Personalized Medicine and Imaging

Enumeration and Molecular Characterization ofTumor Cells in Lung Cancer Patients Using aNovel In Vivo Device for Capturing CirculatingTumor CellsTobias M. Gorges1, Nicole Penkalla2,Thomas Schalk2, Simon A. Joosse1, Sabine Riethdorf1,Johannes Tucholski3, Klaus L€ucke3, Harriet Wikman1, Stephen Jackson4, Nora Brychta5,Oliver von Ahsen5, Christian Schumann2,6, Thomas Krahn5, and Klaus Pantel1

Abstract

Purpose: The use of circulating tumor cells (CTC) as "liquidbiopsy" is limited by the very low yield of CTCs available forsubsequent analyses. Most in vitro approaches rely on smallsample volumes (5–10 mL).

Experimental Design: Here, we used a novel approach, theGILUPI CellCollector, which enables an in vivo isolation of CTCsfrom peripheral blood. In total, 50 lung cancer patients werescreened in two subsequent device applications before and aftertherapy (n ¼ 185 applications).

Results: By in vivo isolation, 58% (108/185) of the patientswere positive for �1 CTC (median, 5 CTCs; range, 1–56 cells) as

compared with 27% (23/84; range, 1–300 cells) using the FDA-cleared CellSearch system. Furthermore, we could show thattreatment response during therapywas associatedwith significantdecreases in CTC counts (P ¼ 0.001). By dPCR, mutations in theKRAS and EGFR genes relevant for treatment decisions could bedetected in CTCs captured by in vivo isolation and confirmed inthe primary tumors of the same patients.

Conclusions: In vivo isolation of CTCs overcomes bloodvolume limitations of other approaches, which might help toimplement CTC-based "liquid biopsies" into clinical decisionmaking. Clin Cancer Res; 22(9); 2197–206. �2015 AACR.

IntroductionAlthough tumor cells detached from primary tumors or metas-

tases into the blood (circulating tumor cells; CTC) have beendiscovered almost 150 years ago, specific detection andmolecularexamination have only recently gained heightened attention. Thismay be explained by the fact that technology improvements haveonly recently become available to enable more consistent CTCisolation. However, despite encouraging research progress, it isstill a tremendous challenge to enrich sufficient amounts of CTCsfrom peripheral blood of cancer patients (1, 2). Detection and

molecular characterization of CTCs is challenging because CTCsare rare events, with a frequency of approximately 1 to 10 CTCs inthe background of 6 � 106 leukocytes, 2 � 108 platelets and 4 �109 erythrocytes per mL of patient-derived blood sample (3).Thus, despite promising results demonstrating that enumerationand characterization of CTCs might provide important informa-tionon the individual risk of cancer patients, there is still anurgentdemand to increase the yield of capturing CTCs.

Lung cancer is one of themost frequently occurring and deadlydiseases (4). A sensitive CTC isolation method holds great prom-ise for early detection of minimal residual disease (MRD). Fur-thermore, CTCs are being evaluated as predictive biomarkers toguide individual cancer treatment strategies (personalized med-icine). Hence, sensitive isolation and profound molecular char-acterization of CTCs could serve as a "liquid biopsy" and facilitateimproved individual treatment regimens for cancer patients. Sofar, several reports have shown that CTC counts are much higherin small cell lung cancer (SCLC) than in non–small cell lungcancer (NSCLC; refs. 4, 5). Using the FDA-cleared CellSearch, CTCvalues in NSCLC seem to be even lower than in other cancerentities (4, 6, 7), pointing to the need for improvedCTC detectionin NSCLC. Moreover, liquid biopsies would be particularly desir-able in NSCLC, where an emerging number of new moleculartargets have been recently discovered that would benefit fromCTC-based patient selection (8).

To date, more than 40 different approaches for CTC detectionhave been developed (9, 10). Various approaches have also beenused to increase the yield of CTCs in NSCLC patient bloodsamples (8); to our best knowledge, none of these assays havebeen sufficiently tested to support FDA approval. In the current

1Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg,Germany. 2Department of Pneumology, Univer-sity Clinic Ulm, Ulm, Germany. 3GILUPI GmbH, Potsdam/Greifswald,Germany. 4Thermo Fisher Scientific, South San Francisco, California.5Bayer Pharma AG, Berlin, Germany. 6Clinic for Pneumology,ThoracicOncology, Sleep, and Respiratory Critical Care, Klinikverbund Kemp-ten-Oberallgaeu, Kempten and Immenstadt, Germany.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

T.M. Gorges, N. Penkalla, and T. Schalk share first authorship.

C. Schumann, T. Krahn, and K. Pantel share last authorship.

Corresponding Author: Klaus Pantel, University Medical Center Hamburg-Eppendorf, Department of Tumor Biology, Center of Experimental Medicine,Martinistrasse 52, 20246 Hamburg, Germany. Phone: 49-40-7410-53503; Fax:49-40-7410-55379; E-mail: pantel@uke.de

doi: 10.1158/1078-0432.CCR-15-1416

�2015 American Association for Cancer Research.

