, is a tumor yet, it has not shown to be clinically relevant in large scale screen- antigen of the mucin family, which are high molecular weight ing trials, which may be due to the difficulty in accurately quan - biomolecules produced by the secretory epithelial cells lining tifying each individual LPA. Thus, further clinical investigation ducts and lumens. Serum concentrations of CA125 are elevated is required with greater numbers of patients to determine its true in 80–90% of advanced-stage ovarian cancers, yet only 47% for diagnostic value. early stage disease. However, the clinical effectiveness of CA125 Human epididymal protein E4 (HE4). Human epididy- as a diagnostic tool is compromised by its lack of specificity and mal protein E4 (HE4), also referred to as Whey-Acidic Protein sensitivity. Elevated concentrations of CA125 are also detected in (WAP) four-disulphide core domain-containing protein 2 other malignancies (pancreatic, breast, bladder, liver and lung), (WFDC2), is a protease inhibitor that serves in protective as well as benign diseases (such as diverticulitis, uterine leiomyo- immunity and is primarily expressed in the reproductive tract mas, endometriosis, benign ovarian cysts, tubo-ovarian abscess, and upper airways. HE4 levels are elevated in ovarian cancer ovarian hyperstimulation syndrome and ectopic pregnancy) and patients but not in common benign gynecological conditions. some physiological conditions (pregnancy, ovulation and men- As a biomarker, it has increased sensitivity for detecting stage 19-21 struation). To overcome this lack of specificity and sensitivity for I ovarian cancer. In comparison to CA125, it appears to early detection, CA125 measurements have been combined with be more specific in detecting ovarian cancer and has a similar ultrasound techniques, such as transabdominal or transvaginal sensitivity. A recent study by Huhtinen et al. measured serum sonography, or other biomarkers, to try to detect early stage EOC. concentrations of HE4 and CA125 in a group of 159 women Meanwhile, the expression of other mucins, such as MUC4, has with multiple gynecologic conditions, such as endometriosis, provided better sensitivity for EOC detection when used in com- ovarian tumors, endometrial cancer and ovarian cancer, as well bination with CA125, with high expression of MUC4 observed as 66 healthy controls. They reported the combination of HE4 in EOC cells present in peritoneal effusions. Additionally, a com- and CA125 had the highest accuracy (96.3%) and sensitivity bined panel of CA125 with MUC1, CA72-4 and macrophage (92.9%) in differentiating patients with ovarian cancer from colony-stimulating factor (M-CSF) was able to improve the sen- healthy controls, as compared to each alone. Moore et al. eval- sitivity of detection of stage I ovarian cancer from 45% (CA-125 uated CA125 and HE4 levels in 531 women with a pelvic mass alone) to 70%, while maintaining 98% first-line specificity. and validated its use as a stratification tool. Analysis with these Higher levels of MUC2, MUC3 and MUC4 mRNA were also biomarkers correctly identified 94% of EOC patients as high detected in early stage EOC compared to late-stage EOC. The risk for ovarian cancer, with half of those misclassified having detailed clinical potential of mucins in diagnosis, prognosis and low malignant potential (LMP) tumors, which are often early therapy of ovarian cancer has been recently reviewed by Singh stage diseases and derive little benet f fi rom adjuvant treatment. et al. The sensitivity and specificity of this test was 93.8 and 75%, Lysophosphatidic acids. Lysophosphatidic acids (LPAs) are a respectively. Montagnana et al. also tested the levels of HE4 in family of phospholipids that act as potent signaling molecules in women with a pelvic mass and noted that the HE4 concentra- the EOC microenvironment. LPA was found to be secreted by tion was ten times higher in ovarian carcinoma versus benign ovarian tumors and clinically elevated in the ascites and serum tumors, with the diagnostic sensitivity of HE4 in early stages 9-11 25 of women with EOC. LPA elicits its function on cells by inter- determined to be 82.7% , compared to only 45.9% for CA125. acting with G-protein coupled receptors, which are often aber- Conversely, a study by Shah et al. revealed that the sensitivity 12,13 rantly expressed in ovarian cancer cells. The presence of LPA to discriminate ovarian cancer from healthy controls in average is known to increase cell proliferation, cell survival, invasion, risk patients was superior with CA125; yet in high-risk patients, migration and production of angiogenic factors. Additionally, HE4 had a higher sensitivity. At 98% specificity, the sensitivity LPA induces a signature of genes in EOC that is associated with of CA125 to identify ovarian cancer in average-risk and high- a reduced progression-free and disease-specific survival, as well as risk patients is 78.4 and 78%, respectively, while the sensitivity an overall worsened prognosis. of HE4 to identify ovarian cancer in average-risk and high-risk Xu et al. first reported LPA’s potential utility as a biomarker patients is 68.6 and 82.9% , respectively. for EOC given that its concentration was significantly higher in Although HE4 has shown promise as a complement to women with malignant gynecologic cancer compared to benign CA-125 for EOC detection, the sensitivity and specificity would www.landesbioscience.com Cancer Biology & Therapy 669 need to be further improved before being an effective popula- either analyte alone in differentiating cancer from healthy con- tion-wide screening test, which requires a minimum sensitivity trols. Similar to OPN, KLKs have shown biomarker utility in of 75% and a specificity of 99.6% to reach a PPV of 10%, as many malignancies but individually lack sufficient specificity or previously stated. The use of HE4 as a biomarker for ovarian can- sensitivity to be clinically useful. Biomarker panels consisting of 27,28 cer has also been recently reviewed by Hellstrom and Li et al. KLKs and other candidate biomarkers may offer improved clini- Osteopontin. Osteopontin (OPN) is an acidic, calcium bind- cally utility. ing glycoprotein found in all body fluids, which was initially Claudins. Claudins, a family of 24 proteins, are the main identified by cDNA microarray techniques. OPN is involved components of tight junctions that function as selective barri- in cell adhesion, inflammation and tumorigenesis and known ers. These proteins control paracellular diffusion and thereby to be associated with the presence and progression of multiple maintain cellular polarity. Transmembrane claudins form bi- or 29,30 cancers. OPN was first identified as a potential biomarker tri-cellular interactions between neighboring cells with occludin, for EOC by Kim et al. in 2002 when they noted OPN mRNA junctional adhesion molecules 1, 2 (JAM 1, 2) and the integral overexpression in ovarian cancer as well as significantly higher membrane protein, tricellulin. Deregulated claudin expression levels of OPN in tissue samples of invasive ovarian cancer and has been observed in gynecological cancers, such as cervical 40-42 borderline ovarian tumors versus benign tumors and healthy and endometrial cancers, as well as in premalignant lesions. 29 43,44 ovarian epithelium. The levels of OPN in plasma were also sig- Claudin-3 and -4 were found to be overexpressed in EOC. nificantly higher in 51 women with EOC compared with those High expression of claudin-7 and -10 has also been observed of 107 healthy controls, 46 patients with benign ovarian disease in ovarian cancer, while claudin-3 expression was found to be 29 45 and 47 patients with other gynecologic cancers. More recently, associated with shorter survival in EOC. In ovarian cancer Nakae et al. measured the preoperative plasma OPN levels in effusions, higher claudin-3 and -7 expression independently pre- 96 gynecologic patients and 31 healthy controls and reported a dicted shorter overall survival. Huang et al. recently reported significant difference in the mean value of OPN between ovar - that silencing the claudin-3 gene with siRNA suppressed ovar- ian cancer patients and all other groups (healthy controls, benign ian tumor growth and metastasis in mouse tumor models. The tumors and other gynecologic cancers). They also showed that use of claudins as diagnostic or prognostic markers for cancers OPN levels were significantly higher in stage IV patients than and gynecologic cancers has been reviewed by Morin et al. in 30 44,47 stage I and II. However, the sensitivity and specificity of OPN 2005, and most recently by Szabo et al. in 2009. As potential alone were inferior to that of CA125, and the combination of markers for ovarian cancer, the specificity of claudins in differ - OPN with CA125 decreased sensitivity although the specific - entiating EOC from normal and benign diseases remains to be ity increased. OPN levels were also evaluated in multiplexed determined. assays with other molecules, such as prolactin and leptin, show- Other potential serum biomarkers. A recent study evaluating 31,32 ing promising results. Unfortunately, the use of OPN as an 300 Japanese women (100 with EOC, 100 with benign tumors, EOC biomarker is thwarted since its levels have been found to 100 healthy controls) suggested that nidogen-2 may be a new be elevated in multiple cancers as well as benign conditions as serum biomarker. Nidogen-2 is a basement membrane protein reviewed by Johnston et al. that functions in the organization of the extracellular matrix. It Kallikreins. Kallikreins (KLK) are serine proteases that regu- is involved in regulating cell polarity, migration and invasion due late proteolytic cascades. Their activity may promote or inhibit to its role in facilitating interactions between cells and basement cancer cell growth, angiogenesis, invasion and metastasis by pro- membranes. Nidogen-2 serum levels were significantly elevated teolytic processing of various proteins, such as growth factors, in early and late stage EOC, compared to both healthy controls angiogenic factors and extracellular matrix components. Most and benign disease, although no difference in concentration was of the 16 family members are overexpressed in cancer and have