reducing ovarian cancer mortality through early detection ...cancer prevention research | minireview...

CANCER PREVENTION RESEARCH | MINIREVIEW

Reducing Ovarian Cancer Mortality Through EarlyDetection: Approaches Using Circulating BiomarkersCamille V. Trinidad1, Ashley L. Tetlow1, Leonidas E. Bantis2, and Andrew K. Godwin1,3

ABSTRACT◥

More than two-thirds of all women diagnosed withepithelial ovarian cancer (EOC) will die from the disease(>14,000 deaths annually), a fact that has not changedconsiderably in the last three decades. Although the 5-yearsurvival rates for most other solid tumors have improvedsteadily, ovarian cancer remains an exception, making itthe deadliest of all gynecologic cancers and five timesdeadlier than breast cancer. When diagnosed early, treat-ment is more effective, with a 5-year survival rate of up to90%. Unfortunately, most cases are not detected until afterthe cancer has spread, resulting in a dismal 5-year survivalrate of less than 30%. Current screening methods forovarian cancer typically use a combination of a pelvic

examination, transvaginal ultrasonography, and serumcancer antigen 125 (CA125), but these have made minimalimpact on improving mortality. Thus, there is a compel-ling unmet need to develop new molecular tools that canbe used to diagnose early-stage EOC and/or assist in theclinical management of the disease after a diagnosis, giventhat more than 220,000 women are living with ovariancancer in the United States and are at risk of recurrence.Here, we discuss the state of advancing liquid-basedapproaches for improving the early detection of ovariancancer.See all articles in this Special CollectionHonoring Paul

F. Engstrom, MD, Champion of Cancer Prevention

IntroductionOvarian cancers are a heterogeneous group of malignancies

arising from or involving the ovary and/or fallopian tubes (1).Ovarian cancers are broadly classified into nonepithelial ovar-ian cancer and epithelial ovarian cancer (EOC) and that thelatter is attributed to the majority of ovarian cancer–relateddeaths (2). Overall, EOC is the fifth leading cause of cancer-related death among American women, making it the deadliestcancer of the reproductive system (3). EOC is a heterogeneousdisease, comprising several histotypes (serous, �70%; endo-metrioid, �10%; clear cell, �10%; and mucinous, �3%–10%)with distinct epidemiologic, molecular, and clinical features.High-grade serious carcinomas (HGSC) are estimated to be50% to 60% of all ovarian malignancies and account for half ofall EOCs.EOCs are diagnosed predominantly at an advanced stage

with widespread metastases throughout the peritonealcavity (4–6). Manifestations of the disease are typically vagueand do not become apparent until the disease is advanced and

difficult to treat. Thus, ovarian cancer is caught at an early stageonly in about one-fifth of all cases according to the NationalOvarian Cancer Coalition. Furthermore, no reliable screeningtests are currently available for ovarian cancer, and currentdiagnostic tools for early detection remain inadequate. Earlydetection of ovarian cancer at a localized stage (stages IA andIB) results in far better disease prognosis: according to theAmerican Cancer Society (2019), the projected 5-year survivalrate for these patients is about 92%. However, only 15% of allovarian cancers are successfully diagnosed at this stage. Patientswith advanced-stage HGSC have a worst prognosis (�32% and�15% for 5- and 10-year survival, respectively) compared withpatients diagnosed with early-stage HGSC [�71% and �53%(5- and 10-year survival, respectively); ref. 7]. Moreover,30 years of statistics gathered by the NIH Surveillance, Epide-miology, and End Results (SEER) Program indicate thatapproximately 70% of patients diagnosed with ovarian cancerwill experience disease recurrence after initial benefit fromchemotherapy (8). These statistics clearly emphasize the needto develop tools to detect ovarian cancer at an earlier stage.The twomost commonly used screening tests in the clinic are

transvaginal ultrasound (TVUS) and blood tests for elevatedcancer antigen-125 (CA125) protein levels. It is difficult todistinguish benign from malignant tumors by TVUS and,hence, the need for more invasive biopsies. On the other hand,CA125 protein levels are typically not elevated in the serum ofup to 50% of patients with stage I ovarian cancer; thereby, it isnot as reliable for early detection (9). Also, blood tests quan-tifying CA125 protein level are not specific to ovarian cancer, asCA125 elevation can be potentiated by many factors (10).Unlike breast cancer in which a biopsy can be performedfor diagnosis, EOC screening requires invasive surgery tomake

1Department of Pathology and Laboratory Medicine, University of KansasMedical Center, Kansas City, Kansas. 2Department of Biostatistics, Universityof KansasMedical Center, Kansas City, Kansas. 3The University of Kansas CancerCenter, Kansas City, Kansas.

