Clinical Cancer Research - CCR FOCUS · The Institute of Cancer Research, 15 Cotswold Road, Sutton,...

Circulating Tumor Cells: A Multifunctional Biomarker

Timothy A. Yap1,2, David Lorente1,2, Aurelius Omlin3, David Olmos4, and Johann S. de Bono1,2

AbstractOne of the most promising developments in translational cancer medicine has been the emergence of

circulating tumor cells (CTC) as aminimally invasivemultifunctional biomarker. CTCs in peripheral blood

originate from solid tumors and are involved in the process of hematogenous metastatic spread to distant

sites for the establishment of secondary foci of disease. The emergence of modern CTC technologies has

enabled serial assessments to be undertaken at multiple time points along a patient’s cancer journey for

pharmacodynamic (PD), prognostic, predictive, and intermediate endpoint biomarker studies. Despite the

promise of CTCs as multifunctional biomarkers, there are still numerous challenges that hinder their

incorporation into standard clinical practice. This review discusses the key technical aspects of CTC

technologies, including the importance of assay validation and clinical qualification, and compares existing

and novel CTC enrichment platforms. This article discusses the utility of CTCs as a multifunctional

biomarker and focuses on the potential of CTCs as PD endpoints either directly via the molecular

characterization of specific markers or indirectly through CTC enumeration. We propose strategies for

incorporatingCTCs as PDbiomarkers in translational clinical trials, such as thePharmacological Audit Trail.

We also discuss issues relating to intrapatient heterogeneity and the challenges associated with isolating

CTCs undergoing epithelial–mesenchymal transition, as well as apoptotic and small CTCs. Finally, we

envision the future promise of CTCs for the selection and monitoring of antitumor precision therapies,

including applications in single CTC phenotypic and genomic profiling and CTC-derived xenografts, and

discuss the promises and limitations of such approaches.

See all articles in this CCR Focus section, "Progress in Pharmacodynamic Endpoints."

Clin Cancer Res; 20(10); 2553–68. �2014 AACR.

Disclosure of Potential Conflicts of InterestT.A. Yap reports receiving speakers bureau honoraria from Janssen. A. Omlin is a consultant/advisory board member for Pfizer, Astellas,

AstraZeneca, and Janssen. D. Olmos reports receiving speakers bureau honoraria from Veridex. J.S. de Bono reports receiving speakers

bureau honoraria from Johnson & Johnson. No potential conflicts of interest were disclosed by the other authors.

CME Staff Planners' Disclosures

The members of the planning committee have no real or apparent conflict of interest to disclose.

Learning ObjectivesUpon completion of this activity, the participant should have a better understanding of the strategies currently under investigation for the

development of circulating tumor cells as a minimally invasive multifunctional biomarker in patients with a range of different cancers.

Acknowledgment of Financial or Other SupportThis activity does not receive commercial support.

IntroductionThe advent of rationally designed molecular therapeutics

that inhibit specific tumoral molecular aberrations has ledto a paradigm shift in our understanding of cancer anddrug discovery (1). In contrast with previous drug deve-lopment strategies, there is now a general acceptance thatmaximum-tolerated doses may not necessarily correlatewith the biologic efficacy of therapy, and therefore the needfor molecular tools or biomarkers that can accurately assessthe underlying mechanisms of action and pharmacody-namic (PD) effects of the drug has emerged (2).

Such PD biomarkers, together with pharmacokinetic(PK) parameters, provide confirmation that pharmacologic

Authors' Affiliations: 1Division of Clinical Studies, The Institute of CancerResearch; 2DrugDevelopmentUnit, RoyalMarsdenNHSFoundation Trust,Sutton, Surrey, United Kingdom; 3Kantonsspital St. Gallen, Department ofMedical Oncology, Gallen, Switzerland; and 4Spanish National CancerResearch Centre, Madrid, Spain

Note: T.A. Yap and D. Lorente contributed equally to this article.

Corresponding Author: Johann S. de Bono, Division of Clinical Studies,The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM25NG, United Kingdom. Phone: 44-20-8722-4302; Fax: 44-20-8642-7979;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2664

�2014 American Association for Cancer Research.

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effects of a novel antitumor compound on its intendedtarget, pathway, and associated functions have taken place(3). Such PD biomarkers may be used to define the quan-titative extent and duration of target inhibition required inthe clinic for biologic and therapeutic effects, andultimatelyinmaking "go" or "no-go" drug development decisions (1).Although indirect PD readouts such as mechanism-basedtoxicities, for example, rash with EGF receptor (EGFR)inhibitors, are useful general indicators of pharmacologicblockade, modern technologies now permit the directquantification of protein phosphorylation knockdown orother related parameters in tumor biopsies. The recentdevelopment of minimally invasive PD assays may reducethe risks and issues associated with repeated tumor biopsiesand enable serial determinations of drug effects, thus min-imizing the impact of inter- and intrapatient variability onsuch results.

One of the most promising developments has involvedthe emergence of circulating tumor cells (CTC) as a min-imally invasive biomarker in cancer medicine. This reviewfocuses on the utility of CTCs as PD endpoints in themonitoring of novel molecularly targeted therapeutics. Wealso discuss the technical aspects of CTC assays and detailexisting and novel CTC enrichment technologies. We pro-pose strategies for incorporating CTCs as PD biomarkersinto early-phase clinical trials, discuss the promises andlimitations of CTCs, and envision the future utility of CTCsas multifunctional biomarkers in modern clinical studies.