noted between controls and benign disease. More studies are shown some utility as prognostic markers. Specifically, KLK6 warranted to determine nidogen-2 utility as a new serum bio- and KLK10 were elevated in ovarian cancer tissues that had low marker for EOC. levels of CA125, while KLK8 was associated with early stage Folate receptor alpha (FRα), is overexpressed in solid tumors disease and a favorable prognosis. Preliminary data also indicate to meet the increased demands for DNA synthesis and cell that KLK14 may be a potential biomarker for ovarian cancer. growth. The amount of FRα shed into the blood increases in Recently, KLK5 levels were measured in 167 women (50 with tumors expressing high levels of FRα and is subsequently detect- EOC) and found to be significantly higher in EOC patients ver - able in serum samples. A recent clinical trial measured the FRα sus all other groups (healthy controls, benign tumors and bor- levels in 30 women with ovarian cancer and 30 matched healthy derline tumors). KLK5 has a specificity of 95% to differentiate controls. Circulating FRα was found to be comparable between EOC from healthy controls and a sensitivity of 52%, however, early and late stage disease; yet, significantly higher than healthy the specificity dropped to 88% when differentiating cancer from controls. borderline tumors. Other members of the Kallikrein family, Recently, a correlation between L-Asparaginase (L-ASP) KLK6 and KLK13, have also been evaluated for their potential as efficacy and asparagine synthetase (ASNS) expression has been ovarian cancer biomarkers. The combination of KLK6, KLK13 shown in the ovarian subpanel of the NCI-60 cells. Lorenzi et and CA125 mRNA expression in 106 ovarian tumors and eight al. reviewed the potential of developing ASNS as a biomarker for healthy controls proved to be more sensitive and specific than ovarian cancer treatment. 670 Cancer Biology & Therapy Volume 9 Issue 9 In addition to the markers mentioned above, patient derived resulted in a diminished response of the EOC to cis-platinum tumor reactive antibodies have shown to be promising biomark- while overexpression of let-7i restored the cancer’s response to ers for EOC. Tumor antibodies are found in the circulation soon cis-platinum. This group also found that low let-7i expres- after initial tumor development and prior to detection of circulat- sion significantly correlated with decreased survival. miRNA ing antigens or palpable tumors. These proteins are stable and, expression profiling has also identified the miRNA-200 family unlike other serum markers, less sensitive to confounding factors as a potential prognostic indicator. Hu et al. compared miRNA such as stress, time of blood draw and sample manipulation. In a expression profiles of 58 EOC tissue samples with disease outcome recent study, the intensity of the antibody immunoblot correlated and showed that miR-200a expression was significantly associ - with the stage of EOC, with late stage samples having a greater ated with recurrence-free survival and overall patient survival 51 58 number of bands at a greater intensity. Additionally, early stage independent of other clinicopathologic features. Conversely, disease displayed unique bands above 100 kD, while late stage Nam et al. found that high levels of miR-200a or miR-200c were 51 59 disease had unique bands less than 40 kD. Of the identifiable significantly correlated to shortened survival. These discrepan- bands, all cancer patients made antibodies to cathepsin D, p53 cies could be a caused by varied miRNA screening methods and and SSX; however, only ovarian cancer patients (stage II and indicate a need for greater investigation into miRNA profiling. beyond) made antibodies to placental type alkaline phosphatase. Of particular benet fi , miRNA can be detected in the serum of Interestingly, the combination of nucleophosphmin, cathepsin EOC patients. Taylor and Gercel-Taylor first identified miRNA D, GRP78 and SSX autoantibodies appeared to be specific to within circulating tumor exosomes in EOC patients and con- 51 60 stage I disease. firmed the presence of previously observed miRNA signatures. Currently, although a full spectrum of serum biomarkers Lodes et al. demonstrated that sufficient miRNA is obtainable has been identified and explored as possible adjuncts to CA125, from serum to distinguish cancer patients from normal samples. limited clinical success has been achieved. They have isolated miRNA from the serum of 36 cancer patients (ovarian, prostate, lung, colon and breast cancer) and 13 normal Epigenetic Aberrations as Biomarkers in EOC controls. Hierarchical clustering of the microarray data resulted in discrimination of the cancer and normal specimens, demon- Epigenetic aberrations, including DNA methylation, histone strating the ability to identify patients as cancer or normal solely modifications and micro-RNA dysregulation, have been found from their serum miRNA profile. Therefore, serum miRNA to be intimately associated with ovarian cancer. These relatively can potentially serve as a new set of biomarkers for detection and stable aberrations are associated with distinct disease subtypes therapy of ovarian cancer. and are present in circulating serum; thus, they are promis- ing diagnostic and prognostic biomarkers. Inhibition of DNA Emerging Technologies for Ovarian Cancer methylation or histone-modifying enzymes has shown promising Detection and Biomarker Discovery results when used in combination with one another or with con- ventional chemotherapy in preclinical trials, as well as in current In addition to traditional techniques used for identification clinical trials. The best studied epigenetic aberration in ovarian and evaluation of differentially expressed protein biomarkers in cancer is BRCA1. BRCA1 hypermethylation occurs in 10–15% serum, new technologies are emerging and showing great prom- of sporadic disease cases, associates with loss of BRCA1 RNA ise for EOC biomarker discovery. and protein, and significantly correlates with poor patient out - come. The most recently discovered epigenetic regulation is that Comparative Genome Hybridization (CGH) by microRNA (miRNA), which are short single stranded RNAs that interfere with protein translation via complementary bind- Comparative genome hybridization is a technique used to assay ing to the 3' UTR of the target mRNA. miRNAs are deregulated the whole genome to detect gains or losses in gene copy number. in every cancer examined and have been discovered to act as both Many regions with abnormal copy numbers have been identi- tumor suppressors and oncogenes. Specifically, expression pro - fied in EOC. Additionally, there is evidence that certain dele - files of miRNA in EOC cell lines and patient specimens have tions or gains may be associated with the progression of tumors led to the identification of several miRNAs associated with EOC from benign to borderline and eventually malignancy. In a prognosis, patient survival and staging and chemo-resistance. recent study, 28 primary ovarian tumor samples were analyzed Dahiya et al. examined the miRNA profiles of 34 ovarian for high frequency gains and losses in their chromosomes and cancer tissue specimens and ten ovarian cancer cell lines rela- initial analysis revealed that chromosomes 1, 8 and 17 showed tive to an immortalized human ovarian surface epithelium cell the greatest numbers of gains and losses, with late stage disease line and found 56 differentially expressed miRNAs between the showing more gains and losses than early stage. Bruchim et al. neoplastic samples and control. Overall, 94% of the tumors had at analyzed 45 EOC specimens with conventional CGH and least one let-7 family member downregulated. Previously, Shell reported that gains in 5p were associated with increased risk et al. showed that Let-7 regulates the expression of high-mobility of recurrence while gains in 1p or loss of 5q had a protective group A2 (HMGA2) in ovarian cancer and its decreased expres- effect against recurrence. CGH is still quite a new technology sion is a marker for less differentiated cancer. Furthermore, Yang with obvious problems with reproducibility between groups. et al. demonstrated that reduction of let-7i expression in vitro Nevertheless, the information obtained from these analyses have www.landesbioscience.com Cancer Biology & Therapy 671 potential in identifying changes specific to ovarian cancer and to an increased risk of ovarian malignancy include the presence of discover previously unknown associated genes that can provide solid components, papillary excrescences and increased ovar- 70-74 more insight into EOC pathogenesis. ian volume. Traditionally, ultrasound used 2-D gray-scale images to visualize the ovaries; now, Color Doppler technology Proteomics allows for the characterization of flow within the vasculature of the ovary by identifying the shape of its waveform (arterial vs. Proteomic technologies were championed to EOC by Petricoin et venous), its direction (toward or away from the transducer) and 65 75-78 al. who discovered unique proteomic patterns specic t fi o EOC. its systolic and diastolic velocities. We previously reported Subsequently, many groups have applied mass spectrometry as a that the use of this technology improved diagnostic accuracy of tool to differentiate cancer from benign conditions and controls. ovarian cancer prediction because it better defined morphologic In 2007, Lopez et al. reported discriminating peptides that could and vascular characteristics of ovarian lesions compared to 2D 66 79 accurately identify early stage EOC from healthy controls. These ultrasound. Although imaging by ultrasound helps determine peptides, as a set, had a sensitivity and specic fi ity of 93 and 97%, risk of malignancy, numerous studies have found that transvagi- respectively. Similarly, Wu et al. analyzed 65 samples (30 EOC) nal ultrasound is not effective as a sole screening tool for early 80,81 with SELDI-TOF MS and identie fi d one peak specic t fi o EOC stage EOC in asymptomatic women. and two peaks specic t fi o normal controls. These three identifying Recent developments in ultrasound technology have rekindled peaks had a sensitivity of 84% and specic fi ity of 89%. the hope that