C.V. Trinidad and A.L. Tetlow contributed equally to this article.

Corresponding Author: Andrew K. Godwin, University of Kansas MedicalCenter, 3901 Rainbow Boulevard, MS 3040, Kansas City, KS 66160. Phone: 913-945-6373; Fax: 913-945-6327; E-mail: [email protected]

Cancer Prev Res 2020;13:241–52

doi: 10.1158/1940-6207.CAPR-19-0184

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a definitive diagnosis (11, 12) and therefore false-positivesurgeries must be limited to at most 10 per screen-detectedcancer, that is, a positive predictive value (PPV) exceeding 10%,which for the patient is quite unacceptable. Thus, the lowprevalence of this cancer in the general postmenopausal pop-ulation (1 in 2,500) requires an effective screening strategy tohave a high sensitivity for early-stage disease (>75%) and a veryhigh combined specificity (99.6%) to achieve a PPV exceeding10% (11–19). When CA125 is interpreted longitudinally withthe risk of ovarian cancer algorithm (ROCA) referring 2%annually at highest risk to transvaginal ultrasound (TVUS), this“2-stage screening strategy” achieved a combined specificity of99.8%, a 22% PPV—greater than the 10% lower limit, anddetected an increased but still modest proportion of EOCs inearly stage (11, 20, 21). In a 2017 landmark study, the UnitedKingdom Collaborative Trial of Ovarian Cancer Screeningcompared several screening approaches in more than200,000 postmenopausal women (median follow-up of 11.1years): (i) serum CA125 level evaluated using ROCA (50,640women), (ii) annual TVUS screening (50,639 women), or (iii)no screening (101,359 women). Unfortunately, like otherovarian cancer screening studies primarily focused on mea-suring serum CA125 levels (22–27), this study did not show asignificant decrease in ovarian cancer mortality (28). Thisclinical problem highlights the urgent need to expand the classof liquid-based biomarkers to detect a substantially greaterproportion of cancers in early stage, while maintaining a 98%annual specificity for referral to imaging (e.g., TVUS).

Liquid-Based Biopsies: Can TheseDevelopingMolecular ToolsBeUsedto Improve Early Detection ofCancer?Pathologic analysis of tumor tissue biopsies has been the gold

standard in the initial diagnosis of cancer, but liquid biopsies,which analyze tumor-derivedmaterial circulating in the blood-stream and other bodily fluids, are rapidly gaining traction inthe clinic. Liquid biopsy is a minimal or noninvasive technol-ogy that detects molecular biomarkers using liquid samplewithout the need for costly or invasive procedures. These testshave considerable potential in oncology, e.g., for early detectionof cancer, treatment and recurrence monitoring, and as surro-gates for traditional biopsies with the purpose of predictingresponse to treatments and the development of acquiredresistance. A liquid biopsy can provide information aboutthe genetic landscape of all cancerous lesions (primary andmetastases) as well as offer the opportunity to systematicallytrack genomic evolution. The liquid biopsy biomarker types areprimarily segmented into circulating tumor cells (CTCs), cell-free DNA (cfDNA), and extracellular vesicles (EV; Fig. 1). Thediscovery of CTCs dates back to the 1860s, when cells that weremorphologically identical to the tumor were identified in theblood of a patient with metastatic cancer (29). Their potentialsignificance was not fully realized until the late 2000s, when the