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study, we validated a new strategy. The CellCollector (GILUPICellCollector, GILUPI) enables an in vivo isolation of CTCsdirectly from the arm vein of cancer patients. During the 30-minute incubation in the vein, the device is exposed toapproximately 1 L of blood, which increases the chance tocapture CTCs. Our present investigation includes (i) the anal-ysis of a large cohort of lung cancer patients to assess thedistribution of CTCs, (ii) consecutive applications of the CTCdevice to demonstrate reproducibility of blood sampling forCTCs (which has rarely been shown in any of the >16,000 CTCpublications in PubMed), (iii) monitoring of lung cancerpatients during treatment for clinical response correlated toCTC counts, (iv) comparison of CellCollector (catheter-based)results to the FDA-cleared CellSearch system (blood sample-based), and (v) molecular characterization of CTCs capturedby the medical device for mutations relevant to cancer therapyand comparison to the primary tumor of the same patients.This in-depth validation will support the use of in vivo CTCcapture as a new diagnostic principle for future clinical trialstesting the relevance of "liquid biopsies" to direct cancertherapies.

Materials and MethodsIn vitro application of the CellCollector

The GILUPI CellCollector (termed as catheter-based or wire-based enrichment of EpCAM-positive cells) is based on a sterilestainless steel medical wire, covered with 2-mm gold and ahydrogel layer at its functionalized tip. This hydrogel layer iscovalently coupled with antibodies against the EpCAM protein(humanized HEA 125, GILUPI GmbH; Fig. 1A). For our in vitrostudies, the wire was incubated together with unauthenticatedlung cancer cells (NCI-H332, NCI-H3122, NCI-H1650, 32M1,NCI-H1975, and A549) spiked into blood (EDTA) from healthyindividuals for 30 minutes on a rotator. After the incubation, thewire was washed twice and fixed with acetone (10 minutes,room temperature). Captured cells were permeabilized (0.1%Triton X-100/PBS, 10 minutes), blocked (3% BSA/PBS, 30 min-utes), and stained against EpCAM (Acris, clone HEA125-FITC)and pan-keratins (Exbio, clone C11-Alexa488; Millipore, cloneLP5K-FITC; Exbio, clone A53-B/A2-Alexa488). Pan-keratinswere stained to improve the detection rate of clinically relevant

CTCs (11). CD45 staining was performed to exclude unspecificleucocytes (Exbio, clone MEM-28-Alexa647). Nuclear counter-staining was done using Hoechst33342 (Invitrogen) at a con-centration of 1 mg/mL for 2 minutes. Next, the medical wire wasplaced into a special holder and subsequently screened for CTCsunder the microscope (Fig. 5A). Images were obtained using theAxio Imager A1 microscope equipped with an AxioCam digitalcamera and the AxioVision 4.6 software (Zeiss). A tumor cell wascounted as positive when its morphological features (�4 mm,intact nucleus) and staining patterns were consistent with thoseof an epithelial cell (Hoechst33342-/pan-keratin- and/orEpCAM-positive/CD45-negative). The cell line A549 was pur-chased from CLS (Cat. No. 300114) and cultured in 45%Dulbecco's MEM (Biochrom; Cat. No. F0425) þ 45% HAM'sF-12 Medium (Biochrom; Cat. No. FG0815) þ 10% FBS (Bio-chrom; Cat. No. S0615) þ 2 mmol/L L-Glutamin (Biochrom;Cat. No. K0282) at 37�C with a 5% CO2 atmosphere. The cellline NCI-H1975 was purchased from the ATCC (Cat. No. CRL-5908) and cultured in 90% RPMI-1640 (Biochrom; Cat. No.FG1235) þ 10% FBS (Biochrom; Cat. No. S0615) at 37�C with a5% CO2 atmosphere. Cell line cells (NCI-H1650; Cat. No. CRL-5883 and NCI-H322; Cat. No. CRL-5806) were bought fromATCC. The cell line NCI-H3122 was kindly provided by C. Costa,PhD (Pangaea Biotech), and Dr. E. Weber (University of Halle)kindly provided the cell line 32M1. The cell lines NCI-H1650,NCI-H322, NCI-H3122, and 32M1 were cultured in 90% RPMI-1640 (Biochrom; Cat. No. FG1235) þ 10% FBS (Biochrom; Cat.No. S0615) at 37�C with a 5% CO2 atmosphere. Initially to theexperiments, all cell lines were tested negative for Mycoplasmausing the Venor GeM Mycoplasma Detection Kit (MinervaBiolabs; Cat. No. 11-1100).

Study design and clinical informationThis prospective, blinded, single-center clinical study (in accor-

dance with article 23b MDA) investigated the isolation andmolecular characterization of CTCs from the peripheral bloodof lung cancer patients using the CE-certified medical wire. Thestudy was conducted at the University Medical Center Ulm andapproved by the Ethics Committee of the University of Ulm in2012. The study included 50 patients with newly diagnosed andlocally advanced or metastatic lung cancer. The most predomi-nant histology was NSCLC (n ¼ 48, 96%), among them theadenocarcinoma subtype (n ¼ 30, 60%). All patients were ableto receive systemic anticancer treatment according current recom-mendations (12). A detailed summary of all patient data, includ-ing the CTC counts, is disclosed in Table 1 and SupplementaryTable S1.