ultrasound can be used to detect vascular changes Although proteomic patterns can be used as “finger prints” associated with early stage disease. Harmonics and pulse inversion to differentiate cancer from normal cases, biomarker discovery technology have improved signal-to-noise ratios and structural requires protein identicfi ation within the patterns. Thanks to conspicuity while the introduction of IV contrast agents (akin higher resolution mass spectroscopy and the incorporation of to those used with CT and MRI) has helped to more accurately immuno-MS, reverse phase protein arrays (RPPA) and nanome- detect, characterize and quantify aberrant vascularity and blood 82-84 trology-based procedures, researchers are currently identifying flow. The contrast enhancement of ultrasound helps to visu- hundreds of proteins that are differentially expressed in EOC alize tumor neovascularization in developing microscopic (not patients by analyzing ascites, urine and plasma. One recent study macroscopic) tumors by delineating the aberrant leaky vascular evaluated urine and blood samples from 209 women who had channels associated with early malignancy. The incompetent exploratory laparotomies for ovarian neoplasms. Of the 21 sig- extracellular matrix of neovascularization allows extravasation of nic fi ant peaks identie fi d, the three most signic fi ant peptides were RBCs (5–7 microns) and contrast agents (1–10 microns in diam- purie fi d and identie fi d as: b fi rinogen α , collagen α-1 (III) frag- eter), which can then be detected by ultrasound. The enhanced ment and fibrinogen β -N terminal fragment. These peptides ability to accurately visualize vascular changes specific to early alone did not have adequate discriminatory power; however, when stage EOC may help to detect early ovarian cancer with greater combined with CA125, had a higher sensitivity and specicfi ity sensitivity and specificity. Quantification of contrast enhance - than either alone. Gunawardana et al. identie fi d eight unique ment kinetics obtained with pulse inversion harmonic ultrasound extracellular and membrane bound proteins associated with EOC has shown potential to accurately differentiate benign from by using MS to analyze conditioned media from four cell lines. malignant ovarian tumors with a sensitivity of 100% and speci- 85,86 ELISA measurement of the proteins’ concentration in serum from ficity of 96.2%. Our findings suggest that contrast-enhanced ten EOC patients and 20 healthy women indicated that clus- ultrasound may have a high enough sensitivity and specificity terin and IGFBP6 expression was signic fi antly different between to differentiate benign disease from malignant, and thus, shows the two groups. Follow-up validation PCR revealed that clus- great promise as a potential imaging technique for ovarian cancer 85,86 terin mRNA expression is increased in EOC, while IGFBP6 is detection. decreased. Proteomic technologies have introduced a wealth of infor- Signaling Pathways and Therapeutic Targets mation that researchers can mine for possible early detection in Ovarian Cancer biomarkers. Unfortunately, as a costly, rapidly evolving, sophis- ticated technology, it is wrought with multiple problems associ- Since the 1990s, the foundation of ovarian cancer chemotherapy ated with specimen handling, which affect reproducibility and has included the combination of platinum based agents and tax- specificity. The discriminatory power of the results obtained anes. Over the past 20 y, refinements to these first-line agents have from these small studies must be confirmed with large multi-in - greeted patients with only slight increases in survival and quality stitutional prospective validation trials. Despite these criticisms, of life. Major improvements in these parameters will require the there is much anticipation that proteomics will be able to identify development of therapies that target multiple biologic processes clinically valid biomarkers for EOC early detection. associated with the ovarian cancer microenvironment, namely growth, angiogenesis, invasion and metastasis. Cancer cells are Contrast-Enhanced Ultrasonography genetically deranged normal cells that have aberrantly expressed and functioning biological pathways. The identification of spe - Today, transvaginal ultrasound is the best modality to image and cific pathways has allowed for the emergence of a new class of examine the ovaries. Ultrasound characteristics associated with therapeutics—molecularly targeted therapies, which interact 672 Cancer Biology & Therapy Volume 9 Issue 9 Figure 1. Schematic view of major signaling pathways shown to be involved in ovarian carcinoma growth and metastasis. These pathways have been important targets for the development of molecular therapeutics currently in clinical trials. 90,91 with and inhibit pathways to affect tumor growth and metastasis 52% of persistent/recurrent patients having stable disease. (Fig. 1). In the following section, we discuss important pathways Furthermore, there are a handful of trials underway combining associated with EOC and detail recent clinical trials. Bevacizumab with other chemotherapeutics. One earlier study combining Bevacizumab with cyclophosphamide in recurrent Vascular Endothelial Growth Factor (VEGF) ovarian cancer patients achieved a 28% response rate with a 57% 6-mo progression-free survival. The optimistic results from VEGFs are known to be integral players in the process of these Phase II trials have lead to the development of three ongo- angiogenesis, which is required for the survival, growth and ing Phase III trials—GOG218, ICON7 and GOG213. Of note, metastasis of cancer. The expression of VEGF and its recep- although Bevacizumab is generally well tolerated, it appears to tors, especially, VEGF Receptor-2 (VEGFR-2), are known to have a relatively high gastrointestinal perforation rate, quoted as be involved in the malignant progression of ovarian cancer, as 5.4%, which is more than double that of colorectal cancer. The well as the formation of ascites. Additionally, overexpression cause of this event needs to be further elucidated with additional of either is associated with decreased survival. Consequently, data from upcoming trials. many agents, such as small molecule inhibitors and monoclonal Cediranib (Recentin, AZD2171), a small molecule inhibitor of antibodies to VEGF and VEGFR have been developed to target VEGFR1-3 as well as PDGFR-β and c-kit, is currently in a Phase this signaling pathway. III trial in Europe for patients with recurrent, platinum-sensitive Bevacizumab (Avastin), a monoclonal antibody against VEGF, disease. A prior Phase II trial by Hirte et al. revealed that 41% has shown great clinical efc fi acy in Phase III trials for colorec - of patients with platinum-sensitive disease responded to mono- 87 88 89 tal, lung and breast cancer, and is showing great promise in therapy, while there was a 29% response rate in platinum-resistant Phase II trials for ovarian cancer. As a monotherapy for patients. The median time to progression was noted to be 4.1 mo patients with persistent/recurrent ovarian cancer or platinum- with the median overall survival being 11.9 mo. Matulonis et al. resistant disease, Bevacizumab has a 16–21% response rate, with also conducted a Phase II trial of Cediranib for recurrent EOC www.landesbioscience.com Cancer Biology & Therapy 673 and sited a 17% partial response rate with 13% of patients having known to stimulate cell growth by producing matrix metallo- stable disease. proteinases and to facilitate drug resistance. Additionally, b-Raf Another novel inhibitor of this pathway is the VEGF trap, and c-Raf, two isoforms of Raf, are associated with ovarian which is a fusion protein comprised of the VEGFR1 and cancer—high levels of b-Raf correlate with improved survival VEGFR 2 extracellular domains fused to the Fc portion of human while high concentrations of c-Raf are associated with a poor 96 107,108 IgG. This agent is able to bind to VEGF-A as well as placental prognosis. growth factor. A current Phase II trial has reported an 11% par- Sorafenib is an oral, multi-kinase inhibitor that targets the tial response rate in women with recurrent, platinum-resistant MAPK pathway as well as VEGFR1-3 and PDGFR-β tyrosine EOC. Other Phase II trials combining this agent with docetaxel kinase. It was initially combined with gemcitabine in a Phase 97,98 are currently underway as well. II trial. This therapy produced a 4.7% partial response rate and 60.4% stable disease, as well as a 5.4 and 13.3 mo progression-free Epidermal Growth Factor Receptor (EGFR) and overall survival. Single agent sorafenib was also tested in a Phase II trial by the GOG in 59 women with recurrent or persis- EGFR is a member of the ErbB family of receptor tyrosine tent EOC, which noted two responders, 20 patients with stable kinases, which also includes ErbB2 (Her2/neu), most com- disease and 30 women with progressive disease. Additionally, a monly associated with breast cancer. Once a ligand binds to recent Phase I trial combining intermittent sorafenib with bev- these receptors, the cytoplasmic tyrosine domains dimerize and acizumab yielded a 12% partial response rate and 53% stable subsequently lead to the phosphorylation of specific tyrosines. disease rate. Given these promising results, a Phase II trial This phosphorylation leads to the activation of multiple intracel- combining sorafenib with the clinically active bevacizumab is lular signaling pathways, such as the mitogen-activated protein currently underway. kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)- AKT pathways, which are involved in cell proliferation and sur- Phosphatidylinositol 3 Kinase (PI3K) Pathway 99,100 vival. EGFR has been noted to be overexpressed in 35–70% of EOC and is associated with a poor prognosis; consequently, The PI3K pathway is known to mediate angiogenesis and vascu- several inhibitors have been developed against this target. lar permeability, namely through its effectors, AKT and mTOR. Erlotinib, an oral EGFR tyrosine kinase inhibitor, was studied This pathway is known to be involved in the growth related as a monotherapy in women with refractory, recurrent, EGFR- functions of cells, such as transcription, translation, protein positive EOC. There was a 6% response rate with 43% of patients degradation and reorganization of the actin cytoskeleton. Forty having stabilized disease. This inhibitor has also been tested in percent of EOC cases have shown amplification or mutations combination with carboplatin and taxanes with promising results in PI3K or AKT, which subsequently leads to mTOR activa- 110,111 (NCI 5886). Additionally, its use as a maintenance therapy is tion and tumor survival. A Phase II trial (NCT00429793) being evaluated in a randomized Phase II trial (EORTC-55041). is currently underway to determine the clinical efficacy of Another small EGFR tyrosine kinase inhibitor, Get fi inib, which Temsirolimus, an inhibitor of mTOR, in refractory or recurrent binds to EGFR’s ATP binding site to prevent activation, is also ovarian cancer. in clinical trials. An initial trial of 30 women found a 13% pro- gression-free survival greater than 6 mo, along with one objec- Nuclear Factor KappaB (NFκB) Pathway tive response in a woman who had a mutation in the catalytic domains of EGFR. Multiple studies with this drug as a mono- The NFκB signaling pathway is activated by multiple upstream therapy and in combination with other drugs in Phase II studies effectors, such as chemokines, cytokines and growth factors. 