CTCs: Emergence as a MultifunctionalBiomarker

It is now widely accepted that CTCs found in peripheralblood originate from solid tumors and are involved in theprocess of hematogenous metastatic spread by sheddingfrom such cancers and migrating to distant sites for theestablishment of secondary foci of disease (4). CTCs areultimately rare events, with a frequency of approximately 1to 100 million cells in the bloodstream (4). Although theirexistence has been known since the nineteenth century, it isonly the recent development of modern technologic plat-forms that has permitted the reliable capture and charac-terization of CTCs. A major advantage of CTCs comparedwithotherminimally invasive assays is their potential utilityas a liquid biopsy serving multiple critical functions (5).Although beyond the scope of this article, important con-sideration should be given to CTCs, not just as PD end-points, but also as prognostic markers, predictive andintermediate endpoint biomarkers of response (6).

For example, studies have validated the prognostic sig-nificance of CTC counts in several tumor types, includingbreast, colorectal, and prostate cancers, providing furtherevidence of their tumoral origins (7–9). The U.S. Food andDrug Administration (FDA) approval of baseline CTCcounts, as assessed with CellSearch technology as a prog-nostic marker in metastatic breast, colorectal, and prostatecancers, heralded the emergence of CTC enumeration as ananalytically validated tool in clinical practice.

Although their utility as predictive and intermediateendpoint (or surrogate) biomarkers remains under inves-tigation, they represent areas of great promise andpotential.For example, Maheswaran and colleagues were able todetect the emergence of the acquired EGFR kinase domainT790M drug-resistance mutation in CTCs in the majority ofpatients with metastatic non–small cell lung cancer(NSCLC) who had clinical tumor progression while receiv-ing tyrosine kinase inhibitor treatment, suggesting that themolecular analysis of CTCs offers the possibility of moni-toring changes in tumor genotypes during treatment (10).Such potential to detect secondary genetic aberrations thatmay lead to drug resistance with CTCs suggests a possiblerole as putative predictive biomarkers.

In addition, during the COU-AA-301 phase III clinicaltrial of the CYP17 inhibitor abiraterone (Zytiga; JanssenBiotech) in post-docetaxel–treated patients with castration-resistant prostate cancer (CRPC), CTCs were enumerated atseveral time points (baseline, weeks 4, 8, and 12) duringtreatment. CTC conversion (�5 to

collaborative studies. An analytically validated assay shouldthen undergo clinical validation in the setting of clinicaltrials, aiming to link the information the biomarker pro-vides to specific biologic or clinical outcomes. For example,clinical studies evaluating CTC protein expression as PDbiomarkers will need to establish the baseline variabilityassociated with the assay through the use of technical andbiologic duplicates, to determine if the PD effects observedare indeed due to the drug being assessed, or if CTCheterogeneity is a potential issue. Furthermore, it will becritical for any new CTC technologies, which are notapprovedby the FDA, toundertake robust healthy volunteerstudies to assess the false-positive CTC detection rates.

Isolation and enrichment of CTCsSeveral methods have been developed for the evaluation,

isolation, and enrichment of CTCs in blood, based on thephysical and chemical properties of these cells. BecauseCTCs are extremely rare cells in the bloodstream, enrich-ment techniques have been used for separation fromperipheral blood cells. Identification of CTCs is then per-formed, through either immunofluorescence, reverse tran-scription PCR (RT-PCR), or other techniques involvingsophisticated software and microscopy. Enrichment andisolation technologies that are validated or which are cur-rently in development are summarized in Table 1.Affinity binding approaches use antibodies that either

bind to the surface of cells expressing specific antigens(positive selection by capturing EpCAM or CK 9, CK19-positive cells, or negative selection by specifically eliminat-ing cells that express the leukocytic antigenCD45), or attachto the magnetic beads for separation based on magneticfields (immunomagnetic assays; Fig. 1). Microfluidic plat-forms (CTC-chips) are based on devices with antibody-coated microstructures, which allow the mixing of bloodcells through the generationofmicrovortices to significantlyenhance the number of interactions between target CTCsand the antibody-coated chip surface (14–16). Such anapproach enables the capture of large numbers of viableCTCs in a single step fromwhole bloodwithout the need foran initial enrichment step.Other CTC capture platforms are based on other physical

properties such as size (using microfilters that isolate CTCsbased on their greater size), density, or decreased deform-ability of CTCs compared with erythrocytes and leukocytes(Fig. 1; ref. 17). Another development has been the use ofnanodetectors bound with EpCAM antibodies, which areinserted into a peripheral vein, thus increasing the volumeof blood that is in contact with the detector and therebyallowing the capture of greater numbers of CTCs (18). Adifferent approach uses dielectrophoretic methods, basedon the assumption that CTCs have different electric prop-erties and can therefore be separated from normal cells byapplying electric fields (19). Recently, novel assays thattarget a combination of physical (size) and biologic (immu-nomagnetic) properties of CTCs have been developed, suchas the CTC-iChip, which is capable of sorting rare CTCsfromwhole blood at a rate of 10 million cells per second in

both epithelial and nonepithelial cancers (20). The Euro-pean consortium CTCTrap has also developed a platforminvolving a functionalized antibody-containing three-dimensional (3D) matrix that combines immunocaptureand size-based separation for CTC enumeration and char-acterization, including CTC culture (21).

Identification of CTCsMost CTC assays use an immunofluorescence-based

method that defines CTCs as nucleated cells [positive forthenucleardye40,6-diamidino-2-phenylindole (DAPI)] thatare positive for epithelial markers [cytokeratins (CK) andEpCAM] and negative for the leukocyte markers (CD45).These need to be undertaken by trained operators selectingCTCsbasedonfluorescentmicroscopy, althoughautomateddigital microscopy systems are able to detect CTCs in arelatively reliable and efficient fashion (22, 23). The HD-CTC platform (Epic Sciences, Inc.) is a novel platform thatdoes not rely on any single protein enrichment strategy.Instead, all nucleated cells are retained and immunofluor-escently stained with anti-CK, anti-CD45, and anti-DAPIantibodies and imaged in a high definition scanner. Thisenables multiple parameters to be analyzed for the charac-terization of specific populations of CTCs (Fig. 2; ref. 24).