In vivo application of the wirePrior to the use of the device, a 20G peripheral venous catheter

was placed into the median cubital vein of the patient. The wirewas carefully inserted into the vein through the catheter until thetip of the device was extended 2 cm into the vein. To assess thereproducibility of measurements with the medical wire, twosubsequent applications were performed during the same day(visit) for every cancer patient participating into the study.Patients underwent two subsequent device applications bothbefore and after 12weeks of therapy. Fourteenpatients underwentonly the pretherapy but not the following applications. Exceedingthe initial studydesign, 7 patients had an additional third visit andone of them a fourth visit, each visit with double applications.

Translational Relevance

The CellCollector used in this study enables an in vivoisolation of circulating tumor cells (CTC) directly from thearm vein of cancer patients. In this study, 50 lung cancerpatients were screened in two subsequent device applicationsbefore and after therapy (n ¼ 185 applications). This devicecaptured CTCs from lung cancer patientsmore frequently thanthe FDA-cleared CellSearch system. Our findings indicate thatin vivo isolation of CTCs is a promising approach to isolateCTCs in sufficient amounts for treatment monitoring andfurther downstream molecular analyses in lung cancerpatients. This new approach can now be tested in interven-tional clinical studies where the number and/or characteristicsof CTCswill be used to stratify patients to particular treatmentsand to assess established endpoints, including survival.

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This led to 16 additional device applications and a total of 185analyzable applications. After the in vivo application, the wire wastreated as described above. Cells captured by the device werestained and counted by different operators who were blinded tothe patient's history and the CellSearch results.

CellSearch analysisFor method comparison, 7.5 mL of venous blood were col-

lected into CellSave tubes immediately before the application ofthe wire. Blood was sent to the University Medical Center Ham-burg-Eppendorf and processed within 96 hours as recommendedby Janssen Diagnostics. The operator was blinded to the catheter-based results.

Statistical analysisClinical factors (T, N, M, and UICC stage) were tested for

associationwith CTC count bymultivariate analysis using ordinallogistic regression. Paired analyses between replicate measure-ments were tested for (i) difference in median using Wilcoxonsigned rank test, (ii) difference for negative (0 CTCs) or positive(�1 CTC) using Liddell's exact test, and (iii) correlation betweenordinal values ofCTCsusingKendall's tau. Thedifference betweenmedian number of CTCs as detected by the wire and CellSearchwas assessed by theWilcoxon signed rank test, and the agreementand correlation between the results of the two systems wasassessed using Cohen's kappa and Kendall's tau, respectively. Thedifferences in CTC count (visit 2� visit 1) for catheter-based and

Figure 1.A, schematic overview of the wire and the in vivo application. The wire is coated with anti-EpCAM antibodies and placed into the arm vein of cancer patients for 30minutes. During the application EpCAM-positive cells bind to the device. Afterward, the tumor cells are stained for EpCAM and keratins. Nuclear counterstainis done using Hoechst33342. CD45 staining is necessary to classify false positive events (leukocytes). B, schematic overview of the study design. Theschedule of the study was designed with the first visit directly before therapy initiation and the second visit 12 weeks after treatment started. Patients had theopportunity to have additional CTC investigations at a third and fourth visit (always after a 12-week period in between). Patients underwent the same CTCinvestigation procedures at each visit (7.5 mL blood draw for CellSearch analysis and two wire applications).

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blood sample–based system results were tested for associationwith therapy response using the Kruskal–Wallis test.

Mutational analysis of tumor cells captured by in vivo isolationCTC-positive wires from2patients (fromdifferent applications

and visits) with known mutational status of the primary tumorwere used for further molecular characterization of the CTCs bychip-based digital PCR (dPCR). Briefly, the wire was fragmentedinto small pieces into a 0.2 mL PCR-tube filled with 4.8 mL PBS(Qiagen). Whole genome amplification (WGA) was performed

using the REPLI-g Single Cell Kit (Qiagen) using a 1.2� reactionvolume. Amplified gDNA was quantified with the QuantiFlourONE dsDNA System (Promega) and a fluorescence reader (Fluor-oskanAscent; Thermo Fisher) at 485 nmEx/538 nmEm. For chip-based dPCR the ThermoFisher QuantStudio 3D system was used.Fifty nanogramsofWGADNA(2mL)was combinedwith 7.5mLof2 � dPCR Mastermix and 0.75 mL of 20 � SNP assay for theappropriate allele. The total volume was brought to 15 mL withwater, applied to the chip and sealed according to the manufac-turer's instructions. The chip was placed on a flat-block PCR