103,104 showed no significant clinical efficacy. This transcription factor plays an important role in the induc- Monoclonal antibodies to EGFR have also been developed with tion of the inflammatory response and may provide the link similarly unremarkable results. Cetuximab (Erbitux), a chimerized between inflammation and oncogenesis. The NFκB molecule monoclonal antibody against EGFR showed the most promise in is composed of several subunits and in its inactive form, is local- colorectal and head and neck cancers. In a Phase II trial, cetux- ized to the cytoplasm. To become active, its inhibitory unit, imab in combination with carboplatin in 28 women with recur- IκB, must be phosphorylated so that the NFκB nuclear local- rent platinum-sensitive ovarian and primary peritoneal carcinoma ization sequence can be activated. reported that 32% had objective responses with an additional eight In an ovarian cancer model, inhibition of NFκB resulted patients having stable disease. Another Phase II trial combining in decreased VEGF and IL8 expression, which correlated with cetuximab with carboplatin and paclitaxel evaluated 38 women decreased tumorigenicity, decreased vascularization, decreased and showed a progression-free survival of 39% at 18 mo. formation of malignant ascites and prolonged survival of mice. Mabuchi et al. reported that NFκB inhibitor increased Mitogen Activated Protein Kinase (MAPK) Pathway the therapeutic efficacy of cisplatin when used in combina- tion. Additionally, Liu et al. revealed that the use of antioxi- The MAPK (Raf/MEK/ERK) pathway is activated by recep- dants that block paclitaxel-induced NFκB activation leads to tor tyrosine kinases as well as other receptors for cytokines increased sensitivity to paclitaxel treatment and increased cell and integrins. It is an evolutionarily conserved pathway that is apoptosis. 674 Cancer Biology & Therapy Volume 9 Issue 9 As of date, there are no NFκB inhibitors in current ovarian discovering and identifying hundreds of interesting and poten- cancer clinical trials; however, the above results indicate that it tially clinically relevant proteins. The identification of these pro- may show promise as an adjunct to current regimens. teins will not only help to discover proteins to be used as early detection biomarkers but will also provide clues regarding the Conclusions and Future Perspectives tumor microenvironment and targets for EOC specific therapeu - tics. Additionally, it is anticipated that biomarkers will benet fi In order to decrease the morbidity and mortality associated with from state-of-the-art ultrasound technologies, which are increas- EOC, we must shift from an era of advanced stage detection ing the imaging capabilities of at-risk ovaries and have the poten- to that of early stage diagnosis. Unfortunately, we have yet to tial to accurately differentiate benign from malignant tumors. discover and clinically validate the ideal EOC early detection Our hope is that a clinically validated combination panel of screening test that is based upon the molecular, genetic and bio- multiple analytes, which together have the adequate sensitivity chemical events regulating carcinogenesis, invasion and meta- and specificity to detect EOC at an early stage, will be developed. static dissemination. The quest for identifying this marker is A few groups have already embarked on this task. Mor et al. have proving to be more difficult than initially anticipated for multiple tested a multiplex panel including leptin, prolactin, OPN, IGF-II, reasons. First, EOC has a rather low incidence rate in the general macrophage inhibitory factor (MIF) and CA125 on hundreds of 31,32 population compared to other common conditions; therefore, serum samples. The highest sensitivity and specificity reached 31,32 the screening test needs to be both highly sensitive and specific with these panels has been 95.3 and 99.4%, respectively. to limit the number of invasive procedures performed. Also, Once these prototypical panels are further developed and vali- the shared pathophysiologic events that occur between all solid dated, we hope that the results will allow patients to be triaged cancers and other prevalent non-malignant conditions serve to into either high-risk or low-risk groups and subsequently, sent to decrease biomarker specificity. Additionally, we must gather more cancer centers where they can be imaged by emerging ultrasound information about the molecular mechanisms underlying EOC technologies. Following these tests, gynecological oncologists can initiation, progression and metastasis—with this information we more confidently assess the risk of ovarian cancer and thus, shift may be able to identify those unique, specific biomarkers that are the paradigm from advanced to early stage disease and greatly only associated with EOC. Other major obstacles to clinically improve women’s healthcare. valid biomarkers include the molecular heterogeneity between Acknowledgements tumors, the frequency of benign disease that reduces biomarker specificity for cancer, and low biomarker concentrations, espe - Sonia Dutta is a Howard Hughes Medical Institute Medical cially for early stage disease. Research Training Fellow. 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Bevacizumab in the treatment of tor expression: a Gynecologic Oncology Group study. ovarian cancer. Expert Rev Anticancer Ther 2007; Gynecol Oncol 2008; 108:493-9. 7:1339-45. www.landesbioscience.com Cancer Biology & Therapy 677 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

Biomarkers for ovarian cancer detection and therapy

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Copyright © 2010 Landes Bioscience
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Abstract

re VIew Cancer Biology & Therapy 9:9, 668-677; May 1, 2010; © 2010 Landes Bioscience Biomarkers for ovarian cancer detection and therapy Sonia Dutta, Feng-qiang w ang, Adrien Phalen and David A. Fishman* Department of Obstetrics, Gynecology and r eproductive Sciences; Mount Sinai School of Medicine; New York, NY USA Key words: epithelial ovarian cancer, early detection, serum biomarkers, signaling pathways, contrast enhanced ultrasound, proteomics novel technologic advances. Through the continuous efforts of Despite advances in surgical technologies and the develop- many investigators, hundreds of biomarkers that may potentially ment of more effective chemotherapeutics, epithelial ovarian serve to detect early stage EOC have been reported; however, cancer (eOC) remains the leading cause of death in women with to date, not a single biomarker has been proven to be clinically gynecologic malignancies. The high mortality and morbidity effective in detecting early stage EOC with adequate sensitivity associated with eOC is mostly attributed to the inability to and specificity. detect the disease before it is widely disseminated throughout In this article we will discuss current ovarian cancer biomark- the abdominal cavity. In the past decade, tremendous efforts have been taken to search for novel biomarkers that will detect ers that show promise to become potential screening markers, eOC at an early stage, which may also serve as new targets for as well as emerging technologies available to identify new EOC the prevention and control of metastasis. Here, we review biomarkers. In addition, we will discuss the signaling pathways recent developments in eOC early detection and targeted involved in ovarian cancer initiation and progression, in particu- therapy, as well as new technologies for the discovery of novel lar, those associated with EOC invasion and metastasis. biomarkers. Biomarkers for Ovarian Cancer Early Detection For the past several decades, the efforts to detect ovarian cancer Introduction at an early stage have been hampered by the lack of an effective screening tool. Ideally, this screening method would be a simple Ovarian cancer is deemed the “most lethal gynecologic malig- blood test with both a high sensitivity and a high specificity to nancy” in the United States given that more women die from decrease the number of false positive and false negative results. this disease than all other gynecologic malignancies combined. Unfortunately, an increase in the sensitivity of a test is often asso- The most common form of ovarian cancer (≥80%), thought to ciated with a reduction in its specificity and vice versa. Specificity derive from ovarian surface epithelium, is called epithelial ovar- of screening tools is of great importance in EOC because the ian carcinoma (EOC). Despite improvements in median survival majority of women with a positive result will require surgical through surgical advances and new chemotherapeutic regimens, intervention. For example, a screening test for postmenopausal the overall survival for women with late stage (III/IV) EOC has women would require a 99.6% specificity to yield an approximate remained relatively unchanged for the past 40 y, holding steady positive