Functional assays are also currently available, basedon thedetection of secreted proteins by CTCs, and can potentiallyspecifically detect viable cells and discard apoptotic ones(EPISPOT; ref. 25). Other approaches for CTC identificationare based on targeting specific mRNAs with RT-PCR (26).This strategy requires semi-quantitative assays, as nontu-moral cells are able to express the targeted transcripts, albeitat a reduced level. The detection of tumor-specific DNAaberrations in CTCs has also been explored and couldpotentially be themost specificapproach, althoughconcernsexist about intra- and interpatient tumor heterogeneity (27).

The CellSearch SystemThe only FDA-cleared assay to date is the CellSearch

System, an immunomagnetic system that has been devel-oped for the quantification ofCTCs inwhole blood samples(Fig. 3). The CellSearch system defines a CTC according toits size, positivity for EpCAM and CK, and negativity ofCD45 expression. CTC enrichment is performed usingimmunomagnetic antibodies against EpCAM. CTC identi-fication is performed by a trained operator with a fluores-cence microscope, after inmunofluorescent labeling withantibodies against CD8, 18, 19, and 45, and DAPI. Resultsare expressed as the number of CTCs per 7.5 mL of wholeblood (28).

High reproducibility of the assay, as shown by an inter-assay variation coefficient (CV) of 12% and an interinstru-ment CV of

Table 1. CTC assays classified by their respective underlying mechanisms

Assay Developer Comments (reference)

1. Immunomagnetic assays: positive selection—EpCAM antibodiesCellSearch� Janssen

DiagnosticsEpCAM-coated beads based positive selection using magnetic beads followed by

staining and image analysis (92)Clinically validated in metastatic breast, colorectal, and prostate cancer

AdnaTest AdnaGen Immunomagnetic enrichment (EpCAM, MUC-1, Mesothelin) and subsequentmolecular profiling (93)

Anti-EpCAM/anti-CK antibodyCTC enrichment

Glenn Deng, StanfordUniversity

CTC enrichment assay using the combination of anti-CK and anti-EpCAMantibodies (13)

MACS Miltenyi Biotec Bound by antibodies against a ligand of asialoglycoprotein receptor, andsubsequently magnetically labeled by magnetic beads (94)

DynabeadsEpithelial Enrich

Life Technologies Uses the monoclonal antibody BerEP4 against the EpCAM antigen (95)

CellCollector Gilupi Functionalized structured medical wire coated with anti-EpCAM antibodies placeddirectly into the blood stream of a patient via an indwelling catheter, remains inthe arm vein for 30 minutes and thus enables the capture of CTCs in vivo (96)

Biofluidica CTCDetection System

Biofluidica EpCAM-coated chip to capture followed by release of cells and electricalcounting (97)

2. Immunomagnetic assays: negative selection—CD45 antibodiesEPISPOT Laboratoire de Virologie,

Hôpital Lapeyronie,CHU Montpellier

Detects proteins secreted/released/shed from viable cells after the depletion ofCD45þ and selecting based on the expression of EpCAM (98)

Aviva CTCEnrichment Kit

AVIVA Biosciences Combination of size-based RBC depletion and WBC depletion (99)

Precelleon Precelleon Red cell lysis step followed by immunomagnetic labeling, and subsequentdepletion, of CD45þ cells (100)

DynabeadsCD45

Life Technologies Paramagnetic beads covalently coupled to anti-human CD45 antibody that enableisolation or depletion of CD45þ leucocytes (95)

NegativeEnrichment OMS

Jeffrey Chalmers,Cleveland Clinic

Red cell lysis, immunomagnetic labeling, and subsequent depletion of CD45þ

cells (13)RARE(RosetteSep-Applied ImagingRare Event)

StemcellTechnologies

Negative selection technique where tetrameric antibody complexes cross-linkCD45-expressing leukocytes to RBCs in whole blood (101)

3. Microfluidic chipsOncoCEE Biocept Biotin-tagged antibodies that bind selectively to CTCs (102)ClearCell FXSystem

Clearbridge Label-free technology that uses lateral traps to capture tumor cells based on sizeand deformability (103)

ClearID Cynvenio High-throughput microfluidic sheath flow isolation technology by ferrofluid withcell staining plus downstream DNA analysis via NGS or qPCR (104)

Isoflux Fluxion Biosystems Microfluidic chip based on immunomagnetic capture (105, 106)CTCChip Daniel Haber and

Mehmet Toner,Dana-Farber and MGH

Microfluidic fitted with anti-EpCAM antibodies (10)

Herringbone-Chip

Daniel Haber andMehmet Toner,Dana-Farber and MGH

Microvortices are used to significantly increase the number of interactions betweentarget CTCs and the antibody-coated chip surface (15)

4. SizeScreenCell ScreenCell Microporous membrane filter allows size selective isolation of CTCs (107)CellSieve Creatv Microtech Lithographically fabricated filters with precision pore dimensions (108)CellOptics Ikonysis Automated imaging platform combined with size-based isolation (109)ISET RareCells Size-based enrichment with track-etched polycarbonate membrane (110)

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with amedian 14%variabilitywith themanual process, andhas been used to evaluate the significance of morphologicchanges in CTCs (30, 31).

The Utility of CTCs as PD BiomarkersIn general, CTCs have been used as PD biomarkers

through two main strategies, either indirectly via CTCenumeration or directly through the molecular characteri-zation of CTCs.