number of CTCs in the bloodstream were shown to haveprognostic significance in various different tumor types, forexample, metastatic breast, metastatic castration-resistantprostate, and metastatic colorectal cancer (30). Another waythat tumors release biomarker information is through tumorcell necrosis and the release of dead cells or cell fragments.These cells are engulfed by phagocytes that process the tumorcell DNA into small fragments (160–180 base pairs in length) ofnucleic acids, which is then released into the blood-stream (31, 32). Although levels of tumor DNA have beenshown to mirror tumor burden, interpretation is often com-plicated by the presence of other cfDNA derived from non-tumor cells. Therefore, a number of highly sensitive methodshave been developed to detect aberrations found in circulatingtumor DNA (ctDNA), including mutation, amplification,chromosomal rearrangement, and hypermethylation. A thirdbiomarker, which has been known for years, has reemerged andis showing great promise. This member of the EV family,known as exosomes or small EVs, are nano-sized vesicles(30–150 nm) of endocytic origin, which are produced andreleased by most cell types under normal physiologic and indiseased states (33, 34). Once considered little more thangarbage cans whose job was to discard unwanted cellularcomponents, recent discoveries have sparked considerableinterest in exosomes as circulating biomarkers. Exosomes areinformative molecules that carry cargo representative of theiroriginating cell including nucleic acids, cytokines, membrane-bound receptors, and a wide assortment of other, biologicallyactive lipids and proteins (35–38). This cargo remains func-tional upon entry or fusion with a recipient cell, thus exosomaltransfer is now considered an important form of cell–cellcommunication in normal and pathologic states, such ascancer. Because exosomes can travel systemically throughoutthe body, efforts are underway to exploit them as potentialbiomarkers to detect and monitor disease states. In this review,we will focus on the current methods and advancements usingliquid-based biopsies that have shown promising potential inthe early detection of ovarian cancer.

Circulating Tumor CellsFirstobserved in1869byThomasAshworth in thebloodof an

individualwithmetastaticdisease,CTCshave longbeenthoughtto be an important component of tumor dissemina-tion (29, 39, 40). CTCs are cancer cells that have detached fromthe tumor and are found at extremely low levels in the blood-stream. Modern research on CTCs has focused on CTC enu-meration and characterization. Some speculate that tumor cellshedding is an early event in tumorigenesis andmight be usefulfor early detection, but few studies have shown utility in thisclinical setting. As a blood-based, FDA-approved biomarker,CTCs were thought useful for determining the prognosis ofpatients with metastatic breast, colorectal, and prostatecancers (41–45). As the only FDA-cleared device for the enu-meration of CTCs inwhole blood, CellSearch (Menarini SiliconBiosystems, Inc.) was originally used for the evaluation of CTC

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numbers in patients with cancer to help predict prognosis andoverall survival (30). However, clinical enumeration of thesecirculating cells has become less useful and costly, due to the factCTC tests are rarely reimbursed by insurance. Regardless, theinterest in CTCs continues and many different platforms forCTC isolation have been developed for research as well asclinical use in recent years (46). Nevertheless, CTC isolationand characterization remain technically challenging and prog-ress has been hampered by the difficulty in rapidly and effec-tively isolating pure populations of CTCs using liquid biopsy.The three main categories of isolation techniques are

immune-affinity–based methods, size-/density-based meth-ods, and microfluidic device–based methods (ref. 47; Fig. 2).The earliest approaches utilized positive and negative selectionfor commonCTC surface antigens or organ-specificmarkers todifferentiate CTCs from other cell types. In these studies,researchers used antibody-coated beads to positively selectCTCs from the cellular milieu or deplete the milieu of othercell types with a negative selection marker. EpCAM, a celladhesion protein found on epithelial cells, is often used as apositive selection marker for CTCs (48). The most commonnegative selection marker is CD45 because it is found only on

cells of hematopoietic origin. First reported in 2000, the ISET(isolation by size of epithelial tumor cells) system is based oncell size filtration and has been shown to effectively recoverCTCs for diagnostic cytopathologic analysis (49). However,smaller CTCs less than 8 mm in diameter can be lost using thismethod, due to the selective pore size.Microfluidic devices offerseveral attractive advantages for CTC-based studies, such ascontinuous sample processing using a small volume to reduceloss and integration of downstream analyses on the chip itself.These devices can also reduce hands-on time and increaseconsistency of heterogeneous CTC profiles (50). Some micro-fluidic devices integrate existing CTC isolation techniqueswithin their design, such as the size-dictated immunocapturechip (SDI-Chip; ref. 51). The SDI-chip is composed of twomirrored anti-EpCAMantibody-coatedmicropillar arrays thatselectively enhance the interaction of CTCs with the selectionantibodies, resulting in efficient capture of CTCs based on theprinciple of deterministic lateral displacement (52). The dis-advantage to the EpCAM-based capture is that not all CTCsexpress EpCAM (53). In an effort to move away from the biasintroduced by affinity-based isolation, researchers have begunto focus on label-free surface expression–independent

Figure 1.