predictive value (PPV) of only 10%, where a surgeon at approximately 30% . In sheer contrast, women diagnosed with would perform ten operations for each case of EOC detected, EOC when the disease is confined to the ovary (stage I) not only given that the prevalence of EOC in this population is approxi- require less morbid surgical interventions and a decreased need for mately 1 in 2,500. However, lower specificities may be consid - adjuvant chemotherapy, but most importantly, have an overall 5-y ered acceptable in higher risk populations (such as those with a survival of approximately 93%, in addition to a much improved personal history of breast cancer or a recognized familial cancer quality of life. Unfortunately, the majority of women (70–75%) syndrome with an ovarian cancer predisposition) because as inci- continue to be diagnosed with advanced stage EOC because of dence increases, the specificity required to achieve a given PPV the rather asymptomatic nature of early stage disease and the lack decreases. of an adequate early detection screening method. To address this Currently physicians typically use a combination of pelvic problem, numerous researchers have focused on the identifica - exam, quantitative serum CA125 levels and transvaginal sonog- tion and validation of novel biomarkers for the early detection of raphy to monitor and screen women. Together, these techniques EOC with their increased knowledge of cancer biology and using are inadequate to detect early stage EOC due to their inability to delineate the subtle changes associated with the initial transfor- mation and their lack of specificity and sensitivity. Despite recent *Correspondence to: David A. Fishman; Email: david.s fi [email protected] Submitted: 02/19/10; Accepted: 02/24/10 advances in ovarian cancer biomarker research, no simple blood Previously published online: test with the required sensitivity and specificity has yet been clin - www.landesbioscience.com/journals/cbt/article/11610 ically validated. Fortunately, recent studies have identified and 668 Cancer Biology & Therapy Volume 9 Issue 9 re VIew re VIew validated a variety of new biomarkers or biomarker panels that disease and healthy controls. Their assay correctly identified have shown improved sensitivity and specificity over the current 47/48 patients with EOC, specifically 9/10 women with stage “gold standard”—CA125. Several biomarkers, in combination I disease, in comparison to only 2/9 early stage patients when with CA125, have enhanced the diagnostic accuracy of CA125 analyzed by CA125 alone. These results indicated that LPA had alone. Following, is a list of ovarian cancer biomarkers of recent a relatively high sensitivity and specificity of 95 and 89% , respec - interest. tively. However, follow-up studies by Meleh et al. reported no significant difference between LPA levels in benign and malig - Serum Protein Markers nant tumors. On the other hand, a later study by Sedlakova et al. corroborated with Xu’s in all respects and further added CA125 and the mucin family. To date, serum CA125 remains that LPA levels were associated with FIGO stage and histological the most useful tumor marker for the detection of recurrence after type. Although LPA appears to be a promising biomarker, as primary treatment. CA125, also known as MUC16, is a tumor yet, it has not shown to be clinically relevant in large scale screen- antigen of the mucin family, which are high molecular weight ing trials, which may be due to the difficulty in accurately quan - biomolecules produced by the secretory epithelial cells lining tifying each individual LPA. Thus, further clinical investigation ducts and lumens. Serum concentrations of CA125 are elevated is required with greater numbers of patients to determine its true in 80–90% of advanced-stage ovarian cancers, yet only 47% for diagnostic value. early stage disease. However, the clinical effectiveness of CA125 Human epididymal protein E4 (HE4). Human epididy- as a diagnostic tool is compromised by its lack of specificity and mal protein E4 (HE4), also referred to as Whey-Acidic Protein sensitivity. Elevated concentrations of CA125 are also detected in (WAP) four-disulphide core domain-containing protein 2 other malignancies (pancreatic, breast, bladder, liver and lung), (WFDC2), is a protease inhibitor that serves in protective as well as benign diseases (such as diverticulitis, uterine leiomyo- immunity and is primarily expressed in the reproductive tract mas, endometriosis, benign ovarian cysts, tubo-ovarian abscess, and upper airways. HE4 levels are elevated in ovarian cancer ovarian hyperstimulation syndrome and ectopic pregnancy) and patients but not in common benign gynecological conditions. some physiological conditions (pregnancy, ovulation and men- As a biomarker, it has increased sensitivity for detecting stage 19-21 struation). To overcome this lack of specificity and sensitivity for I ovarian cancer. In comparison to CA125, it appears to early detection, CA125 measurements have been combined with be more specific in detecting ovarian cancer and has a similar ultrasound techniques, such as transabdominal or transvaginal sensitivity. A recent study by Huhtinen et al. measured serum sonography, or other biomarkers, to try to detect early stage EOC. concentrations of HE4 and CA125 in a group of 159 women Meanwhile, the expression of other mucins, such as MUC4, has with multiple gynecologic conditions, such as endometriosis, provided better sensitivity for EOC detection when used in com- ovarian tumors, endometrial cancer and ovarian cancer, as well bination with CA125, with high expression of MUC4 observed as 66 healthy controls. They reported the combination of HE4 in EOC cells present in peritoneal effusions. Additionally, a com- and CA125 had the highest accuracy (96.3%) and sensitivity bined panel of CA125 with MUC1, CA72-4 and macrophage (92.9%) in differentiating patients with ovarian cancer from colony-stimulating factor (M-CSF) was able to improve the sen- healthy controls, as compared to each alone. Moore et al. eval- sitivity of detection of stage I ovarian cancer from 45% (CA-125 uated CA125 and HE4 levels in 531 women with a pelvic mass alone) to 70%, while maintaining 98% first-line specificity. and validated its use as a stratification tool. Analysis with these Higher levels of MUC2, MUC3 and MUC4 mRNA were also biomarkers correctly identified 94% of EOC patients as high detected in early stage EOC compared to late-stage EOC. The risk for ovarian cancer, with half of those misclassified having detailed clinical potential of mucins in diagnosis, prognosis and low malignant potential (LMP) tumors, which are often early therapy of ovarian cancer has been recently reviewed by Singh stage diseases and derive little benet f fi rom adjuvant treatment. et al. The sensitivity and specificity of this test was 93.8 and 75%, Lysophosphatidic acids. Lysophosphatidic acids (LPAs) are a respectively. Montagnana et al. also tested the levels of HE4 in family of phospholipids that act as potent signaling molecules in women with a pelvic mass and noted that the HE4 concentra- the EOC microenvironment. LPA was found to be secreted by tion was ten times higher in ovarian carcinoma versus benign ovarian tumors and clinically elevated in the ascites and serum tumors, with the diagnostic sensitivity of HE4 in early stages 9-11 25 of women with EOC. LPA elicits its function on cells by inter- determined to be 82.7% , compared to only 45.9% for CA125. acting with G-protein coupled receptors, which are often aber- Conversely, a study by Shah et al. revealed that the sensitivity 12,13 rantly expressed in ovarian cancer cells. The presence of LPA to discriminate ovarian cancer from healthy controls in average is known to increase cell proliferation, cell survival, invasion, risk patients was superior with CA125; yet in high-risk patients, migration and production of angiogenic factors. Additionally, HE4 had a higher sensitivity. At 98% specificity, the sensitivity LPA induces a signature of genes in EOC that is associated with of CA125 to identify ovarian cancer in average-risk and high- a reduced progression-free and disease-specific survival, as well as risk patients is 78.4 and 78%, respectively, while the sensitivity an overall worsened prognosis. of HE4 to identify ovarian cancer in average-risk and high-risk Xu et al. first reported LPA’s potential utility as a biomarker patients is 68.6 and 82.9% , respectively. for EOC given that its concentration was significantly higher in Although HE4 has shown promise as a complement to women with malignant gynecologic cancer compared to benign CA-125 for EOC detection, the sensitivity and specificity would www.landesbioscience.com Cancer Biology & Therapy 669 need to be further improved before being an effective popula- either analyte alone in differentiating cancer from healthy con- tion-wide screening test, which requires a minimum sensitivity trols. Similar to OPN, KLKs have shown biomarker utility in of 75% and a specificity of 99.6% to reach a PPV of 10%, as many malignancies but individually lack sufficient specificity or previously stated. The use of HE4 as a biomarker for ovarian can- sensitivity to be clinically useful. Biomarker panels consisting of 27,28 cer has also been recently reviewed by Hellstrom and Li et al. KLKs and other candidate biomarkers may offer improved clini- Osteopontin. Osteopontin (OPN) is an acidic, calcium bind- cally utility. ing glycoprotein found in all body fluids, which was initially Claudins. Claudins, a family of 24 proteins, are the main identified by cDNA microarray techniques. OPN is involved components of tight junctions that function as selective barri- in cell adhesion, inflammation and tumorigenesis and known ers. These proteins control paracellular diffusion and thereby to be associated with the presence and progression of multiple maintain cellular polarity. Transmembrane claudins form bi- or 29,30 cancers. OPN was first identified as a potential biomarker tri-cellular interactions between neighboring cells with occludin, for EOC by Kim et al. in 2002 when they noted OPN mRNA junctional adhesion molecules 1, 2 (JAM 1, 2) and the integral overexpression in ovarian cancer as well as significantly higher membrane protein, tricellulin. Deregulated claudin expression levels of OPN in tissue samples of invasive ovarian cancer and has been observed in gynecological cancers, such as cervical 40-42 borderline ovarian tumors versus benign tumors and healthy and endometrial cancers, as well as in premalignant lesions. 