Table 1. Patient characteristics

StagingCTC count: CellCollector

A-1/A-2 (sum)CTC count:CellSearch

Patient Histology T N M UI CC V1 V2 V1 V2 Treatment response

001 Squamous cell carcinoma x 3 1b IV 2/0 (2) 0/3 (3) 4 0 SD002 Adenocarcinoma 2b 1 1b IV 0/0 (0) 0/0 (0) n.a. n.a. n.a.003 SCLC 2 3 1b IV 3/3 (6) 6/2 (8) 3 0 SD004 Squamous cell carcinoma 4 2 1a IV 10/0 (10) 0/0 (0) 2 n.a. n.a.005 Adenocarcinoma 4 3 0 IIIB 3/0 (3) 0/0 (0) 0 n.a. n.a.006 Adenocarcinoma 3 2 0 IIIA 1/6 (7) 4/9 (13) 0 0 PR007 Adenocarcinoma 1b 3 1b IV 1/0 (1) 45/7 (52) 0 n.a. SD008 Large cell carcinoma 4 2 1b IV 0/5 (5) 27/6 (33) 300 2 PD009 Adenocarcinoma 3 2 1b IV 1/0 (1) 5/5 (10) 0 0 PD010 Adenocarcinoma 4 3 1b IV 0/0 (0) 1/11 (12) 0 0 SD011 Squamous cell carcinoma 2 3 1b IV 0/0 (0) 11/6 (17) 0 0 SD012 Adenocarcinoma 2a 0 1b IV 0/0 (0) 2/14 (16) 2 0 SD013 Adenocarcinoma 4 3 1b IV 3/5 (8) 1/1 (2) 8 24 PD014 Adenocarcinoma 4 3 1b IV 3/11 (14) 8/18 (26) 0 0 SD015 Squamous cell carcinoma 3 2 0 IIIA 0/2 (2) 4/8 (12) 1 0 SD016 Adenocarcinoma 4 3 1b IV 42/29 (71) 0/0 (0) 0 n.a. PR017 Squamous cell carcinoma 4 3 1b IV 0/0 (0) 0/0 (0) 0 n.a. n.a.018 Squamous cell carcinoma 4 1 0 IIIA 5/13 (18) 0/0 (0) 0 0 n.a.019 Squamous cell carcinoma 4 3 0 IIIB 6/25 (31) 0/6 (6) n.a. 0 PR020 Adenocarcinoma 4 3 1b IV 26/24 (50) 0/0 (0) 1 n.a. SD021 Adenocarcinoma 4 2 0 IIIB 10/46 (56) 5/8 (13) 0 0 PR022 Adenocarcinoma 4 3 1b IV 20/22 (42) 0/0 (0) 2 n.a. SD023 Squamous cell carcinoma 3 3 1b IV 35/13 (48) 0/3 (3) 0 0 CR024 Adenocarcinoma x x 1b IV 41/56 (97) 0/0 (0) 4 n.a. n.a.025 Adenocarcinoma 4 2 1a IV 17/1 (18) 1/0 (1) 0 0 PR026 Adenocarcinoma 4 3 1b IV 2/0 (2) 0/0 (0) 0 0 SD027 Adenocarcinoma 2a 3 1b IV 2/1 (3) 0/0 (0) 1 0 PD028 Adenocarcinoma 3 2 1b IV 0/4 (4) 0/0 (0) 0 n.a. n.a.029 Adenocarcinoma 4 3 1b IV 1/0 (1) 0/0 (0) 3 n.a. n.a.030 Squamous cell carcinoma 4 2 1b IV 2/1 (3) 0/0 (0) 0 0 SD031 Adenocarcinoma 2 2 1b IV 14/4 (18) 0/0 (0) 0 n.a. n.a.032 Squamous cell carcinoma 3 2 1b IV 6/0 (6) 0/0 (0) 0 0 SD033 Squamous cell carcinoma 4 2 0 IIIB 6/6 (12) 3/4 (7) 0 1 SD034 Adenocarcinoma 4 2 0 IIIB 0/30 (30) 0/0 (0) 0 0 SD035 Adenocarcinoma 4 3 1b IV 1/2 (3) 0/0 (0) 0 1 SD036 Not otherwise specified 4 3 1b IV 1/6 (7) 0/0 (0) 0 n.a. n.a.037 Squamous cell carcinoma 4 3 0 IIIB 0/2 (2) 0/0 (0) 0 0 SD038 Adenocarcinoma 4 3 1b IV 0/0 (0) 0/0 (0) 0 n.a. n.a.039 Adenocarcinoma 4 3 1a IV 0/0 (0) 6/0 (6) n.a. 0 PR040 Squamous cell carcinoma 3 2 0 IIIB 0/0 (0) 0/0 (0) 0 0 SD041 Adenocarcinoma 1a 2 1b IV 2/0 (2) 0/0 (0) 4 n.a. n.a.042 Adenocarcinoma 4 3 1b IV 2/2 (4) 1/3 (4) 0 0 SD043 Not otherwise specified 3 3 1b IV 0/0 (0) 2/0 (2) 1 0 SD044 Squamous cell carcinoma 3 2 1b IV 0/0 (0) 0/0 (0) 1 0 PD045 SCLC 3 2 n.a. III 1/8 (9) 0/0 (0) n.a. n.a. n.a.046 Adenocarcinoma 4 2 1a IV 0/8 (8) n.a./0 (0) 2 1 SD047 Adenocarcinoma 2 3 1b IV 0/1 (1) 0/12 (12) 8 0 SD048 Adenocarcinoma 3 2 1a IV 0/0 (0) 1/2 (3) 0 0 PR049 Adenocarcinoma 4 2 1a IV 16/0 (16) 1/1 (2) 0 0 SD050 Squamous cell carcinoma 4 2 1a IV 0/29 (29) 0/4 (4) n.a. 0 SD

NOTE: Patient data and CTC counts from individual patients per visit (A-1/A-2¼wire application 1 or 2, V¼ visit, SD ¼ stable disease, PR¼ partial remission, PD¼progressive disease, n.a. ¼ not analyzed).

