targeted dual-modality imaging in renal cell carcinoma: an ... · marlene c.h. hekman1,2, otto c....

Personalized Medicine and Imaging

Targeted Dual-Modality Imaging in Renal CellCarcinoma: An Ex Vivo Kidney Perfusion StudyMarl�ene C.H. Hekman1,2, Otto C. Boerman1, Mirjam de Weijert2,Desir�ee L. Bos1, Egbert Oosterwijk2, Hans F. Langenhuijsen2,Peter F.A. Mulders2, and Mark Rijpkema1

Abstract

Purpose: Antibodies labeled with both a near-infrared fluo-rescent dye and a radionuclide can be used for tumor-targetedintraoperative dual-modality imaging. Girentuximab is a chi-meric monoclonal antibody against carbonic anhydrase IX(CAIX), an antigen expressed in 95% of clear cell renal cellcarcinoma (ccRCC). This study aimed to assess the feasibilityof targeted dual-modality imaging with 111In-girentuximab-IRDye800CW using ex vivo perfusion of human tumorouskidneys.

Experimental Design: Seven radical nephrectomy specimensfrom patients with ccRCC were perfused during 11 to 15 hourswith dual-labeled girentuximab and subsequently rinsed during2.5 to 4 hours with Ringer's Lactate solution. Then, dual-modalityimaging was performed on a 5- to 10-mm-thick lamella of thekidney. Fluorescence imaging was performed with a clinicalfluorescence camera set-up as applied during image-guidedsurgery. The distribution of Indium-111 in the slice of tumor

tissue was visualized by autoradiography. In two perfusions, anadditional dual-labeled control antibody was added to demon-strate specific accumulation of dual-labeled girentuximab inCAIX-expressing tumor tissue.

Results: Both radionuclide and fluorescence imaging clearlyvisualized uptake in tumor tissue and tumor-to-normal tissueborders, as confirmed (immuno)histochemically and by gammacounting. Maximum uptake of girentuximab in tumor tissue was0.33% of the injected dose per gram (mean, 0.12 %ID/g; range,0.01–0.33 %ID/g), whereas maximum uptake in the normalkidney tissue was 0.04 %ID/g (mean, 0.02 %ID/g; range, 0.00–0.04 %ID/g).

Conclusions:Dual-labeled girentuximab accumulated speci-fically in ccRCC tissue, indicating the feasibility of dual-modal-ity imaging to detect ccRCC. A clinical study to evaluateintraoperative dual-modality imaging in patients with ccRCChas been initiated. Clin Cancer Res; 22(18); 4634–42. �2016 AACR.

IntroductionIn oncological surgery, radical tumor resection is crucial for

treatment outcome and patient survival (1–6). During surgery,differentiation of tumor tissue from nontumorous tissue with thenaked eyemay be challenging. Intraoperative imaging techniquesthat can distinguish tumor from normal tissue will help thesurgeon to achieve complete tumor resection.

One of these techniques, radio-guided surgery, has alreadybeen implemented in clinical practice, for example, to detectsentinel lymph nodes (7–9). The high tissue penetration depthof gamma radiation allows accurate localization of tumors,almost regardless of tissue depth. However, exact tumor delin-eation with a gamma probe is difficult, and for precise real-timetumor delineation during surgery, an optical signal may be

more beneficial. For this purpose, a fluorescent probe can beused, but because the penetration depth of light in biologictissue is limited, fluorescence imaging is mainly useful for thedetection of superficially located tumors (10, 11). Therefore,dual-modality image-guided surgery, combining the advan-tages of radioguided and fluorescence-guided surgery, maybe a synergistic combination (12–15). A dual-labeled tumor-targeting imaging probe could allow intraoperative tumordetection, tumor delineation, and assessment of resection mar-gins and remnant disease. Results from preclinical studies arepromising (16–19), and tumor-targeted dual-modality imagingis awaiting its first use in patients.