29 43,44 ovarian epithelium. The levels of OPN in plasma were also sig- Claudin-3 and -4 were found to be overexpressed in EOC. nificantly higher in 51 women with EOC compared with those High expression of claudin-7 and -10 has also been observed of 107 healthy controls, 46 patients with benign ovarian disease in ovarian cancer, while claudin-3 expression was found to be 29 45 and 47 patients with other gynecologic cancers. More recently, associated with shorter survival in EOC. In ovarian cancer Nakae et al. measured the preoperative plasma OPN levels in effusions, higher claudin-3 and -7 expression independently pre- 96 gynecologic patients and 31 healthy controls and reported a dicted shorter overall survival. Huang et al. recently reported significant difference in the mean value of OPN between ovar - that silencing the claudin-3 gene with siRNA suppressed ovar- ian cancer patients and all other groups (healthy controls, benign ian tumor growth and metastasis in mouse tumor models. The tumors and other gynecologic cancers). They also showed that use of claudins as diagnostic or prognostic markers for cancers OPN levels were significantly higher in stage IV patients than and gynecologic cancers has been reviewed by Morin et al. in 30 44,47 stage I and II. However, the sensitivity and specificity of OPN 2005, and most recently by Szabo et al. in 2009. As potential alone were inferior to that of CA125, and the combination of markers for ovarian cancer, the specificity of claudins in differ - OPN with CA125 decreased sensitivity although the specific - entiating EOC from normal and benign diseases remains to be ity increased. OPN levels were also evaluated in multiplexed determined. assays with other molecules, such as prolactin and leptin, show- Other potential serum biomarkers. A recent study evaluating 31,32 ing promising results. Unfortunately, the use of OPN as an 300 Japanese women (100 with EOC, 100 with benign tumors, EOC biomarker is thwarted since its levels have been found to 100 healthy controls) suggested that nidogen-2 may be a new be elevated in multiple cancers as well as benign conditions as serum biomarker. Nidogen-2 is a basement membrane protein reviewed by Johnston et al. that functions in the organization of the extracellular matrix. It Kallikreins. Kallikreins (KLK) are serine proteases that regu- is involved in regulating cell polarity, migration and invasion due late proteolytic cascades. Their activity may promote or inhibit to its role in facilitating interactions between cells and basement cancer cell growth, angiogenesis, invasion and metastasis by pro- membranes. Nidogen-2 serum levels were significantly elevated teolytic processing of various proteins, such as growth factors, in early and late stage EOC, compared to both healthy controls angiogenic factors and extracellular matrix components. Most and benign disease, although no difference in concentration was of the 16 family members are overexpressed in cancer and have noted between controls and benign disease. More studies are shown some utility as prognostic markers. Specifically, KLK6 warranted to determine nidogen-2 utility as a new serum bio- and KLK10 were elevated in ovarian cancer tissues that had low marker for EOC. levels of CA125, while KLK8 was associated with early stage Folate receptor alpha (FRα), is overexpressed in solid tumors disease and a favorable prognosis. Preliminary data also indicate to meet the increased demands for DNA synthesis and cell that KLK14 may be a potential biomarker for ovarian cancer. growth. The amount of FRα shed into the blood increases in Recently, KLK5 levels were measured in 167 women (50 with tumors expressing high levels of FRα and is subsequently detect- EOC) and found to be significantly higher in EOC patients ver - able in serum samples. A recent clinical trial measured the FRα sus all other groups (healthy controls, benign tumors and bor- levels in 30 women with ovarian cancer and 30 matched healthy derline tumors). KLK5 has a specificity of 95% to differentiate controls. Circulating FRα was found to be comparable between EOC from healthy controls and a sensitivity of 52%, however, early and late stage disease; yet, significantly higher than healthy the specificity dropped to 88% when differentiating cancer from controls. borderline tumors. Other members of the Kallikrein family, Recently, a correlation between L-Asparaginase (L-ASP) KLK6 and KLK13, have also been evaluated for their potential as efficacy and asparagine synthetase (ASNS) expression has been ovarian cancer biomarkers. The combination of KLK6, KLK13 shown in the ovarian subpanel of the NCI-60 cells. Lorenzi et and CA125 mRNA expression in 106 ovarian tumors and eight al. reviewed the potential of developing ASNS as a biomarker for healthy controls proved to be more sensitive and specific than ovarian cancer treatment. 670 Cancer Biology & Therapy Volume 9 Issue 9 In addition to the markers mentioned above, patient derived resulted in a diminished response of the EOC to cis-platinum tumor reactive antibodies have shown to be promising biomark- while overexpression of let-7i restored the cancer’s response to ers for EOC. Tumor antibodies are found in the circulation soon cis-platinum. This group also found that low let-7i expres- after initial tumor development and prior to detection of circulat- sion significantly correlated with decreased survival. miRNA ing antigens or palpable tumors. These proteins are stable and, expression profiling has also identified the miRNA-200 family unlike other serum markers, less sensitive to confounding factors as a potential prognostic indicator. Hu et al. compared miRNA such as stress, time of blood draw and sample manipulation. In a expression profiles of 58 EOC tissue samples with disease outcome recent study, the intensity of the antibody immunoblot correlated and showed that miR-200a expression was significantly associ - with the stage of EOC, with late stage samples having a greater ated with recurrence-free survival and overall patient survival 51 58 number of bands at a greater intensity. Additionally, early stage independent of other clinicopathologic features. Conversely, disease displayed unique bands above 100 kD, while late stage Nam et al. found that high levels of miR-200a or miR-200c were 51 59 disease had unique bands less than 40 kD. Of the identifiable significantly correlated to shortened survival. These discrepan- bands, all cancer patients made antibodies to cathepsin D, p53 cies could be a caused by varied miRNA screening methods and and SSX; however, only ovarian cancer patients (stage II and indicate a need for greater investigation into miRNA profiling. beyond) made antibodies to placental type alkaline phosphatase. Of particular benet fi , miRNA can be detected in the serum of Interestingly, the combination of nucleophosphmin, cathepsin EOC patients. Taylor and Gercel-Taylor first identified miRNA D, GRP78 and SSX autoantibodies appeared to be specific to within circulating tumor exosomes in EOC patients and con- 51 60 stage I disease. firmed the presence of previously observed miRNA signatures. Currently, although a full spectrum of serum biomarkers Lodes et al. demonstrated that sufficient miRNA is obtainable has been identified and explored as possible adjuncts to CA125, from serum to distinguish cancer patients from normal samples. limited clinical success has been achieved. They have isolated miRNA from the serum of 36 cancer patients (ovarian, prostate, lung, colon and breast cancer) and 13 normal Epigenetic Aberrations as Biomarkers in EOC controls. Hierarchical clustering of the microarray data resulted in discrimination of the cancer and normal specimens, demon- Epigenetic aberrations, including DNA methylation, histone strating the ability to identify patients as cancer or normal solely modifications and micro-RNA dysregulation, have been found from their serum miRNA profile. Therefore, serum miRNA to be intimately associated with ovarian cancer. These relatively can potentially serve as a new set of biomarkers for detection and stable aberrations are associated with distinct disease subtypes therapy of ovarian cancer. and are present in circulating serum; thus, they are promis- ing diagnostic and prognostic biomarkers. Inhibition of DNA Emerging Technologies for Ovarian Cancer methylation or histone-modifying enzymes has shown promising Detection and Biomarker Discovery results when used in combination with one another or with con- ventional chemotherapy in preclinical trials, as well as in current In addition to traditional techniques used for identification clinical trials. The best studied epigenetic aberration in ovarian and evaluation of differentially expressed protein biomarkers in cancer is BRCA1. BRCA1 hypermethylation occurs in 10–15% serum, new technologies are emerging and showing great prom- of sporadic disease cases, associates with loss of BRCA1 RNA ise for EOC biomarker discovery. and protein, and significantly correlates with poor patient out - come. The most recently discovered epigenetic regulation is that Comparative Genome Hybridization (CGH) by microRNA (miRNA), which are short single stranded RNAs that interfere with protein translation via complementary bind- Comparative genome hybridization is a technique used to assay ing to the 3' UTR of the target mRNA. miRNAs are deregulated the whole genome to detect gains or losses in gene copy number. in every cancer examined and have been discovered to act as both Many regions with abnormal copy numbers have been identi- tumor suppressors and oncogenes. Specifically, expression pro - fied in EOC. Additionally, there is evidence that certain dele - files of miRNA in EOC cell lines and patient specimens have tions or gains may be associated with the progression of tumors led to the identification of several miRNAs associated with EOC from benign to borderline and eventually malignancy. In