A graphical representation of a variety of approaches ovarian cancer researchers can take to evaluate blood-based liquid biopsies for cancer-associated biomarkersto aid in early detection of malignancy.

Reducing Ovarian Cancer Mortality Through Early Detection

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microfluidic devices to isolate CTCs. A label-free approach isoptimal because CTCs are very heterogeneous and fluid in theirgene expression, making any affinity-based approach biasedtoward cells expressing a certainmarker. Several label-freeCTCseparation techniques have arisen in recent years includinga microfluidic flow fractionation approach, acoustic-basedseparation, and deterministic lateral displacement separa-tion (52, 54, 55). The Labyrinth device developed by Lin andcolleagues in 2017 is a label-free circular microfluidic chamberthat uses a hydrodynamic maze to isolate CTCs by size (56).The device functions by flowing blood through a labyrinth(hence the name) of 56 sharp turns, which results in size-based

enrichment of CTCs depleted of hematopoietic cells. Improve-ments can be made on the purity of resultant CTCs by doubleLabyrinth separation. Labyrinth output can even be used withvarious downstream single-cell analysis tools including Bio-Mark Real-Time PCR System (Fluidigm), DEPArray (SiliconBiosystems), and droplet digital PCR (Rain-dance).CTC enumeration data can provide only limited insight into

the underlying biology of malignant disease. More recentresearch efforts have been focused on the molecular character-ization and functional analysis of CTCs. Using less than 1mL ofblood, Racila and colleagues demonstrated that 12 of 14 patientswith clinically organ-confined breast cancers and 3 of 3 patientswith organ-confined prostate cancers had excess epithelial cellsdetected in their blood (57). However, the diagnostic utility ofCTCs in ovarian cancer has been decidedly underwhelming. Inthe first studies led by us, CTCs (as circulating endothelial cells/circulating endothelial progenitors) were found to be rare inpatients with ovarian cancer and were not overly informativewhen evaluated along with activity of molecular targeted ther-apies (58, 59). In a 2018 study, CTCs were detected in only 5 outof 29 patients (17.2%) with proven primary ovarian cancer(Table 1). CTCs were shown to be more prevalent in patientswith metastases to the ovary, rather than in primary ovariancancer (43). Although CTCsmay not be useful as early detectionbiomarkers, CTC status in ovarian cancer is associated withoverall survival and progression-free survival according to arecent meta-analysis of 11 publications and 1,129 patients (60).

Circulating Tumor DNAIt was reported for the first time in 1977 by Leon and

colleagues that patients with cancer have increased levels of

Figure 2.

Examples of methods to capture circulating tumor cell from whole blood.

Table 1. Examples of liquid-based biopsy studies to detect ovarian cancer using ctDNA and CTCs.

Platformused

Selectionparameters Case size

Controlsize Sensitivitya Specificitya CI (95%) References

CTCs CellSearch� EpCAM Histologicdiagnosis ofovarian cancer

n ¼ 29 n ¼ 17a

Benigndisease

25.7% 100% Se ¼ 12.5% � 43.3%Sp ¼ 76.8% � 100%

43

Cancer withmetastasesto the ovary

n ¼ 5

CirculatingtumorDNA(ctDNA)

Microarray(MethDet56)

Promoters:RASSF1A,CALCA,EP300

Benign disease n ¼ 30 n ¼ 30 90% 86.7% Se ¼ 80% � 100%Sp ¼ 66.7% � 96.7%

75Ovarian cancer n ¼ 30

Microfluidicdigital PCR

TP53-mutantallelefraction

Ovarian n ¼ 32 n ¼ 5 71% 88% Se ¼ 42% � 92%Sp ¼ 64% � 99%

71Primary peritoneal n ¼ 5Fallopian tube n ¼ 3

Ultra-highcoveragebi-sulfitesequencing

Unspecified3 DNAmemarkerpanel

Ovarian cancer n ¼ 41 n ¼ 21 41.4% 90.7% Se ¼ 24.1% � 60.9%Sp ¼ 84.3% � 94.8%

76Other cancer n ¼ 37Nonepithelial tumors n ¼ 5Borderline n ¼ 27Benign tumors n ¼ 119

Note: Pairs of sensitivity and specificity in this table are not directly comparable with each other because they are calculated on the basis of different cutoffsthroughout.aSensitivity (Se) and specificity (Sp) pair values listed correspond to a specific decision cutoff utilized in each study.