Monoclonal antibodies against tumor-associated antigenstagged both with a radiolabel and a fluorophore may be used asprobes during image-guided surgery (20). Girentuximab, a chi-meric monoclonal IgG1 antibody, is an excellent vehicle to targetclear cell renal cell carcinoma (ccRCC). It specifically recognizescarbonic anhydrase IX (CAIX), a cell surface antigen that isexpressed abundantly in more than 95% of ccRCC and is absentinnormal kidney tissue (21, 22). Previous researchhas shown thatafter administration of radiolabeled girentuximab, both primarytumors and metastases can be detected by PET/CT or SPECT/CTin patients (23–25). Animal studies using 111In-girentuximab-IRDye800CW have illustrated the potential of dual-modalityimaging (17, 18). Dual-labeled girentuximab accumulateshighly and specifically in CAIX-expressing SK-RC-52 subcutane-ous tumors (18). Next, a proof-of-principle study was performed

1Department of Radiology and Nuclear Medicine, Radboudumc,Nijmegen, the Netherlands. 2Department of Urology, Radboudumc,Nijmegen, the Netherlands.

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

Corresponding Author: Marl�ene C.H. Hekman, Radboudumc, Geert Groote-plein-Zuid 10, 6525 GA Nijmegen, the Netherlands. Phone: 024-3619097, Fax:024-3618942; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2937

�2016 American Association for Cancer Research.

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to show the feasibility of image-guided surgery using an intra-peritoneal tumor model (17). After administration of dual-labeled girentuximab, submillimeter CAIX-expressing tumornodules could be detected preoperatively with SPECT/CT. Sub-sequently, during surgery, superficially located tumor nodulescould be visualized and resected based on the fluorescent signal.

However, animal studies only partly reflect the clinical sit-uation. To test the translation of dual-modality imaging to theclinic, models using clinical imaging systems and intact tumorsobtained from patients are required. In the present study,tumorous kidneys resected from patients with ccRCC wereconnected ex vivo to a perfusion system via the renal artery ashas been described previously (26). Subsequently, the tumor-ous kidneys were perfused with dual-labeled girentuximab andinvestigated using the clinical imaging system to be used duringimage-guided surgery. In preparation of the first targeted dual-modality image-guided surgery study in patients, this studyaimed to show the feasibility of tumor-targeted dual-modalityimaging in renal cell carcinoma using dual-labeled girentux-imab in an ex vivo kidney perfusion model.

Materials and MethodsStudy design

The aim of this study was to assess the feasibility of dual-modality imaging in patients with ccRCC in an ex vivo kidneyperfusion study. Patients suspected of ccRCC and scheduled for aradical nephrectomy that signed informed consentwere included.Exclusion criteria were a known subtype other than ccRCC or theadministration of a radioisotope within ten physical half-livesprior to surgery. The first human tumorous kidney was perfusedwith DOTA-girentuximab-IRDye800CW not labeled with Indi-um-111, to estimate the radiation safety risks of the procedure.Next, four human tumorous kidneys were perfused ex vivo withdual-labeled girentuximab. To demonstrate specific binding ofgirentuximab, two additional tumorous kidneys were perfused

with a mixture of dual-labeled 131I-girentuximab-IRDye800CWand a 125I-labeled control antibody-IRDye800CW. After perfu-sion, radionuclide and fluorescence imaging were performed on a1-cm lamella of the specimen using clinical and preclinicalimaging systems. Subsequently, samples of tumor and normaltissue were taken for further analysis: quantification of antibodyaccumulation, fluorescence imaging, autoradiography, CAIXstaining, and hematoxylin and eosin (H&E) staining. The primaryoutcome measure was the ability to detect a fluorescent andradioactive signal in tumor tissue. Secondary outcome measureswere the antibody accumulation in tumor and normal kidneytissue expressed as percentage of injected dose per gram (%ID/g)and as the tumor-to-kidney tissue ratio. This study was approvedby the regional ethical review board (CMO, region Arnhem-Nijmegen).

Dual-labeled antibody productionChimeric girentuximab (IgG1) was a kind gift from Wilex

AG. The isotype-matched control human Immunoglobulin(IgG1) was purchased from Biotrend Chemikalien. The fluor-ophore IRDye800CW-NHS ester was purchased from LI-CORbiosciences. IRDye800CW is a near-infrared dye that can bestably coupled to monoclonal antibodies (27). It emits 789-nmphotons when excited at 774 nm. Near-infrared fluorescentdyes (700–1,000 nm) have a higher tissue penetration depthcompared with dyes in the visible light range (28). The chelatorDOTA-NHS ester (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester) waspurchased from Macrocyclis.