Enumeration of CTCs for PD studiesAlthough an indirect marker of drug-associated effects,

changes in CTC levels before and during treatment havebeen used to reflect active doses of a novel antitumor agentin different tumor types, including breast, colorectal, andprostate cancers. In CRPC, enumeration of CTCs has beenused as an indirect PD biomarker for both androgen recep-tor (AR)- and non-AR–targeting drugs. For example, CTCenumeration before and during treatmentwas incorporatedin single-agent abiraterone and enzalutamide phase I/IIclinical trials to demonstrate active doses of drug. TheseCTC declines not only confirmed PD effects of these drugs,but were also an early indicator of antitumor activity, asdemonstrated by their correlation with significant prostate-

specific antigen (PSA) declines (32–34). CTCs are especiallyuseful in such a setting as patients with advanced CRPCgenerally have bone-predominant disease and thereforelack objectively measurable soft tissue disease (34).

CTC enumeration was also used as a PD and potentialintermediate endpoint biomarker in a recent phase II trial ofthe novel MET/VEGFR2 inhibitor cabozantinib, where anadaptive design was used to determine the lowest activedaily dose of drug to administer to patients with meta-static CRPC. In this study, although PSA responses byPCWG2 criteria only occurred in 1 of 34 (3%) patientswith advanced CRPC, 58% of patients who received alower dose of 40 mg of cabozantinib had a CTC conver-sion from unfavorable to favorable categories, providingconfirmation of the PD effects of cabozantinib at thatdose, which was also associated with clinical benefit,supporting its use as an intermediate endpoint biomarker(35).

Because individual patients will have different CTCcounts, future studies should also consider using the relativechanges of CTC counts to monitor PD effects (e.g., >30%reduction to indicate treatment response), rather than justlooking at changes fromunfavorable (�5) or favorable (

Molecular characterization of CTCs for PD studiesPD studies of antitumor molecular therapeutics have

involved the characterization of drug effects on cell mem-brane antigens on CTCs and/or the selective reduction ofgenetically distinct subpopulations of CTCs. The molec-ular characterization of CTCs has involved a range ofdifferent techniques, including the assessment of proteinexpression by immunofluorescence or immunohis-tochemistry (6, 37). There are a range of protein-basedassays, including HER2 (38), g-H2AX (39), EGFR (40),and insulin-like growth factor-I receptor (IGF-IR; 41)expression, as well as AR signaling (42) on CTCs, whichhave been incorporated as exploratory PD biomarkers inclinical trials and are highlighted here in this section.

HER2 expression. HER2 expression on CTCs has beenextensively tested in patients with breast cancer in differentdisease settings. During theGeparQuattro clinical trial, CTCHER2 expression was evaluated in patients with HER2-positive early breast cancer before and after neoadjuvantchemotherapy/trastuzumab treatment using the CellSearchsystem (43). Initial validation studies were undertakenwithbreast cancer cell lines with known HER2 gene amplifica-tion status. CTCs were considered to overexpress HER2 if atleast one CTC showed strong (3þ) HER2 immunofluores-

cence. Fourteen of 58 (24%) patients were found to over-express HER2 on CTCs, including 8 (14%) patients withHER2-negative primary tumors and 3 (5%) patients aftertrastuzumab treatment. Interestingly, CTCs thatwere scoredHER2-negative or weakly HER2-positive before or aftertreatment were present in 11 of 21 patients with HER2-positive primary tumors.

In view of the potential for HER2 status to changeduring disease recurrence or progression in patients withbreast cancer, reevaluation of HER2 expression on CTCsmay be an important strategy. Different CTC assays havebeen evaluated, with varying results. For example, theCellSearch and AdnaTest BreastCancer assays were pro-spectively evaluated for HER2 expression in 221 patientswith metastatic breast cancer (44). Overall, only 62(28%) of 221 patients were CTC-positive in both assaysand, of these, 13 (21%) had HER2-positive CTCs withboth platforms. Concordance in HER2 status betweenboth assays was observed in only 31 (50%) of these 62CTC-positive patients (P ¼ 0.96). The authors attributedthis lack of correlation in CTC HER2 assessment toseveral reasons, including technical differences betweenboth assays. For example, while the CellSearch assayevaluates the HER2 status of individual CTCs by

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Figure 1. Schematic depiction of CTC processing methods. Enrichment techniques are necessary to separate the extremely rare CTCs from peripheral bloodcells (erythrocytes and leukocytes). Identification of CTCs is then performed generally through immunofluorescence or RT-PCR techniques. RBC, red bloodcells;WBC,white blood cells. Reproduced fromArya et al. (120) by permission of theRoyal Society of Chemistry. Access themost recent version of this articleat: http://dx.doi.org/10.1039/C3LC00009E.

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immunofluorescence, the AdnaTest BreastCancer assaydetermines the average HER2 expression of all tumorcells, and is therefore unable to detect heterogeneity inHER2 expression between different CTCs and to establishthe percentage of HER2-positive CTCs (44).Other studies have indicated that CTC counts seem to be

higher in patients with estrogen receptor–positive (ERþ)breast cancer, in contrast to HER2-positive and triple-neg-ative breast cancers, which could potentially be explainedby low EpCAM expression and a more mesenchymal phe-notype of tumors belonging to the basal-like molecularsubtype of breast cancer, and therefore not detectable bymost currentmethods (45, 46). A novel HER2-basedmicro-fluidic device for the isolation of CTCs from peripheralblood of patients with HER2-expressing solid tumors hasrecently been developed as an alternative to EpCAM-basedCTC capture methods (47).A phase III clinical trial (DETECT III) is ongoing that

evaluates CTC counts before first- to third-line treatmentsin patients with metastatic breast cancer found to be HER2-negative in the primary tumor. Patients with �1 HER2-positive CTC before the start of a new line of chemotherapywill be randomized to chemotherapy alone versus chemo-therapy plus lapatinib with a primary endpoint of progres-sion-free survival (PFS) superiority (NCT01619111;ref. 48).g-H2AX expression and other markers of apoptosis. The

expression of the nuclear DNA double-strand break dam-age biomarker g-H2AX on CTCs has been assessed inpatients with a range of tumors receiving cytotoxic che-motherapies and PARP inhibitors. The evaluation of PDchanges was undertaken through CTC enumeration and

the assessment of percentage of g-H2AX–positive CTCs.Out of 11 of 15 patients with CTCs identified, g-H2AX–positive CTCs were detected in 6 patients (% of g-H2AX–positive CTCs among CTCs: 1.6%–31%; ref. 49). The PDeffects of chemotherapies with or without a PARP inhib-itor were assessed over time in 5 patients. There wereincreased g-H2AX–positive CTCs found in all patientsafter treatment [mean of 2% (range, 0%–6%) at baseline;38% (range, 22%–64%) posttreatment].