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machine and cycled: 45�C 1:00minute, 98�C2:30minutes (95�C1:00 minute, 53.5�C 0:1 minute with 30% ramp, 57.5�C 0:30minutes) for 40 cycles, and held at either 10�C or 20�C untilread. Chips were read on the QuantStudio 3D reader andanalyzed by QuantStudio 3D AnalysisSuite and MicrosoftExcel. SNP assays were designed using the ThermoFisher Cus-tom SNP design pipeline (Life Technologies) without modifi-cations. PBMCs (1,000) from a healthy donor subjected to thesame WGA protocol were used as a negative control to provethe specificity of the assay.

Mutational analysis of the primary tumorMutational analysis of the primary tumor tissue was performed

in the institute of pathology at theUniversityMedical CenterUlm.EGFR (exons 18, 19, 20, and 21) mutational analysis was donewith Sanger sequencing with a lower limit of 10%mutated DNA.KRAS (exons 2, 3, and 4) mutational analysis was done with pyrosequencing with a lower limit of 5% mutated DNA.

ResultsStudy design and patient characteristics

Fifty patients with newly diagnosed and locally advanced ormetastatic lung cancer (stage III, n ¼ 11; stage IV, n ¼ 39) with aperformance status of ECOG 0 or 1 and who were able to receivea systemic antitumor treatment (platinum-based doublet)according to current clinical guidelines (12) were enrolled intothis study. Tumor histology included squamous cell carcinoma(n¼ 15), adenocarcinoma (n¼ 30), small cell carcinoma (n¼ 2),and large cell carcinoma (n ¼ 1), as well as 2 carcinoma patientswith unknown histology (Table 1). Staging for distant overtmetastases was available from 49 patients; 39 (78%) patientshad distant overt metastases (stage M1), whereas 10 patients(20%) had no clinical or radiological signs of overt metastases(stage M0). Local tumor extension (T stage) ranged from T1 to T4with the majority of patients showing large T3 or T4 tumors(80%). No significant differences were seen with regard to CTCnumbers and clinicopathologic parameters such as TNMor UICCstage (Supplementary Table S2).

A schematic overview of the wire during in vivo application ispresented in Fig. 1A. In total, 185 in vivo applications wereperformed and screened for CTCs (Fig. 1B). Briefly, the wire—coated with anti-EpCAM antibodies—was placed into the armvein of cancer patients for 30minuteswhere EpCAM-positive cellscan bind to the device. Afterward, the wire was placed into aspecial holder under the microscope and tumor cells were iden-tified based on epithelial markers (EpCAM- and/or pan-keratin-positive) and nuclear counterstain (Hoechst33342-positive) byfluorescence microscopy. CD45 staining is necessary to discrim-inate possible leukocytes bound to the wire.

CTC counts and correlations to clinicopathologiccharacteristics

In total, 58% (108/185) of the analyzed wires were positive for�1 CTC (range, 1–56 cells; Fig. 2A). Most of the identified cellswere single CTCs. However, in 20 of 185 analyzed wires CTCclusters (range, 3–7 cells) could also be detected. Representativeimage galleries of tumor cells from spiking experiments, CTCs,CTC clusters, and leukocytes (range, 0–�1,000 per wire) aredisclosed in Fig. 2B and C. No "CTC-like" events were found onthe wires placed into the arm vein of healthy individuals (n¼ 10;data not shown).

For assessing clinical correlation to CTC counts, the sum of theCTC counts of the double applications were used in our multi-variate analysis. First, an ordinal scale with four categories for CTCvalues was created (negative, 0 CTCs; low, 1–4 CTCs; intermedi-ate; 5–17 CTCs, high, �18 CTCs) such that every category hadapproximately the same number of cases. The resulting regressionrevealed that none of the clinical factors (T,N, orM stage aswell asUICC) was significantly correlated with the CTC categories,although the tumor stage was close to significant (P ¼ 0.09).

Reproducibility of wire applicationsTo assess the reproducibility of measurements with the wire,

the results of the two subsequent CTC analyses performed at eachvisit from each cancer patient (Table 1) were compared andstatistically evaluated for potential differences.

At the first (pre-therapeutic) visit, CTC counts of �1 weredetected in 78% of the study participants (combined analysis).As expected, the paired analyses resulted in some differencesin CTC counts; however, these differences were not significant(P ¼ 0.33, Wilcoxon signed rank test). Furthermore, the numberof CTC-positive (�1 CTCs) and -negative (0 CTCs) cases was alsonot significantly different between the two analyses (P ¼ 0.59,relative risk 95% CI, 0.35–2.84, Liddell exact test). And finally, apositive and significant correlation could be detected betweenCTC values of the paired analyses of the first visit (P ¼ 0.003,t ¼ 0.36, Kendall rank correlation).

At the second visit, 60% (20/33) and 56% (20/36) of thepatients were identified to be CTC positive with repeated wireanalyses (combined positivity rate 72%). Comparing the resultsof the two analyses, no significant difference between the numberof identified tumor cells could be detected (P ¼ 0.42, Wilcoxonsigned rank test). Furthermore, the number of CTC negative andpositive patients was comparable (P ¼ 0.64, Liddell's exact test).Finally, the correlation between the number of CTCs detectedwith the two analyses was also significantly correlated (P ¼0.0015, t ¼ 0.50, Kendall rank correlation).

Performing the same statistical analyses on the two wire anal-yses of visit 3, the number of CTCs and the number of CTC-positive and -negative patients were not significantly different (P¼ 0.88, Wilcoxon signed rank test; P ¼ 0.25, Liddell exact test);however, the correlation between the CTC values was not signif-icant anymore because almost all cases were CTC negative (P ¼0.59, t ¼ 0.27, Kendall rank correlation). Visit 4 was excludedfrom this analysis because only one case was analyzed.