DOTA-girentuximab-IRDye800CW was produced under met-al-free conditions as described previously (29). In short, gir-entuximab (5 mg/mL) was incubated in 0.1 mol/L NaHCO3,pH 8.5, at room temperature (RT) for 1 hour with a threefoldmolar excess of the IRDye800CW-NHS ester. Then, the mixturewas incubated in NaHCO3, pH 9.5, for 1 hour with a 10- to 20-fold molar excess of the DOTA-NHS ester. After conjugation, thereaction mixture was transferred into a Slide-A-Lyzer cassette(molecular weight cut-off: 10,000 or 20,000 Da; Thermo Sci-entific) and extensively dialyzed against 0.25 mol/L ammoniu-macetate, pH 5.5, for 3 days with buffer changes to removethe unconjugated IRDye800CW and DOTA. The average num-ber of IRDye800CW molecules that was conjugated to theantibody (substitution ratio or SR) was determined spectro-photometrically with the Ultrospec 2000 UV/Visible spectro-photometer (Pharmacia Biotech) and ranged from 1.3 to 1.5.The immunoreactive fraction of dual-labeled girentuximab wasdetermined as described by Lindmo (30), within 2 weeks ofeach experiment and always exceeded 70%. DOTA-girentuxi-mab-IRDye800CW was stored in the dark at 4�C until use.

For the control experiments, girentuximab-IRDye800CW andcontrol IgG-IRDye800CW were prepared as described above.The SR of girentuximab-IRDye800CW and the irrelevant controlIgG-IRDye800CW were 2.0 and 2.1, respectively.

Radiolabeling of antibodiesOn the day of the experiment, 1.2 mg DOTA-girentuximab-

IRDye800CW was labeled with 3.5 to 7.0 MBq 111InCl3(Mallinckrodt Pharmaceuticals) in two volumes of 0.5 mol/L2-(N-morpholino)ethanesulfonic acid (MES). After 40 minutesof incubation at 45�C, 50 mmol/L ethylenediaminetetraacetic

Translational Relevance

Intraoperative dual-modality imaging may provide the sur-geon with valuable information about tumor localization andresection margins, and therefore may improve locoregionalcontrol and patient outcome. In the current study, we bridgethe gapbetween preclinical studies and the clinical applicationof intraoperative dual-modality imaging using radiolabeledand fluorescently labeled tumor-targeting antibodies. In an exvivo perfusion study of human kidneys with renal cell carci-noma, dual-modality imaging was tested in a translationalsetting. Real-time fluorescence images acquired with a clinicalfluorescence camera system clearly visualized uptake in tumortissue and tumor-to-normal tissue borders, as confirmed(immuno)histochemically and by gamma counting. Theseresults demonstrate the clinical potential of dual-modalityimaging and have led to the initiation of the first targeteddual-modality image-guided surgery study in clear cell renalcell carcinoma patients (NCT02497599). Targeted dual-modality imaging has the potential to revolutionize oncologicsurgery.

Targeted Dual-Modality Imaging in Renal Cell Carcinoma

www.aacrjournals.org Clin Cancer Res; 22(18) September 15, 2016 4635




acid (EDTA) was added to a final concentration of 5 mmol/L tochelate unincorporated Indium-111. Preparations were puri-fied on a PD10 disposable gelfiltration column eluted withPBS. Radiochemical purity was determined by Instant ThinLayer Chromatography (ITLC) on silicagel, using 0.1 mol/Lsodiumcitrate buffer, pH 6.0, as mobile phase. Radiochemicalpurity of the Indium-111–labeled preparations exceeded95%. After purification, the DOTA-girentuximab-IRDye800CWamount was adjusted to a total protein amount of 1.2 mg.Standards of the ID were prepared in triplicate to be able toquantify antibody accumulation corrected for radioactivedecay. The injected activity dose was determined in a dosecalibrator and ranged from 3.4 to 5.1 MBq.