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free DNA in their blood serum (61). Cell-free DNA broadlyrefers to all circulating DNA, whereas ctDNA is specificallytumor-derived as the name suggests. Unfortunately, typicalDNA isolation methods do not distinguish DNA by its origin,so scientists have developed several strategies to increase theproportion of ctDNA isolated from serum or plasma. Enrich-ment for ctDNA is optimal because it increases the likelihood ofuncovering biomarkers specific to the cellular heterogeneity ofthe tumor. Apoptotic DNA fragmentation is thought to be themain source of ctDNA due to its average size of 70 to 200 basepairs, which corresponds to the length of DNA around anucleosome (62). The amount of ctDNA can be highly variable.ctDNA can comprise approximately 0.01% � 90% of totalcirculating DNA (63–65). Even so, ctDNA has been used todetect tumor-specific mutations, loss of heterozygosity, DNAintegrity, microsatellite alterations, and epigenetic altera-tions (62, 66). Importantly, ctDNA can be used to quantifythe level of disease burden and reveal the genomic landscape ofthe tumor. Researchers have estimated that the half-life ofctDNA ranges from 16 minutes to 2.5 hours, based on thedegradation kinetics of DNA in circulation (67–69). Thismakes ctDNA a temporal snapshot of tumor status. Even withthis short half-life, promising results have been shown incolorectal cancer, in which >60% of early-stage tumors canbe detected through sensitive mutational analysis of DNAfragments in plasma (64). Figure 3 depicts typical analysismethods used to evaluate ctDNA and its required sensitivity aswell as various applications.As we move toward the future, more and more advance-

ments are taking place to increase the sensitivity of currentDNA-based analysis methods such as real-time PCR, micro-

fluidic digital-drop PCR, BEAMing, Safe-SeqS, CAPP-Seq, andother next-generation sequencing techniques (70). In a recentretrospective study, researchers determined that detection ofmutations in TP53 using high-grade serous ovarian carcinomapatient plasma has the potential to assess clinical prognosis andresponse. This study also demonstrated that the amount ofctDNA correlated with tumor volume as determined by three-dimensional (3D) volume reconstruction fromCT images (71).Overall, all patients with cancer evaluated in this study with atumor volume greater than 20 cm3 had reliably detectableTP53mutations; however, the role of ctDNAmutation screening forearly detection remains unproven. Using a mathematical mod-el, researchers postulate that tumors in themillimeter diameterrange can only be detected using secreted blood biomarkersunder ideal conditions of extremely high rates of biomarkersecretion and essentially zero background from healthycells (72, 73). Thus, very sensitive technology is necessary todetect ctDNA from early-stage ovarian cancer.Epigenetic changes can also be reliably detected in ctDNA.

Aberrant methylation in circulating DNA has been shown inmany cancer types (74). Cancer cells use promotermethylationto deregulate gene expression and can be informative as anoninvasive biomarker. The hypermethylation of importantDNA repair genes is often an early step in carcinogenesis. In2011, Liggett and colleagues determined that themethylation ofthree promoters differentiated patients with ovarian cancerfrom healthy controls with a sensitivity of 90% and a specificityof 86.7% (75). These methylated genes, RASSF1A, CALCA,and EP300, are associated with tumor suppression, calciumregulation, and histone acetylation, respectively. In addition,promoter methylation of RASSF1A and PGR-PROX was

Figure 3.