a prognosis, patient survival and staging and chemo-resistance. recent study, 28 primary ovarian tumor samples were analyzed Dahiya et al. examined the miRNA profiles of 34 ovarian for high frequency gains and losses in their chromosomes and cancer tissue specimens and ten ovarian cancer cell lines rela- initial analysis revealed that chromosomes 1, 8 and 17 showed tive to an immortalized human ovarian surface epithelium cell the greatest numbers of gains and losses, with late stage disease line and found 56 differentially expressed miRNAs between the showing more gains and losses than early stage. Bruchim et al. neoplastic samples and control. Overall, 94% of the tumors had at analyzed 45 EOC specimens with conventional CGH and least one let-7 family member downregulated. Previously, Shell reported that gains in 5p were associated with increased risk et al. showed that Let-7 regulates the expression of high-mobility of recurrence while gains in 1p or loss of 5q had a protective group A2 (HMGA2) in ovarian cancer and its decreased expres- effect against recurrence. CGH is still quite a new technology sion is a marker for less differentiated cancer. Furthermore, Yang with obvious problems with reproducibility between groups. et al. demonstrated that reduction of let-7i expression in vitro Nevertheless, the information obtained from these analyses have www.landesbioscience.com Cancer Biology & Therapy 671 potential in identifying changes specific to ovarian cancer and to an increased risk of ovarian malignancy include the presence of discover previously unknown associated genes that can provide solid components, papillary excrescences and increased ovar- 70-74 more insight into EOC pathogenesis. ian volume. Traditionally, ultrasound used 2-D gray-scale images to visualize the ovaries; now, Color Doppler technology Proteomics allows for the characterization of flow within the vasculature of the ovary by identifying the shape of its waveform (arterial vs. Proteomic technologies were championed to EOC by Petricoin et venous), its direction (toward or away from the transducer) and 65 75-78 al. who discovered unique proteomic patterns specic t fi o EOC. its systolic and diastolic velocities. We previously reported Subsequently, many groups have applied mass spectrometry as a that the use of this technology improved diagnostic accuracy of tool to differentiate cancer from benign conditions and controls. ovarian cancer prediction because it better defined morphologic In 2007, Lopez et al. reported discriminating peptides that could and vascular characteristics of ovarian lesions compared to 2D 66 79 accurately identify early stage EOC from healthy controls. These ultrasound. Although imaging by ultrasound helps determine peptides, as a set, had a sensitivity and specic fi ity of 93 and 97%, risk of malignancy, numerous studies have found that transvagi- respectively. Similarly, Wu et al. analyzed 65 samples (30 EOC) nal ultrasound is not effective as a sole screening tool for early 80,81 with SELDI-TOF MS and identie fi d one peak specic t fi o EOC stage EOC in asymptomatic women. and two peaks specic t fi o normal controls. These three identifying Recent developments in ultrasound technology have rekindled peaks had a sensitivity of 84% and specic fi ity of 89%. the hope that ultrasound can be used to detect vascular changes Although proteomic patterns can be used as “finger prints” associated with early stage disease. Harmonics and pulse inversion to differentiate cancer from normal cases, biomarker discovery technology have improved signal-to-noise ratios and structural requires protein identicfi ation within the patterns. Thanks to conspicuity while the introduction of IV contrast agents (akin higher resolution mass spectroscopy and the incorporation of to those used with CT and MRI) has helped to more accurately immuno-MS, reverse phase protein arrays (RPPA) and nanome- detect, characterize and quantify aberrant vascularity and blood 82-84 trology-based procedures, researchers are currently identifying flow. The contrast enhancement of ultrasound helps to visu- hundreds of proteins that are differentially expressed in EOC alize tumor neovascularization in developing microscopic (not patients by analyzing ascites, urine and plasma. One recent study macroscopic) tumors by delineating the aberrant leaky vascular evaluated urine and blood samples from 209 women who had channels associated with early malignancy. The incompetent exploratory laparotomies for ovarian neoplasms. Of the 21 sig- extracellular matrix of neovascularization allows extravasation of nic fi ant peaks identie fi d, the three most signic fi ant peptides were RBCs (5–7 microns) and contrast agents (1–10 microns in diam- purie fi d and identie fi d as: b fi rinogen α , collagen α-1 (III) frag- eter), which can then be detected by ultrasound. The enhanced ment and fibrinogen β -N terminal fragment. These peptides ability to accurately visualize vascular changes specific to early alone did not have adequate discriminatory power; however, when stage EOC may help to detect early ovarian cancer with greater combined with CA125, had a higher sensitivity and specicfi ity sensitivity and specificity. Quantification of contrast enhance - than either alone. Gunawardana et al. identie fi d eight unique ment kinetics obtained with pulse inversion harmonic ultrasound extracellular and membrane bound proteins associated with EOC has shown potential to accurately differentiate benign from by using MS to analyze conditioned media from four cell lines. malignant ovarian tumors with a sensitivity of 100% and speci- 85,86 ELISA measurement of the proteins’ concentration in serum from ficity of 96.2%. Our findings suggest that contrast-enhanced ten EOC patients and 20 healthy women indicated that clus- ultrasound may have a high enough sensitivity and specificity terin and IGFBP6 expression was signic fi antly different between to differentiate benign disease from malignant, and thus, shows the two groups. Follow-up validation PCR revealed that clus- great promise as a potential imaging technique for ovarian cancer 85,86 terin mRNA expression is increased in EOC, while IGFBP6 is detection. decreased. Proteomic technologies have introduced a wealth of infor- Signaling Pathways and Therapeutic Targets mation that researchers can mine for possible early detection in Ovarian Cancer biomarkers. Unfortunately, as a costly, rapidly evolving, sophis- ticated technology, it is wrought with multiple problems associ- Since the 1990s, the foundation of ovarian cancer chemotherapy ated with specimen handling, which affect reproducibility and has included the combination of platinum based agents and tax- specificity. The discriminatory power of the results obtained anes. Over the past 20 y, refinements to these first-line agents have from these small studies must be confirmed with large multi-in - greeted patients with only slight increases in survival and quality stitutional prospective validation trials. Despite these criticisms, of life. Major improvements in these parameters will require the there is much anticipation that proteomics will be able to identify development of therapies that target multiple biologic processes clinically valid biomarkers for EOC early detection. associated with the ovarian cancer microenvironment, namely growth, angiogenesis, invasion and metastasis. Cancer cells are Contrast-Enhanced Ultrasonography genetically deranged normal cells that have aberrantly expressed and functioning biological pathways. The identification of spe - Today, transvaginal ultrasound is the best modality to image and cific pathways has allowed for the emergence of a new class of examine the ovaries. Ultrasound characteristics associated with therapeutics—molecularly targeted therapies, which interact 672 Cancer Biology & Therapy Volume 9 Issue 9 Figure 1. Schematic view of major signaling pathways shown to be involved in ovarian carcinoma growth and metastasis. These pathways have been important targets for the development of molecular therapeutics currently in clinical trials. 90,91 with and inhibit pathways to affect tumor growth and metastasis 52% of persistent/recurrent patients having stable disease. (Fig. 1). In the following section, we discuss important pathways Furthermore, there are a handful of trials underway combining associated with EOC and detail recent clinical trials. Bevacizumab with other chemotherapeutics. One earlier study combining Bevacizumab with cyclophosphamide in recurrent Vascular Endothelial Growth Factor (VEGF) ovarian cancer patients achieved a 28% response rate with a 57% 6-mo progression-free survival. The optimistic results from VEGFs are known to be integral players in the process of these Phase II trials have lead to the development of three ongo- angiogenesis, which is required for the survival, growth and ing Phase III trials—GOG218, ICON7 and GOG213. Of note, metastasis of cancer. The expression of VEGF and its recep- although Bevacizumab is generally well tolerated, it appears to tors, especially, VEGF Receptor-2 (VEGFR-2), are known to have a relatively high gastrointestinal perforation rate, quoted as be involved in the malignant progression of ovarian cancer, as 5.4%, which is more than double that of colorectal cancer. The well as the formation of ascites. Additionally, overexpression cause of this event needs to be further elucidated with additional of either is associated with decreased survival. Consequently, data from upcoming trials. many agents, such as small molecule inhibitors and monoclonal Cediranib (Recentin, AZD2171), a small molecule inhibitor of antibodies to VEGF and VEGFR have been developed to target VEGFR1-3 as well as PDGFR-β and c-kit, is currently in a Phase this signaling pathway. III trial in Europe for patients with recurrent, platinum-sensitive Bevacizumab (Avastin), a monoclonal antibody against VEGF, disease. A prior Phase II trial by Hirte et al. revealed that 41% has shown great clinical efc fi acy in Phase III trials for colorec - of patients with platinum-sensitive disease responded to mono- 87 88 89 tal, lung and breast cancer, and is showing great promise in therapy, while there was a 29% response rate in platinum-resistant Phase