Wang and colleagues recently presented data on theutility of CTC-based PD biomarkers in phase I and IIstudies of targeted therapies conducted by the NCI (50).Only 30% of patients participating in eight NCI phase Iand II studies in a variety of solid tumors were statisticallyevaluable because of insufficient CTCs collected at base-line. Also, when considering specific PD biomarkers frommultiple studies involving topoisomerase-1 and PARPinhibitors, the g-H2AX–positive CTC baseline level wasless than 20% in 34 of 50 patients. The fraction of CTCsexpressing g-H2AX—independent of changes in the totalCTC count—increased in patients receiving treatmentwith different topoisomerase-1 inhibitors either alone orin combination with other drugs. Interestingly, correla-tions between g-H2AX levels and antitumor responseswere observed in patients with refractory cancers in aphase II randomized trial of the combination of velipariband cyclophosphamide (50).

Other markers, such as RAD51, M30, and phosphorylat-ed histone H3, may also potentially be used as functionalreadouts of apoptosis but are limited by the number ofCTCs that have to be analyzed. Suchbiomarkersmay enablethe monitoring of the apoptotic-inducing and DNA-

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DAPI CK CD45 ARFigure 2. Traditional and novel CTCcandidates. The top two panelsdemonstrate "traditional" CTCs(CKþ, CD45�, intact andmorphologically distinct DAPI) withAR expression and AR subcellularlocalization using the Epic CTCplatform. In addition, novel CTCcandidates, including CK� CTCs(CK�, CD45�, intact andmorphologically distinct DAPI),small CTCs [CKþ, CD45�, intactDAPI butmorphologically similar towhite blood cells (WBC)], andapoptotic CTCs (CKþ, CD45�,fragmented and morphologicallydistinct DAPI) may also beidentified (bottom three panels). AllCTC subpopulations can becharacterized for proteinexpression and subcellularlocalization such as AR. Imagescourtesy of Ryan Dittamore, EpicSciences, Inc.

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damaging effects of drugs such as PARP and ATM inhibitorsover time (37).

EGFR expression. EGFR expression on CTCs has beendemonstrated in a number of tumor types using the Cell-

Search� system, including patients with advanced breast,prostate, colorectal, and lung cancers (40, 45, 51, 52). Forexample, serial sampling of a patient with advanced colo-rectal cancer treated with the EGFR inhibitor panitumumabhad a decrease in the number of EGFR-positive CTCs (40).Nevertheless, heterogeneity and variability in EGFR expres-sion onCTCshave been issues that have hampered its utilityas a PD biomarker. In addition, a lack of concordance inEGFR expression has been observed between CTCs and theprimary tumor and associatedmetastases. For example, of 9patients with EGFR-negative CTCs, 6 had EGFR-positivemetastases, while the available primary tumor specimensfor 3 patients were also EGFR-positive (53).

Procurement of tissue representative of tumor is a par-ticular issue in lung cancer, where accessibility to the pri-mary cancer is often inaccessible. There is thus great interestin the utility of a number of different sources of surrogatetissues including CTCs and lung lavage specimens. Lunglavage and blood samples were collected frompatients withNSCLC and analyzed on the VerIFAST platform (54). Signal

intensity of EGFR expression seemed to be less heteroge-neous among CTCs than the lavage specimens, althoughthis could reflect differences in tumor burden.

IGF-IR expression. We have previously incorporated theevaluation of IGF-IR–positive CTCs as an exploratory end-point in patients treated on a phase I study with the IGF-IRmonoclonal antibody figitumumab (CP-751,871; Pfizer;ref. 41). The CellTracks system was adapted to incorporateantibodies to detect IGF-IR immunofluorescence. The assaywas initially validated using cell lines with known levels ofIGF-IR expression and spiked blood samples from healthyvolunteers, before incorporation in three phase I trials offigitumumab administered as a single agent or with chemo-therapy involving patients with metastatic cancers. Impor-tantly, the diagnostic antibody was shown not to interferewith figitumumab as they target different IGF-IR epitopes.One of the limitations of this assay was the binary classifi-cation of CTCs as either positive or negative for IGF-IR, andthus further work will be required to enable better quanti-fication of IGF-IR immunofluorescence.

AR signaling. Miyamoto and colleagues molecularlycharacterized CTCs that were isolated using microfluidicchip technology and subsequently analyzed by immuno-fluorescence for PSA and prostate-specific membrane

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Figure 3. Examples ofimmunofluorescence images ofCTCs and disseminated tumorcells (DTC) from patients withCRPC. Top two panelsdemonstrate CTCs enriched withthe CellSearch CTC Test; third andfourth panels demonstrate CTCsisolated using the ISET filtrationdevice; bottom, DTCs from a bonemarrow trephine biopsy specimen.ISET, isolation by size of epithelialtumor cells. Images were obtainedusing an automated fluorescencemicroscope scanning systemBioview Duet (Bioview Ltd.).Images courtesy of MateusCrespo, the Institute of CancerResearch (Sutton, Surrey, UnitedKingdom). CD45, leukocytecommon antigen.