Sensitivity, methods, and applications for circulatingtumor DNA analysis. Overview of the most commonapplications and techniques for ctDNA analysis. ctDNArepresented by red DNA strands in blood collectiontubes (adapted from ref. 102).





informative for distinguishing ovarian cancer from benignovary disease (sensitivity 80%, specificity 73.3%; ref. 75). Meth-ylation patterns found in ctDNAhave even shown the potentialto diagnose a subset of ovarian cancers up to 2 years in advanceof elevated CA-125 levels (Table 1; ref. 76).It is thought that solid malignant neoplasms shed DNA into

the circulatory system most often by necrosis, rather thanapoptosis as in normal tissue (77). Necrosis typically resultsin larger and less uniform DNA fragments than apoptosis.Because of this, DNA integrity is increased in cancer and can beanalyzed using ctDNA. Using the most common mobile ele-ment in the human genome, Alu repeats, researchers can derivea DNA integrity index that can differentiate patients withcancer from healthy individuals by isolating circulating DNAfrom plasma. This approach was used byWang and colleaguesto establish a cutoff index value of 0.59, meaning all samplesabove this index are likely malignant (77). This study dem-onstrated inadequacies in sensitivity of the assay (62%);however, specificity was 100% (Table 1). DNA integrityanalysis represents a less-expensive and simpler alternativeto DNA sequencing.

EVs in Ovarian CancerExtracellular vesicles compared with CTCs and cfDNA are

considered a relatively new class of cancer biomarker. EVs aresecreted by cells as a form of intercellular communication.These EVs can shuttle nucleic acids, lipids, and proteins fromtheir cell of origin to surrounding cells to regulate the functionof other cells (78). EVs are classified according to size (from afew nanometers to a few micrometers) and subcellular ori-gin (79). A subtype of EVs, termed exosomes, is endocytic inorigin and includes 60- to 80-nm small exosomes (Exo-S), aswell as 90- to 120-nm large exosomes (Exo-L; ref. 80). Ingeneral, the term “exosomes” is broadly used to refer to aheterogenous mixture of small EVs (sEVs) that are less than200 nm in size; this is due to the fact that widely usedpurification methods (such as differential ultracentrifugation)cannot definitively isolate EV class based on subcellularorigin (81).The role of exosomes in cancer metastasis has been dem-

onstrated in various cancer types (82–85), establishing a ratio-nale for exploring exosome-based diagnostics. In the ovariancancer context, our laboratory has shown that tumor-derivedexosomes from CP30 and CP70, which are both platinum-resistant ovarian cancer cell lines, imparted platinum resistanceto a platinum-sensitive cell line (A2780; ref. 86). In anotherin vitro study, EOC cells were shown to secrete exosomes thataffected the phenotype of mesothelial cells of the peritoneumvia transfer of CD44, a cell surface glycoprotein that plays a rolein cell adhesion and migration. The mesothelial cells, in turn,increased CD44 surface and MMP-9 secretion, resulting in thedegradation of the extracellular matrix and promoting ovariancell invasion (87). Collectively, these studies suggest that exo-somes represent a general mechanism by which one cell type

canmodulate the phenotype and characteristics of neighboringcells.Ovarian tumor–derived EVs are not only rich in proteins

but these contain miRNAs. miRNAs are small noncodingRNAs that can target messenger RNA thereby altering geneexpression. miRNA profiling of EpCAM-captured exosomesfrom ovarian cancer patients’ serum identified miR-21,miR-141, miR200a, miR200b, miR-200c, miR-203, miR-205, and miR-214 to be elevated compared with controls.A high expression of let-7 has been correlated to ovariancancer cell invasiveness in SKOV3 (88). It has been dem-onstrated that miR-1246 expression in ovarian tumor–derived exosomes can aid tumor growth by downregulatingCav1 expression, thereby increasing P-glycoprotein expres-sion in infiltrating pro-tumorigenic immune cells (M2-typemacrophages; ref. 89). This makes the tumor microenviron-ment favorable because this confers drug resistance to pac-litaxel (89). In addition, other studies have shown thatmiRNAs from ovarian cancer tumor–derived EVs can conferdrug resistance through other pathways (82, 90–92).Because there are various evidence of tumor-derived EVs