Comparison of CTC counts obtained with the wire andCellSearch

For a direct comparison of the two CTC assays, 84 analyzableblood samples (7.5mL) were taken prior to the application of thewire and screened ex vivo for CTCs by the FDA-cleared CellSearchsystem. As shown in Fig. 3A, the incidence of CTC-positive lungcancer patients was higher if determined by the wire comparedwith the FDA-cleared technology. Direct comparison of pairedpatient data showed that the wire isolated a median of 3 CTCsmore per patient (P < 0.0001, Wilcoxon signed rank test). Only in11 measurements were more CTCs found with the CellSearchsystem than with the wire.

Taken together, these results indicated that in vivo isolation hasa higher sensitivity in capturing CTCs from lung cancer patients. Itshould be noted that there was no correlation betweenthe positive and negative cases as determined between in vivo

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isolation and CellSearch results (P ¼ 0.76, k ¼ 0.0598, Cohen'skappa) or between the ordinal CTC values (P ¼ 0.99, t ¼ 0,Kendall rank correlation). A direct comparison (weighted scatterplot) between the wire and CellSearch is visualized in Fig. 3B.

Monitoring of CTCs during systemic therapyTo evaluate changes in CTC counts as a potential marker for

monitoring treatment response, patients were grouped intoresponsive, stable, and progressive disease, according to currentRECIST criteria, 1.1 (13). Cancer patients were screened forCTCs using two subsequent applications of the medical wire,both before and after 12 weeks of any systemic treatment.Patients were grouped according to their therapy responsesassessed after initiation of therapy—responder, stable disease,or progressive disease. We could show that patients respondingto the therapy [complete responders (CR) or partial responders(PR)] showed reduced CTC counts with a median decrease of8.25 CTCs after 12 weeks initial first-line therapy (Fig. 4).

Furthermore, patients with stable disease had a median differ-ence of 0 CTCs compared with their previous cell numberinvestigation, whereas patients with progressive disease werediagnosed with a median increase of 4.5 CTCs (P ¼ 0.0013,Kruskal–Wallis test).

Such significant differenceswerenot seenbyCellSearch (Fig. 4),where the median CTC counts between subsequent visits weresimilar in all response groups (P ¼ 0.13, Kruskal–Wallis test).

Mutational analysis of CTCs captured by the medical wireCTCs could be used for "liquid biopsy" to guide individual

treatment decisions by mutational analysis of genes expressingtargets or relevant pathway proteins. Therefore, we performed aproof-of-principle investigation whether CTCs captured by thewire were suitable for further molecular characterization. For thispurpose, captured cells (after staining) from CTC-positivepatients with knownmutational status of the primary tumor wereanalyzed (Fig. 5A–C).

Figure 2.A,CTCcounts of all patients fromall visits. Overall, 185wire sampleswere taken froma total of 50patients, resulting inCTCdetection in 108 samples, as determinedbyimmunofluorescence analysis. B, representative images of 6 different lung cancer cell lines (NCI-H332, NCI-H3122, NCI H1650, 32M1, A549, and NCI-H1975)isolated by the CellCollector in spiking experiments (in vitro). Tumor cells were identified as EpCAM- and/or pan-keratin-positive events (green). C, representativeimages of CTCs and CTC clusters isolated with the wire and 2 leukocytes. Tumor cells were identified as EpCAM- and/or pan-keratin-positive (green)and CD45-negative (red) events. Hoechst33342 (blue) was used for nuclear counterstain. Scale bars, 20 mm.

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CTC-positive wires from the two patients (patients 013 and047) with confirmed primary tumor mutations in the KRAS(KRAS p.G12D, c.35A>D) or EGFR (EGFR p.H773R, c.2318A>G)gene were used for molecular analysis applying the QuantStudio3D Digital PCR system (Life Technologies). Captured CTCs weredetected by immunofluorescence microscopy followed by celllysis, including unspecific adhering blood cells, and WGA. Theamplified genomic DNAwas then analyzed for specific KRAS andEGFR mutations. The same KRAS and EGFR mutations found inthe primary tumors were clearly detectable when 1 to 5CTCswerepresent on the wire. To ensure specificity of the approach, theKRASG12D-mutated sample was also tested for G12Cmutation.Furthermore, the EGFR H773-mutated sample was tested for thealternative activating mutation L858R and the resistance muta-tion T790M. The analyses clearly demonstrated the specificity ofour dPCR approach because no signals were seen when analyzingthe G12C, L858R, or T790M mutations (Fig. 5B and C).

DiscussionIn this study, we demonstrated that in vivo isolation of CTCs is a

promising approach for sensitive capture of CTCs. In vivo isolationdemonstrated higher CTC capture from lung cancer patientscompared with the FDA-cleared CellSearch system. Both devicescapture and identify EpCAM-/keratin-positive CTCs; thus, theobserved differences are most likely due to the new principle ofcapturing CTCs in vivo. Although taking a blood sample is cer-tainly easier than placing the wire into the arm vein for 30minutes, all cancer patients undergomuchmore time consumingand unpleasant diagnostic approaches, including CT, MRI, andeven invasive biopsieswith the risk of severe bleeding or infection.