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antigen (PSMA). In a retrospective analysis of 12 patientstreated with abiraterone, the presence of >10%CTCs with amixed AR signature (PSA- and PSMA-positive) was associ-ated with a worse overall survival compared with patientswith fewer AR-mixed CTCs (42). However, as compelling asthese results may seem, they are associated with severallimitations, namely the nonvalidated CTC isolation assay,CTC selection based on EpCAM, and the fact that cellviability, which may substantially affect such results, wasnot taken into account. An alternative approach that maybe used is to quantify AR expression directly on CTCsusing different platforms, as shown in Figs. 2 and 3. It ispossible to identify varying AR localization, to the nucle-us when ligand bound and activated, or the cytoplasmwhen inactive, even in CTCs from the same patient withCRPC (Fig. 2). Such assessments may be particularlyrelevant in the assessment of novel targeted agents inCRPC, such as enzalutamide, which reduces the efficiencyof AR nuclear translocation.

Considerations When Incorporating CTCs as PDBiomarkersA framework that may be used for the incorporation of

CTCs into early-phase clinical trials is the PharmacologicalAudit Trail (PhAT) that we have previously described(1, 55). The PhAT links all key stages of drug developmentand assesses the risk of failure in a step-wise approach assequential questions are addressed, permitting key "go, no-go" decisions to be made (Table 2). CTC PD studies couldinitially be incorporated as exploratory endpoints in early-phase clinical trials. This will provide investigators withinvaluable insights into their CTC technologies by applyingthe assay in the clinic, without affecting medical or drugdevelopment processes.Implementing CTCs in early-phase clinical trials as PD

endpoint biomarkers requires that several key elements befulfilled, namely (6):

* CTCs need to be detectable in the patient population;* CTCs need to be collected and processed using ananalytically validated standardized assay, for example, theCellSearch platform and using a uniform definition ofwhat is called a CTC;

* CTC viability needs to be assessed (56–59) and apoptoticand nonapoptotic CTCs need to be differentiated (Fig. 2);

* PD endpoints can either be based on CTC enumeration,expression of drug target or a surrogate on CTCs, or acombination of both factors (60, 61);

* Ideally, the PD endpoint should be demonstrated in bothCTCs and tumor biopsies (60).

Looking to the Future: Promises and Limitationsof CTCsPromises of CTCs as PD biomarkersThe main advantage of using CTCs as PD markers lies in

their potential role as the "leukemic phase" of solid tumors

(62). We should thus be able to use CTCs to study the PDinteractions between a novel antitumor therapeutic and itsintended target directly in representative tumor cells with-out requiring invasive tumor biopsies. In the future, isolat-ing such CTCs through technologic advances in single cellprofiling will allow us to go beyond simple cell enumera-tion and the characterization of proteinmarkers onCTCs tomeasure treatment effects. For example, recent studies haveshown that it is possible to monitor tumor genomes byusing array comparative genomic hybridization (CGH) andnext-generation sequencing (NGS) technologies (63). Astumor cell genomes are prone to change in response toantitumor treatments, we could implement this technologyto measure PD and other effects, which may be especiallyrelevant in the identification of genomicmarkers associatedwith treatment resistance (Fig. 4). Other future applicationsof CTCs may also include the study of PD effects of atreatment ex vivo by generating primary cell cultures fromCTCs, also known as CTC-derived xenografts (CDX), whichmay allow the testing andvalidationofmultiple PDmarkersusing unique patient-derived tumor models.

Limitations of CTCs as PD biomarkersDespite recent advances in the field of CTC isolation

and characterization, it has been suggested that CTCstudies may be limited in their role as PD biomarkers asthey may not always be meaningful representations ofbona fide tumor tissue or more aggressive tumor cells. Forexample, immunomagnetic assays are highly specificmethods but can potentially miss CTCs that do notexpress the target antigen (6). Such tumor types includemelanoma and sarcoma, which do not express epithelialsurface antigens; in such cases, CTC enrichment based onalternative cell surface markers, such as CD146 for mel-anoma, or vimentin for sarcoma have been proposed (64,65). Similarly, aggressive tumor cells undergoing epithe-lial–mesenchymal transition (EMT) are known to lose theexpression of epithelial markers and would therefore notbe detected by an EpCAM antibody-based enrichmenttechnique (66). Therefore, using a CTC isolation methodbased on EpCAM-positive cell enrichment may causesome bias against such tumor cells (67). AlthoughEpCAM-negative CTCs and novel CTCs assays tailoredfor specific tumor types may potentially address theseissues, such novel platforms still required analytic vali-dation and/or clinical qualification (Table 1).

Other novel CTC subpopulations that also need furthercharacterization with novel platforms in the future includesmall CTCs (CKþ, CD45�, intact DAPI), which are mor-phologically similar in size to white blood cells (WBC) andcould be missed by filters and other size/density capturedevices (Fig. 3). Such CTC capture platforms that isolateCTCs based on their size, density, or decreased deform-ability compared with erythrocytes and leukocytes arepotentially fast and economic methods, but have the dis-advantage of having an overlap in physical properties withnontumoral cells in the blood (17).





Until such issues are addressed, clinical studies incorpo-rating the use of CTCs should remain focused on currentvalidated platforms, such as the CellSearch platform, whichis based on EpCAM-positive cells enrichment. The use ofCTCs isolated with such methods to study PD treatmenteffects nevertheless poses several challenges, which willneed to be resolved:

1. Viability of CTCs isolated with current platforms. Avariable and significant proportion of CTCs capturedwith the CellSearch assay has been described asapoptotic, and its proportion has been shown to risewith increasing CTC counts (60, 68). However, it isnot clear whether this is a purely an in vivo

phenomenon or if this is due to the multipleprocessing steps undertaken during CTC enrichmentand isolation with the CellSearch platform.Regardless, a comparison of the latter technology withother isolation methods to formally clarify the role ofsample processing in increasing the proportion ofapoptotic CTCs needs to be undertaken.