II trials for ovarian cancer. As a monotherapy for patients. The median time to progression was noted to be 4.1 mo patients with persistent/recurrent ovarian cancer or platinum- with the median overall survival being 11.9 mo. Matulonis et al. resistant disease, Bevacizumab has a 16–21% response rate, with also conducted a Phase II trial of Cediranib for recurrent EOC www.landesbioscience.com Cancer Biology & Therapy 673 and sited a 17% partial response rate with 13% of patients having known to stimulate cell growth by producing matrix metallo- stable disease. proteinases and to facilitate drug resistance. Additionally, b-Raf Another novel inhibitor of this pathway is the VEGF trap, and c-Raf, two isoforms of Raf, are associated with ovarian which is a fusion protein comprised of the VEGFR1 and cancer—high levels of b-Raf correlate with improved survival VEGFR 2 extracellular domains fused to the Fc portion of human while high concentrations of c-Raf are associated with a poor 96 107,108 IgG. This agent is able to bind to VEGF-A as well as placental prognosis. growth factor. A current Phase II trial has reported an 11% par- Sorafenib is an oral, multi-kinase inhibitor that targets the tial response rate in women with recurrent, platinum-resistant MAPK pathway as well as VEGFR1-3 and PDGFR-β tyrosine EOC. Other Phase II trials combining this agent with docetaxel kinase. It was initially combined with gemcitabine in a Phase 97,98 are currently underway as well. II trial. This therapy produced a 4.7% partial response rate and 60.4% stable disease, as well as a 5.4 and 13.3 mo progression-free Epidermal Growth Factor Receptor (EGFR) and overall survival. Single agent sorafenib was also tested in a Phase II trial by the GOG in 59 women with recurrent or persis- EGFR is a member of the ErbB family of receptor tyrosine tent EOC, which noted two responders, 20 patients with stable kinases, which also includes ErbB2 (Her2/neu), most com- disease and 30 women with progressive disease. Additionally, a monly associated with breast cancer. Once a ligand binds to recent Phase I trial combining intermittent sorafenib with bev- these receptors, the cytoplasmic tyrosine domains dimerize and acizumab yielded a 12% partial response rate and 53% stable subsequently lead to the phosphorylation of specific tyrosines. disease rate. Given these promising results, a Phase II trial This phosphorylation leads to the activation of multiple intracel- combining sorafenib with the clinically active bevacizumab is lular signaling pathways, such as the mitogen-activated protein currently underway. kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)- AKT pathways, which are involved in cell proliferation and sur- Phosphatidylinositol 3 Kinase (PI3K) Pathway 99,100 vival. EGFR has been noted to be overexpressed in 35–70% of EOC and is associated with a poor prognosis; consequently, The PI3K pathway is known to mediate angiogenesis and vascu- several inhibitors have been developed against this target. lar permeability, namely through its effectors, AKT and mTOR. Erlotinib, an oral EGFR tyrosine kinase inhibitor, was studied This pathway is known to be involved in the growth related as a monotherapy in women with refractory, recurrent, EGFR- functions of cells, such as transcription, translation, protein positive EOC. There was a 6% response rate with 43% of patients degradation and reorganization of the actin cytoskeleton. Forty having stabilized disease. This inhibitor has also been tested in percent of EOC cases have shown amplification or mutations combination with carboplatin and taxanes with promising results in PI3K or AKT, which subsequently leads to mTOR activa- 110,111 (NCI 5886). Additionally, its use as a maintenance therapy is tion and tumor survival. A Phase II trial (NCT00429793) being evaluated in a randomized Phase II trial (EORTC-55041). is currently underway to determine the clinical efficacy of Another small EGFR tyrosine kinase inhibitor, Get fi inib, which Temsirolimus, an inhibitor of mTOR, in refractory or recurrent binds to EGFR’s ATP binding site to prevent activation, is also ovarian cancer. in clinical trials. An initial trial of 30 women found a 13% pro- gression-free survival greater than 6 mo, along with one objec- Nuclear Factor KappaB (NFκB) Pathway tive response in a woman who had a mutation in the catalytic domains of EGFR. Multiple studies with this drug as a mono- The NFκB signaling pathway is activated by multiple upstream therapy and in combination with other drugs in Phase II studies effectors, such as chemokines, cytokines and growth factors. 103,104 showed no significant clinical efficacy. This transcription factor plays an important role in the induc- Monoclonal antibodies to EGFR have also been developed with tion of the inflammatory response and may provide the link similarly unremarkable results. Cetuximab (Erbitux), a chimerized between inflammation and oncogenesis. The NFκB molecule monoclonal antibody against EGFR showed the most promise in is composed of several subunits and in its inactive form, is local- colorectal and head and neck cancers. In a Phase II trial, cetux- ized to the cytoplasm. To become active, its inhibitory unit, imab in combination with carboplatin in 28 women with recur- IκB, must be phosphorylated so that the NFκB nuclear local- rent platinum-sensitive ovarian and primary peritoneal carcinoma ization sequence can be activated. reported that 32% had objective responses with an additional eight In an ovarian cancer model, inhibition of NFκB resulted patients having stable disease. Another Phase II trial combining in decreased VEGF and IL8 expression, which correlated with cetuximab with carboplatin and paclitaxel evaluated 38 women decreased tumorigenicity, decreased vascularization, decreased and showed a progression-free survival of 39% at 18 mo. formation of malignant ascites and prolonged survival of mice. Mabuchi et al. reported that NFκB inhibitor increased Mitogen Activated Protein Kinase (MAPK) Pathway the therapeutic efficacy of cisplatin when used in combina- tion. Additionally, Liu et al. revealed that the use of antioxi- The MAPK (Raf/MEK/ERK) pathway is activated by recep- dants that block paclitaxel-induced NFκB activation leads to tor tyrosine kinases as well as other receptors for cytokines increased sensitivity to paclitaxel treatment and increased cell and integrins. It is an evolutionarily conserved pathway that is apoptosis. 674 Cancer Biology & Therapy Volume 9 Issue 9 As of date, there are no NFκB inhibitors in current ovarian discovering and identifying hundreds of interesting and poten- cancer clinical trials; however, the above results indicate that it tially clinically relevant proteins. The identification of these pro- may show promise as an adjunct to current regimens. teins will not only help to discover proteins to be used as early detection biomarkers but will also provide clues regarding the Conclusions and Future Perspectives tumor microenvironment and targets for EOC specific therapeu - tics. Additionally, it is anticipated that biomarkers will benet fi In order to decrease the morbidity and mortality associated with from state-of-the-art ultrasound technologies, which are increas- EOC, we must shift from an era of advanced stage detection ing the imaging capabilities of at-risk ovaries and have the poten- to that of early stage diagnosis. Unfortunately, we have yet to tial to accurately differentiate benign from malignant tumors. discover and clinically validate the ideal EOC early detection Our hope is that a clinically validated combination panel of screening test that is based upon the molecular, genetic and bio- multiple analytes, which together have the adequate sensitivity chemical events regulating carcinogenesis, invasion and meta- and specificity to detect EOC at an early stage, will be developed. static dissemination. The quest for identifying this marker is A few groups have already embarked on this task. Mor et al. have proving to be more difficult than initially anticipated for multiple tested a multiplex panel including leptin, prolactin, OPN, IGF-II, reasons. First, EOC has a rather low incidence rate in the general macrophage inhibitory factor (MIF) and CA125 on hundreds of 31,32 population compared to other common conditions; therefore, serum samples. The highest sensitivity and specificity reached 31,32 the screening test needs to be both highly sensitive and specific with these panels has been 95.3 and 99.4%, respectively. to limit the number of invasive procedures performed. Also, Once these prototypical panels are further developed and vali- the shared pathophysiologic events that occur between all solid dated, we hope that the results will allow patients to be triaged cancers and other prevalent non-malignant conditions serve to into either high-risk or low-risk groups and subsequently, sent to decrease biomarker specificity. Additionally, we must gather more cancer centers where they can be imaged by emerging ultrasound information about the molecular mechanisms underlying EOC technologies. Following these tests, gynecological oncologists can initiation, progression and metastasis—with this information we more confidently assess the risk of ovarian cancer and thus, shift may be able to identify those unique, specific biomarkers that are the paradigm from advanced to early stage disease and greatly only associated with EOC. Other major obstacles to clinically improve women’s healthcare. valid biomarkers include the molecular heterogeneity between Acknowledgements tumors, the frequency of benign disease that reduces biomarker specificity for cancer, and low biomarker concentrations, espe - Sonia Dutta is a Howard Hughes Medical Institute Medical cially for early stage disease. Research Training Fellow. 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Bevacizumab in the treatment of tor expression: a Gynecologic Oncology Group study. ovarian cancer. Expert Rev Anticancer Ther 2007; Gynecol Oncol 2008; 108:493-9. 7:1339-45. www.landesbioscience.com Cancer Biology & Therapy 677

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Cancer Biology & Therapy – Taylor & Francis

Published: May 1, 2010

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