Combinedwire applications during the same visit (pre- or post-therapy) revealed CTC positivity rates of 72% to 78%. Similardetection rates could be observed in advanced NSCLC patientsusing an EpCAM-independent filter technology for CTC detectionbased on cell size selection (ISET 80% vs. CellSearch 23%; ref. 6).However, the ISET filter has a pore size of 8 mm (14) and smallerCTCs exist (15) that can be lost. Nevertheless, filter systems aswell

as other EpCAM-independent assays can be helpful in clarifyingthe relevance of "mesenchymal CTCs" with loss of epithelialmarkers. Experimental studies indicated that tumor cells with amesenchymal phenotype have a higher propensity to disseminatethrough the blood stream, but appear to lack the ability to formmetastases at distant sites (16, 17). This would explain why CTCsexpressing (at least some) epithelial markers, such as EpCAMand keratins, are excellent indicators of prognosis in cancerpatients (8).

In this current investigation, we could further demonstrate thatsubsequent applications of the wire during the same visit arefeasible and increase the capture rate of CTCs. Multiple collectorwires can be used for different types of downstream analyses (e.g.,immunostainings and PCR-basedmutation analyses) at the sametime point, which increases the applicability of the wire. More-over, double applications provide an opportunity to analyze thereproducibility of subsequent samples performed with the wire.Although the paired in vivo analyses at the same visit resulted indifferences regarding exact CTC quantification, no significantdifferences between repeated CTC measurements were observ-able, indicating that the CTC results are reproducible. This wasalso shown by the reproducible and specific detection of a KRASmutation using four samples from 1 patient. Regarding the lownumber of CTCs detected, this aspect is of utmost importance andhas been rarely addressed on real clinical specimens using otherCTC assays.

It has been shown that patients who harbor clusters of CTCshave a worse clinical outcome (18). In this study, CTC clusterswere detectable in 20 of 185 wires after in vivo application. Futurestudies are needed to clarify the clinical relevance of CTC clustersfound on the wire. The cellular composition of clusters can beheterogeneous and their biology is still under investigation. It canbe speculated that clusters might contain CTCs from differenttumor clones that are required in a cooperative manner toestablish metastases at distant sites (5, 6).

Monitoring of treatment responses is one of the key applica-tions of CTC assays (19–21). In the present study, we observedsignificant correlation between changes in CTC counts and

Figure 3.A, direct method comparison of CTC detection with the wire and the CellSearch system. Only complete data sets that include results from CellSearch andboth wire applications were used. The respective detection rates are shown below the graph. B, direct comparison (weighted scatter plot) between thewire and CellSearch.

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response to therapy. Decreased CTC counts after therapy initia-tion were frequently seen in lung cancer patients who respondedto systemic treatment, whereas increased CTC numbers wereobserved in most patients with progressive disease. Although thisfinding needs to be validated in larger studies, it suggests that invivo isolation of CTCsmight be useful for monitoring therapies inNSCLC. In contrast, using the ex vivo blood sample-basedapproach to monitor changes in CTC counts did not correlate totreatment response in our present cohort of patients, which seemsto be different fromanother report demonstrating that a change inCellSearch-based CTC counts after treatment was predictive ofprogression-free survival inNSCLC (22). This discrepancymay beexplained by a different selection of patients, and larger multi-center studies should be analyzed to define the value of bothassays in NSCLC.

Besides monitoring of response to therapy, the molecularcharacterization of CTCs can provide important information ontherapeutic targets or resistance mechanisms (23–28). Thus, weevaluated whether the in vivo isolated CTCs also allow subse-quent molecular characterization of genes relevant to therapyof cancer patients. We selected KRAS and EGFR as the twoprominent examples already used in clinical routine for deci-sion making (28). Although only two patients have beenanalyzed, we could demonstrate that the DNA from CTCscaptured by the wire could be isolated, amplified, and analyzedfor mutations in these genes, and—most importantly—theresults matched the gene status in the primary tumor of thesame patients. Control assays for mutually exclusive mutationsin the same genes were negative, proving the specificity of theanalysis. Although this analysis could only be done for 2patients due to the limited clinical data set available, it clearlyindicated the technical feasibility of mutational analysis on (orwith) isolated CTCs. Such results may lead to treatment deci-sions for or against EGFR TKIs based on the presence or absenceof activating or resistance mutations in EGFR or against cetux-imab therapy due to the presence of a KRAS mutation. The

feasibility of mutational analysis based on device-isolated CTCsis an important prerequisite for future use of the wire for"liquid biopsy" analyses in cancer patients. Taking biopsiesfrom lung cancer patients for the assessment of molecularalterations relevant to therapy is an invasive, time-consuming,painful, and risk-associated procedure (e.g., severe bleedingevents). The present investigation suggests that CTCs may offersolutions to this problem in the near future. Genomic infor-mation can also be obtained by the analysis of circulating cell-free DNA released by dying cancer cells into the blood samples,which has recently achieved great attention (29). However, CTCanalysis can be performed on the DNA, RNA, protein level andcapture of sufficient amounts of CTCs to also allow functionalstudies (25, 30, 31).