In the two dual-isotope experiments, 100 mg of girentuxi-mab-IRDye800CWwas radiolabeled with 10 to 15 MBq Iodine-131 (PerkinElmer), and 100 mg of control IgG-IRDye800CWwas labeled with 5 to 7 MBq Iodine-125 (PerkinElmer; ref. 31).Briefly, 100 mg of antibody conjugate was added to 10 mL of0.5 mol/L sodium phosphate, pH 7.4, in a vial coated with100 mg iodogen. After adding the radioactivity, the volumewas adjusted to a total volume of 100 mL with 50 mmol/Lsodium phosphate, pH 7.4. After 10 minutes of incubation atRT, the reaction mixture was transferred to a clean Eppendorfvial together with 100 mL of saturated tyrosine. After purifica-tion on a PD10 column radiochemical purity exceeded 95%and the amount of both antibodies was adjusted to 1.2 mg ofprotein with cold antibody. Standards of the ID were prepar-ed in triplicate. Both Iodine-131-girentuximab-IRDye800CW(1.2 mg, ID 4.6, and 4.1 MBq) and Iodine-125-control IgG-IRDye800CW (1.2 mg, ID 1.0, and 1.2 MBq) were added to theperfusion reservoir.

Kidney perfusion modelIn the operating room, a vessel cannula was inserted on

the bench in the renal artery and connected to a flushing sys-tem with Ringer's Lactate solution to rinse blood (see Supple-mentary Figs. S1 and S2). In case of multiple renal arteries, thetwo largest arteries were connected, and any others were leftopen. Also, the renal vein was left open. Then, the kidneywith the vessel cannula(s) in situ was connected to a recirculat-ing perfusion system, which included a peristaltic pump(30 mL/min). The flow in the system (30 mL/min) is consid-erably lower than the human renal blood flow (500 mL/min).This flow rate was selected based on our experience that at thismaximum flow rate, the risk of leakage of the arterial connec-tion was minimal. After ensuring the leakage-free connectionto the kidney, the afferent and efferent hoses of the pump(Supplementary Fig. S1) were placed in a reservoir with 350 mLRinger's Lactate solution containing 0.1 % BSA. This reservoirwas cooled with ice (0–4�C) and protected against light. Sub-sequently, the dose of Indium-111-DOTA-girentuximab-IRDye800CW (1.2 mg, 3.6–5.2 MBq Indium-111) was addedto the 350 mL reservoir, resulting in an antibody concentra-tion of 3.4 mg/L. This concentration was chosen to mimicthe serum concentration in patients that receive radiolabeledgirentuximab in clinical studies (22–24).

The tumorous kidney was perfused during 11 to 15 hourswith dual-labeled antibody. After this period, the kidney wasrinsed with 5 to 7 liters of nonrecirculating Ringer's Lactatesolution (2.5–4 hours) to wash out unbound antibody. Forsafety reasons, the first experiment was performed without

radioactivity, and therefore antibody uptake was not quantifiedin this first kidney.

Dual-modality imagingA central coronal slice (5–10 mm thick) of the perfused

kidney containing both tumor and normal tissue was obtainedfor further analysis. Fluorescence imaging was performedusing a real-time clinical imaging system (Storz D-light Plaparoscopic setup). Furthermore, fluorescence imaging wasperformed with two nonclinical imaging systems, the IVISLumina closed-cabinet fluorescence imager (Caliper LifeSciences; recording time, 5 to 10 minutes; binning factormedium; F/stop 2; excitation filter 745 nm; emission filterICG; FOV C; autofluorescence correction, 675 nm; and back-ground correction) and the Odyssey CLx flatbed fluorescencescanner (LICOR biosciences; recording time, 20 to 30 minutes;800 nm channel; focus 1.0 mm). Subsequently, the kidneyslice (kept on ice) was exposed for 1 hour to a phosphorimaging plate for autoradiography. This plate was developedusing the Typhoon FLA 7000 Phosphor Imager and analyzedwith Aida Image Analyzer v. 4.21.

Tissue processingAfter dual-modality imaging, 1 cm3 samples of tumorous

and normal tissue were taken from the kidney slice and fixed in4% formalin for further analysis. The amount of Indium-111-DOTA-girentuximab-IRDye800CW in tissue was determinedquantitatively by measuring these samples in a gamma counter(2480 WIZARD2; Perkin Elmer) together with the standards ofthe ID. In the control experiment, a dual-isotope protocol wasused [Iodine-125 35 keV (window 15–85 keV), Iodine-131 360keV (window 260–430 keV), spillover correction was applied].The tracer uptake was expressed as percentage of the ID pergram tissue (%ID/g). In addition, the tumor-to-normal anti-body uptake ratio was calculated.