contributing to the progression of ovarian cancer (87, 93, 94),it is logical to assess the potential of EVs for diagnostics.Recently, several technologies have been developed to cap-ture exosomes from minute volumes of starting material tosurpass the problem of requiring larger sample volumeswhen using conventional methods such as ELISA and immu-noblotting (Table 2). Technologies such as microfluidicchips and the nano-plasmonic exosome (nPLEX) platformcan be conjugated to antibodies to allow for capture andprofiling of exosomes. One study has assessed exosomesderived from ascites of patients with ovarian cancer usingthe nPLEX platform (95). Initially, molecular profiling ofantibodies was used to coat the surface of nanoholes usingselected ovarian cancer markers based from literature. Ovar-ian cell lines and exosomes derived from the same cell lineswere then profiled. The authors found that the combinationof EpCAM and CD24 can distinguish ovarian tumor–derivedexosomes with a detection accuracy of 97%. Furthermore, itwas found that these two markers are highly expressed in theascites of patients with ovarian cancer (n ¼ 20) comparedwith the control population (n ¼ 10), which consisted ofascites from nonmalignant conditions (95). These findingscorrespond with a previous report that identified thatEpCAM and CD24 are found in exosomes of cultured celllines and malignant ascites (96). Our laboratory was the firstto demonstrate the capture of exosomes directly from theplasma of patients with ovarian cancer using a microfluidicchip (97). This was followed by the development of amicrofluidic chip called ExoSearch (Fig. 4) by Zhao andcolleagues (2016), wherein exosomes from plasma of healthycontrols and patients with ovarian cancer were capturedusing anti-CA-125, anti-EpCAM (Epithelial Cell AdhesionMolecule), and anti-CD24 (cluster of differentiation 24 orheat stable antigen CD24; ref. 98). In this study, it was

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observed that exosomal CA125 and EpCAM may be used todiscriminate patients with ovarian cancer from the healthycontrols (98). Most recently, our group together with Zhangand colleagues (2019) has reported the development of anultrasensitive analysis of exosomes using a 3D nanostructuredherringbone chip (nano-HB; ref. 99). In this small case–controlstudy, we report that exo-folate receptor alpha as well as exo-EpCAM and exo-CD24 could be detected in small volumes ofplasma samples (as little as 2 mL) and could significantlydistinguish patients with ovarian cancer from cancer-free con-trols (area under the curve¼ 1; Fig. 5; ref. 99).While these typesof devices have yet to be assessed under the rigor of clinical trialsor in longitudinal samples from asymptotic patients who laterdevelop cancer, these devices clearly hold promise.

ConclusionLiquid-based approaches to detect cancer, although gaining

traction, are not new. Complete blood cell counts, includingwhite blood cells, red blood cells, and platelets, are used to helpdiagnose blood disorders, including leukemia and lymphoma.Prostate-specific antigen is used to help diagnosis prostatecancer, whereas calcitonin, a-fetoprotein, and human chori-onic gonadotropin are used to diagnosis medullary thyroidcancer, liver cancer, and germ cell tumors, such as testicularcancer and ovarian cancer, respectively. Liquid-based biopsiesare likely here to stay and as technologies improve so will theirclinical utility. These types of assays, which are considerablyless invasive when compared with the tissue biopsy procedure,will help further advance personalized targeted therapy and

Table 2. Comparison of studies using circulating EVs to detect ovarian cancer.

Platform used Selection parameters Case size Control size AUC Sensitivitya Specificitya References

nPLEX EpCAM 20 10 0.968 90%b 100%b 95CD24 0.900 80%b 100%b

EpCAM þ CD24 0.995 95%b 100%b

CD63 0.670 75%b 60%b

Microfluidic chip (ExoSearch) EpCAM 15 5 1.00 93%a 100%a 98CA-125 1.00 93%a 100%a

CD24 0.91 93%a 80%a

Microfluidic chip (nanoHB) EpCAM 20 10 1.00 100%a 100%a 99CD24 1.00 100%a 100%a

FR-a 0.995 95%a 100%a

NTA 1.00 69%a 86%a

Note: Pairs of sensitivity and specificity in this table are not directly comparable with each other because they are calculated on the basis of different cutoffsthroughout.aSensitivity and specificity pair values listed correspond to a specific decision cutoff utilized in each study. The sensitivity and specificity correspond to themaximizedYouden index.bAs reported by each study.

Figure 4.

A, Schematic of the ExoSearch chip. Bright-field microscopy images of Y-shaped injector (B), mixer (C), immunomagnetic bead (D), and TEM of exosomes (E)captured on the surface of the immunomagnetic bead (reprinted with permission from ref. 98).





immunotherapy by offering a source of easily obtainablematerial for mutation analysis. Liquid biopsies also have thepotential to be useful in efficacy assessment, especially ifimaging cannot be used or the interpretation is problematic,

and in real-time monitoring of molecular profiles and clonalevolution in patients undergoing cancer therapy to detectmetastatic relapse or metastatic progression as well as mechan-isms of resistance.