Our first validation study of the wire has produced encour-aging results. However, there are still some limitations andopen issues that need to be discussed. The wire application doesnot permit reporting of CTC counts in a given (defined) bloodvolume (i.e., CTCs in 10 mL of blood). Using this device, thevalue can only be reported as CTCs collected in 30 minutes.Because the amount of blood that flows through the vein can bevariable from patient to patient, or even at different time pointsin the same patient, this may make it more difficult to interpretquantitative differences after treatment. However, we couldshow that subsequent applications of the wire during the samevisit are feasible and increase the capture rates for CTCs at whichtime no significant differences between repeated CTC measure-ments were observable, indicating that the CTC results arereproducible. Besides, we had only a limited number of patientswithout metastases, which may also explain why no significantdifferences in CTC rates were observed. Larger studies should beperformed including more early-stage lung cancer patients thatare screened and analyzed for CTCs. Finally, the current anti-EpCAM antibodies used for capturing of CTCs have demon-strated proof-of-principle, but future generations of the deviceshould include a mixture of different capturing antibodies to

Figure 4.Reduction of CTC counts under therapy determined with the wire. Box-and-whisker plot of CTC count difference between different visits (V=visit) for patients withresponsive, stable, and progressive disease.

Gorges et al.

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isolate subsets of EpCAM-negative cancer cells that do notdisplay the epithelial phenotype, e.g., cells that underwentan epithelial to mesenchymal transition (32). This deve-lopment will further increase the capture rate of CTCs via thewire.

In conclusion, our present findings indicate that in vivoisolation of CTCs is a promising approach to capture sufficientamounts of CTCs amenable to downstreammolecular analysesin lung cancer. This new diagnostic tool can now be tested ininterventional clinical studies where the number and/or char-acteristics of CTCs will be used to stratify patients to particulartreatments and established endpoints such as survival will beassessed. There is an increasing number of molecular targetsthat are only present in a small fraction of NSCLC patients(33), which requires stratification based on the molecularcharacteristics of tumor cells in individual patients. Further-more, CTCs have recently been detected in patients withchronic obstructive pulmonary disease without clinicallydetectable lung cancer (34). The implementation of a sensitivein vivo device may now also allow early diagnosis of lungcancer. Thus, future studies are needed to explore the entireclinical potential of the wire.

Disclosure of Potential Conflicts of InterestN. Brychter and O. von Ahsen report receiving commercial research grants

from Bayer Pharma AG. T. Krahn is a consultant/advisory board member forGilupi UK. K. Pantel is a consultant/advisory board member for Gilupi GmbH.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: T. Schalk, S. Riethdorf, K. L€ucke, C. Schumann,T. Krahn, K. PantelDevelopment of methodology: K. L€ucke, S. Jackson, O. von Ahsen,C. Schumann, T. Krahn, K. PantelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Penkalla, T. Schalk, S. Riethdorf, S. Jackson,N. Brychta, C. Schumann, T. Krahn, K. PantelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.M. Gorges, T. Schalk, S.A. Joosse, S. Riethdorf,J. Tucholski, K. L€ucke, H. Wikman, S. Jackson, N. Brychta, O. von Ahsen,C. Schumann, K. PantelWriting, review, and/or revision of the manuscript: T.M. Gorges, N. Penkalla,T. Schalk, S.A. Joosse, S. Riethdorf, K. L€ucke, H. Wikman, O. von Ahsen,C. Schumann, T. Krahn, K. PantelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. Penkalla, T. Schalk, K. L€ucke, T. KrahnStudy supervision: K. PantelOther (conception of the manuscript design): T.M. Gorges

Figure 5.Mutational analysis of the KRAS and EGFR genes in captured CTCs. A, cells isolated with the CellCollector were analyzed microscopically by means ofimmunofluorescence staining according to the staining procedure in the clinical setup. The wire was fragmented and subjected to WGA. Patient materialexaminedby dPCR, CTC count, and primary tumormutation status (NTC¼none template control,WT¼wild-type, andV¼ visit). B, amplifiedgDNAof patient 13wasanalyzed by dPCR for the known primary tumor mutation KRAS G12D and KRAS G12C as specificity control. C, amplified gDNA of patient 47 was analyzedby dPCR for the known primary tumor mutation EGFR H773R and the other EGFR mutations L858R and T790M to control for specificity.

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Other (using the CellCollectors on the patients directly (in vivo) and theirfurther processing): N. Penkalla

AcknowledgmentsTobias M. Gorges, Sabine Riethdorf, and Klaus Pantel thank the City of

Hamburg, Landesexzellenzinitiative Hamburg (LEXI 2012; Tumor targeting viacell-surface molecules essential in cancer progression and dissemination).

Grant SupportPart of this work is supported by the Bundesministerium f€ur Bildung und

Forschung (BMBF) (F€orderkennzeichen 01EZ 0863) and the Ministerium f€urWirtschaft des Landes Brandenburg und der EU (F€orderkennzeichen

80135447). T.M. Gorges, S. Riethdorf, and K. Pantel supported by the ERCAdvanced Investigator Grant DISSECT, TRANSCAN ERA-Network: Grant CTC-SCAN and from the Innovative Medicines Initiative Joint Undertaking undergrant agreement no. 115749, resources of which are composed of financialcontribution from the EuropeanUnion's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 16, 2015; revised October 13, 2015; accepted December 3,2015; published OnlineFirst December 14, 2015.

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Device for CapturingIn VivoLung Cancer Patients Using a Novel Enumeration and Molecular Characterization of Tumor Cells in

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