Then, formalin-fixed, paraffin-embedded 5-mm sections werecut and were analyzed autoradiographically (2 weeks exposure toa phosphor imaging plate) and by fluorescence imaging (Odys-sey CLx flatbed fluorescence scanner; 800 nm channel; recordingtime, 1 to 5 minutes; focus 1.0 mm). Subsequently, sections werestained for CAIX with the murine anti-CAIX antibody M75 (cellline HB-11128 obtained from the ATCC). Also, a classic H&Estaining was performed on all slices.

Statistical analysisStatistical analyses were performed using IBM SPSS 20.0. For

each separate experiment, the antibody accumulation wasexpressed as %ID/g (maximum, mean, and SD). All samplesthat consisted macroscopically at least partly of tumor, alsoborder, central cystic, or necrotic tumor regions, were consid-ered as tumor tissue samples. Only samples macroscopicallyconsisting of normal tissue were considered as normal tissuesamples. Differences in accumulation between tumor and nor-mal tissue samples were tested for significance within the singleexperiment using t tests. Furthermore, in the two controlexperiments, the difference between tumor accumulation ofgirentuximab compared with tumor accumulation of controlIgG was tested for significance using the paired t test. Differ-ences were considered significant at P < 0.05, two-sided. Tumor-to-normal (T:N) ratios were calculated by dividing the mean

Hekman et al.

Clin Cancer Res; 22(18) September 15, 2016 Clinical Cancer Research4636




%ID/g in tumor tissue by the mean %ID/g in normal tissue.Furthermore, a range was given for the T:N ratio: minimum andmaximum antibody accumulation in tumor tissue in relation tothe mean antibody accumulation in normal tissue.

ResultsExperiment characteristics

Between June 2014 and July 2015, seven radical nephrecto-my specimens from patients with ccRCC were perfused ex vivowith dual-labeled girentuximab in Ringer's Lactate solution(experiment 1 was performed without Indium-111). Histopa-thology confirmed that all seven kidneys contained a CAIX-expressing ccRCC. Basic characteristics of these perfusions areshown in Table 1.

Dual-modality imagingReal-time fluorescence imaging of the lamella with the clin-

ical fluorescence camera system revealed images in whichthe tumor was clearly delineated (Fig. 1 and SupplementaryVideo S1). Subsequently, non–real-time fluorescence imagingwas performed with the Odyssey fluorescence flatbed scanner(Figs. 2D and 3B) and the IVIS Lumina fluorescence imager(Fig. 2B). Fluorescence imaging showed preferential accumula-tion of dual-labeled girentuximab in tumor tissue, whereasfluorescence in normal tissue was hardly detectable. As a result,a clear contrast was obtained between tumor and normal tissue.These results were consistent throughout all experiments. Mostimportantly, in one of the perfused kidneys, a small satellitetumor lesion was detected by fluorescence imaging that had notbeen recognized by macroscopic inspection, but proved to beccRCC at histopathologic evaluation (Fig. 3A and B).

After the fluorescence measurements, the accumulation ofIndium-111-girentuximab-IRDye800CW in tumor tissue couldbe clearly visualized by autoradiography of the 1-cm lamella(Fig. 2C, 1h exposure to a Phosphor imaging screen). Visualassessment revealed excellent colocalization of the fluorescentand radioactive signal in regions macroscopically designatedas tumor tissue (Figs. 2 and 3).