Figure 5.

A, Schematic of the 3D nanostructured herringbone chip (nano-HB), method of nano-HB chip fabrication (B), a nano-HB chip with magnified SEM images in themiddle (C), and testing clinical samples on the nano-HB chip (D, left). CD24, EpCAM, and FRa on exosomes derived from the plasma of patients with ovarian cancer(n¼ 20) and control population (n¼ 10) were quantified. Error bars, SD (n¼ 3). E, Plot of the exosomal protein concentration captured on the nano-HB chip versusovarian cancer staging, with stage I/II considered as early and stage III/IV as advanced (reprintedwith permission from ref. 99). BKG, Background; CSA, colloidal self-assembly; SEM, scanning electron microscopy.

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Even with the growing excitement surrounding this area oftranslational research, significant improvements in liquidbiopsy platforms and techniques will be required to advancethe promises of precision cancer medicine. At present, aliquid biopsy test in itself cannot simply replace the goldstandard tissue biopsy–based test; they primarily serve as acomplimentary test. As such, there are several remainingchallenges impeding the wider adoption of liquid biopsy–based clinical tests. First, it is important to determine thelevel of sensitivity of a given liquid-based assay. It is wellestablished that CTCs, ctDNA, and tumor-derived EVs arerelatively rare compared with the number of “other mole-cules” found in a blood or bodily fluid sample; therefore,major hurdles remain to improve the test's detection ability.Furthermore, it is not clear if a given test can accuratelydissect the heterogeneity of a tumor and identify the “badactors” among the other tumor subclones. Second, themajority of liquid biopsy assays lack extensive clinical val-idation resulting in limited reimbursements and utilizationof these next-generation tests within the medical commu-nity (100). It is, therefore, essential to rigorously validate anddemonstrate to both providers and payers the value of liquidbiopsies in the clinical setting before they impact clinicalpractice and are routinely reimbursed. As we move towardthe future, the technologies supporting the development ofliquid biopsy assays will continue to improve and in turn thesensitivity and specificity of the test. Currently, strides arebeing made in improving single CTC capture approachesand platforms. With the adoption of digital drop PCR,researchers are able to increase the sensitivity of detectionof low-abundance mutations in the ctDNA (101). Micro-fluidic technologies are also proving suitable for isolation oflow-abundance cancer-associated EVs/exosomes (99).In summary, developing effective screening tests for early

detection of ovarian cancer remains one of the most significant

unmet needs in the diagnosis and treatment of this disease. It isvital to identify new liquid-based tests and to develop aneffective strategy to identify ovarian cancer at its earliest stagesso that women stand the best chance for successful treatmentand improved survival. To implement a population-wideovarian cancer screening strategy, there must be evidence thatthe test is both sensitive enough to detect the cancer early andspecific enough not to cause harm to healthy people. It is clearthat when cancer, any cancer, is detected late, that is, after it hasmetastasized to other parts of the body, the outcomes aregenerally grim. These new liquid-based platforms representpotentially valuable clinical tools to evaluate various circulatingbiomarkers at a specificity and sensitivity necessary to detectovarian cancer while it is still localized. In the next decade,liquid-based biopsies will become important clinical tools forcancer screening, diagnosis, prognosis evaluation, drugresponse predictions, and disease monitoring. However, theanalytical and clinical utility of such tools must be rigorouslyestablished before this can be applied. Successful “bench tobedside” transition will require extensive clinical validation,which demonstrates a significant decrease in the mortality ofwomen diagnosed with ovarian cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThis report was supported in part by The Kansas Institute

for Precision Medicine (GM130423, to A.K. Godwin), a graduatestudent award from the OVERRUN Ovarian Cancer Foundation (toC. Trinidad) and the Honorable Tina Brozman Foundation, Inc. (Tina'swish to A.K. Godwin). A.K. Godwin is the Chancellors DistinguishedChair in Biomedical Sciences.

Received April 4, 2019; revised June 20, 2019; accepted August 15, 2019;published first March 4, 2020.

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