2. CTC heterogeneity. The heterogeneity of epithelialmalignancies is now well established and CTCs arelikely to represent a subset of cells derived fromheterogeneous primary tumor cells that survive in thecirculation (69–71). The clonal heterogeneity of CTCshas been confirmed at the genomic level through theobservation of a substantial variability in

Table 2. Incorporation of CTCs within the PhAT

PhAT Example CRPC Critical questions

Population identifier Patients with CRPC,post-docetaxel setting

CTC detection* Is CTC enumeration possible?* How many patients will have no CTCsdetectable (e.g., extensive visceral disease?)

* Proportion of apoptotic CTCs?Targeted drug candidate Novel AR-degrading drug

Validated assay for molecularaberration

AR antibody for carboxy- andamino-terminal domains

Drug Target in CTC* Is the drug target expressed in CTCs* Does the assay work in CTC?* How does CTC assay compare with tumorbiopsy assay?

Pharmacokinetics

Pharmacodynamics

Biochemical pathway modulation

Whole blood and plasma PK studies

CTCs, tumor, PRP, hair follicles

Decline in PSA levels

CTC enumeration* Reduction of AR and AR-sv in CTCs?* How does effect in CTC compare with tumorand normal tissue?

Achievement of biologic effect PSA responses by PCWG2Objective soft tissue responsesRadiographic PFS

CTC conversion* Percentage of AR-negative CTCs posttreatment?* Duration of CTC response?

Hypothesis testing usingintermediate endpoints of clinicalresponse

CTC and LDH biomarker panel * Duration of CTC response?* Correlation of CTCs with PSA

Reassessment of molecularalteration at disease progression

AR-dependent or AR-independent(e.g., PI3K and AKT) progression

* Re-appearance or rise in AR-positive or-negative CTC at disease progression

Inhibition of resistant biologicpathway

Reversal of resistance: intrapatientdose escalation, inhibition ofescape pathway

Assay for escape pathway in CTCs?* CTC enumeration?* Characterization of PD effects on CTCs afternew strategy implemented?

NOTE: Left column, the PhAT is a conceptualized framework for successful early drug development (1). Middle column, hypotheticalexample of an AR-degrading compound and its development in CRPC. Right column, potential applications of CTCs in the PhAT usingCTCs as PD biomarkers and early-response biomarker assessments.Abbreviations: AR-sv, AR splice variants; PI3K, phosphoinositide 3-kinase.

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chromosomal abnormalities (68, 72). However, insome cases, such genomic abnormalities may actuallybe homogeneous as in the case of ERG rearrangementsin CTCs from advanced prostate cancer, suggesting apotentially common clonal origin of CTCs andmetastatic disease in ERG-rearranged prostate cancers(60). Phenotypical pleomorphism in CTCs has alsobeen described, and good examples are certain cell-surface markers such as IGF-IR, EGFR, or HER2expression, or intracellular markers such as AR orphosphorylated Histone-H3 with positive andnegative cells coexisting in the same patient(41, 42, 52, 73, 74).

3. Biologic differences between CTCs and solid tumor lesions.In some cases, CTCs may differ in their phenotypebetween primary and metastatic tumors, such as withHER2-positive CTCs in HER2-negative primary andmetastatic breast cancer (75). In addition, there arepotential limitations with the interpretation of theactual impact of antitumor therapies on CTCs versussolid tumor lesions. This may be due to thecontamination of CTCs with another cell populationbecause of drug-related active mobilization oftumor cells to the blood or the passive shed of cellsfrom the tumor surface. There is also higher exposureof the blood compartment to a systemically

CCR Focus

Treatment A

Phenotypic and genomic profiling

CTC-derived xenografts

Patient oncologic treatment journeyTreatment A Treatment A + B Treatment C

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Biologic studiesBiomarker discoveryPerpetual bank

Figure 4. Envisioning the future utility of CTCs in modern oncologic treatment pathways. As CTCmethods and technologies for the characterization of singletumor cells continue to improve, we envision the incorporation of CTCs into clinical studies for the identification and monitoring of tumor heterogeneity,clonal evolution, and treatment selection in individual patients. In the future, it may become possible to treat an individual patient rationally withsequential targeted therapy regimens based on the identification of evolving tumor characteristics, as extrapolated from CTCs, which may be isolated andstudied at multiple time points during the patient's treatment journey. As illustrated by Treatment A in the figure, blood samples may be collected atbaseline for phenotype and genomic profiling studies to dissect tumor heterogeneity, so as to select themost relevant target(s) across all the clones for initialanticancer treatment. Such studies may include the analyses of the tumor mutational landscape, such as NGS technologies, whole genome copy numbervariation studies using array CGH with validation by FISH or quantitative PCR (qPCR), and/or gene expression profiling. To select the most appropriatetreatments, data from these studies will be interrogated with modern bioinformatic tools and compared with accessible cancer genome databases linked totreatment effects and patient outcomes. So as to guide subsequent treatment decisions, blood samples may be drawn again at both early and late timepoints to study antitumor effects, as well as the development of early and late mechanisms of resistance in different tumor clones. Furthermore,coclinical studies with CDX models may be developed by engrafting CTCs in immunosuppressed mouse in parallel to the investigations described above.Following engraftment and an initial growth of the CTC graft, tumors may then potentially be expanded to develop sufficient tumor mass for biobanking,comparative molecular characterization of the CDX with the primary tumor, and for the assessment of a range of treatments to identify mechanismsof resistance to guide future treatment regimens. Adapted from Tentler et al. (121) by permission of Macmillan Publishers Ltd. Access themost recent versionof this article at: http://www.nature.com/nrclinonc/index.html.





administered drug, in contrast to the solid tumorcomponent.

As discussed, CTCs may be reliably detected in themajority of patients with metastatic prostate (41), breast(7), and colorectal cancers (8) using the FDA-approvedCellSearch technology. CTCs have also been detected inpatients with other tumor types, such as small cell lungcancer (76). However, only about 10% of patients withNSCLC have of�5 CTC/7.5 mL for enumeration, limitingthe wider application of CTCs in this patient population(77). Gainor and colleagues (78) discuss such issuesrelating to lung cancer in their article in this in this CCRFocus section.

In the future, another application of CTC technologymay emerge in the field of minimal residual disease diag-nostics after patients have completed curative local treat-ment. For example, in breast cancer, a clinical trial has beeninitiated to investigate the addition of trastuzumab inpatientswithdetectableCTCcounts after adjuvant treatment(NCT01548677).Nevertheless, it remains unclear if residualCTCs have the same phenotype as the primary tumor and ifCTCs represent a primary resistant clone, or if theseCTCs aretruly independent of the targeted pathway (79). Also, atearlier disease stages, CTCs are often absent or only presentat very low frequencies using enrichment anddetection toolscurrently available; sensitivity will thus need to be signifi-cantly improved and platforms appropriately validatedbeforeCTCtechnologycanbeapplied insuchscenarios(80).

Clinical trial designs that incorporate CTCs as PD bio-markers should bear inmind that large numbers of patientsmay be necessary to draw definitive conclusions from suchstudies, which may potentially be hampered by cell hetero-geneity due to the attrition of patients without CTCs or lowviable CTCs counts. Other important limiting factors withthe inclusion of CTCs as PD biomarkers are the significantcosts involved with CTC kits, operator time and complex-ities of CTC capture and evaluation. Nevertheless, in thefuture, it is likely that as CTC platforms become moresensitive and economical, CTC evaluation will increasinglybecome an integral part of translational clinical studies andpatient management.

Other BiomarkersIn addition to CTCs, other blood components are also

excellent sources of information about the cancer and hostthat may be used as potential biomarkers, including PDendpoints. For example, platelet-rich plasmahas been incor-porated successfully in early-phase clinical trials for PDstudies involving selective signaling inhibitors (81). Otherexamples include the isolation of circulating nucleic acidssuch as circulating tumorDNA (ctDNA), as discussed by FiggandNewell in thisCCRFocus edition (82),which is increasedin patients with advanced cancers compare with healthyindividuals (83), micro-RNA or the characterization of geneexpression changes that nonmalignant blood cells undergoin response to micro- and macro-environmental changesinduced by the tumor (84, 85). Recently, two gene expres-

sion signatures with prognostic utility, linked to the inflam-matory and immune response, were developed and validat-ed in CRPC (86, 87). Depending on future testing, thesesignatures may even potentially hold some utility as PD andpredictive biomarkers for immunotherapy treatments. Apartfrom blood-borne biomarkers, van der Veldt and Lam-mertsma (88) discuss the use of in vivo imaging of taxanesas PD biomarkers in this of CCR Focus section. In addition,Hertz andMcLeod(89)alsodiscuss theuseofpharmacogenepanels to detect germline SNP biomarkers, while Low andcolleagues (90) detail genome-wide association studies(GWAS) as a tool to identify common genetic variantsassociated with drug toxicity and efficacy in cancer pharma-cogenomics in this CCR Focus section.

ConclusionsIt is envisioned that the future use of CTCs as PD bio-

markers will not simply be confined to enumeration, butalso include their routine molecular characterization inearly-phase clinical trials. Overall, the assessment of CTC-based PD biomarkers has potential for rapidly demonstrat-ing proof-of-mechanismduring the clinical development ofmolecularly targeted anticancer therapeutics in "real-time."However, clinical trials using CTCs as PD endpoints clearlydemonstrate that the interpretation of data across multiplestudies using different CTC isolation and molecular char-acterization technologies comes with numerous challenges(37, 91). Prospective studies with uniform and standard-ized definitions of CTCs are thus urgently needed. Suchstudies should exploit the full potential of CTCs not just asPD biomarkers, but also as prognostic, predictive, andintermediate endpoint markers.

Authors' ContributionsConception and design: T.A. Yap, D. Lorente, A. Omlin, D. Olmos, J.S. deBonoDevelopment of methodology: T.A. Yap, D. Lorente, J.S. de BonoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T.A. Yap, D. Lorente, J.S. de BonoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): T.A. Yap, D. Lorente, J.S. de BonoWriting, review, and/or revision of themanuscript: T.A. Yap, D. Lorente,A. Omlin, D. Olmos, J.S. de BonoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T.A. Yap, D. LorenteStudy supervision: T.A. Yap, D. Lorente

Grant SupportThe Drug Development Unit of the Royal Marsden NHS Foundation Trust

and the Institute of Cancer Research are supported in part by a program grantfrom Cancer Research UK. Support was also provided by the ExperimentalCancer Medicine Centre (to the Institute of Cancer Research) and the NationalInstitute forHealthResearch (NIHR)Biomedical ResearchCentre (jointly to theRoyal Marsden NHS Foundation Trust and the Institute of Cancer Research).T.A. Yap is recipient of the 2011 Rebecca and Nathan Milikowsky—ProstateCancer Foundation (PCF) Young Investigator Award and is supported by theNIHR.D.Lorente issupportedbytheSpanishMedicalOncologySociety throughBECA SEOM para la Investigacion Traslacional en el Extranjero. D. Olmos is arecipient of a2014Stewart Rhar-ProstateCancer FoundationYoung InvestigatorAward and is supported by Fundaci�on Cientifica de la Asociaci�on Espa~nolacontra el C�ancer (AECC) and Fundaci�on CRIS contra el C�ancer.

Received December 27, 2013; revised March 8, 2014; accepted March 20,2014; published online May 15, 2014.

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