Quantitative analysis of antibody accumulationThe accumulation of Indium-111-girentuximab-IRDye800CW

in tumor and normal tissue kidney samples is shown inTable 2. Indium-111-girentuximab-IRDye800CW accumulatedpreferentially in tumor tissue (mean, 0.12 %ID/g; range, 0.01–

0.33 %ID/g). Accumulation in normal kidney parenchyma waslow (mean, 0.02 %ID/g; range, 0.00–0.04 %ID/g). In the firstkidney perfusion, no radioactivity was used and thereforeantibody accumulation could not be quantified. In experiment2, antibody accumulation in tumor tissue was not significantlyhigher than in normal tissue, most likely because the tumortissue was largely necrotic. In that experiment, high antibodyaccumulation (0.15 %ID/g) was found in the renal vein tumorthrombus. In the other five kidney specimens, accumulation ofgirentuximab in tumor tissue was significantly higher than innormal kidney tissue (P < 0.05). The mean T:N ratio rangedfrom 4 to 14.

Table 1. Characteristics of the seven perfusion experiments

Experiment number HistologyFuhrmangrade

Type ofsurgery T stage

Kidneyweight(kg)

Tumordiameter(cm)

Number ofconnectedarteries

Perfusion/washing (h)

111In-DOTA-girentuximab-IRDye800CW1 ccRCC 2 Right LN pT1b 0.8 8 2 14/32a ccRCC 3 Left ON pT3a 1.0 12 1 14/33 ccRCC 1 Left LN pT1a 0.5 4 1 14/34 ccRCC 3 Left LN pT2a 0.9 7 2 15/45b ccRCC 3 Left ON pT3a 1.5 9 1 11/3

Control experiments: 131I-girentuximab-IRDye800CW and 125I-IgG-IRDye800CW6 ccRCC 4 Left ON pT3a 1.7 13 1 13/37 ccRCC 2 Right ON pT3a 0.5 7 1 13/3

Abbreviations: LN, laparoscopic nephrectomy; ON, open nephrectomy.aRenal vein thrombus (level 0).bON þcavotomy for level 1 thrombus.

Figure 1.Representative images from a kidney lamella after perfusion made withthe clinical laparoscopic fluorescence camera showing the tumor margin.A, visible light image. B, corresponding fluorescence image.






Perfusion of two radical nephrectomy specimens (#6and #7) with a mixture of girentuximab-IRDye800CW anda control antibody-IRDye800CW labeled with I-131 and I-125 showed specific accumulation of dual-labeled girentux-imab in tumor tissue. Uptake of Iodine-131-girentuximab-

IRDye800CW in tumor tissue (mean, 0.14 %ID/g; range,0.01–0.31 %ID/g) was significantly higher than uptake ofthe irrelevant control Iodine-125-IgG-IRDye800CW in tumortissue (mean, 0.02 %ID/g; range, 0.01–0.03 %ID/g, P <0.001). The latter concentration was in the same range as

Figure 2.High accumulation of dual-labeledgirentuximab in tumor tissue can bevisualized with both fluorescence andradionuclide imaging. A, macroscopy.B, fluorescence image acquired withthe IVIS Lumina fluorescence imager.C, autoradiography. D, Odysseyfluorescence flatbed scanner image.

Figure 3.A,macroscopic image of the tumorous kidney tissue slice after ex vivo kidney perfusion, (B) corresponding fluorescence image (Odyssey), and (C) autoradiographyshowing high and specific uptake of the dual-labeled tracer in the tumor region. �� , histologically confirmed satellite ccRCC focus.

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the concentration of girentuximab in normal kidney paren-chyma (mean, 0.02 %ID/g; range, 0.01–0.02 %ID/g; Table 2;Supplementary Figs. S6 and S7).

Microscopic analysisFinally, microscopic analysis of the tissue sections confirmed

that the intratumoral distribution of the radioactive signal wascongruent with that of the fluorescent signal. Most importantly,both signals colocalized with CAIX expression (Fig. 4). In normalkidney parenchyma, the dual-labeled girentuximab was barelydetectable (Supplementary Fig. S4). H&E staining of the sectionsshowed that cell morphology after perfusion was normal, indi-cating that the continued perfusion did not lead to cell damage(Supplementary Fig. S4).

DiscussionThis study demonstrated the feasibility of tumor-targeted

dual-modality imaging in ccRCC using dual-labeled girentux-imab ex vivo. Tumors were clearly distinguishable from normalkidney parenchyma both by fluorescence and radionuclideimaging when tumor-bearing kidneys were perfused withdual-labeled girentuximab. The specific localization of thedual-labeled antibody in CAIX-expressing tumor tissue allowedexcellent tumor visualization. Colocalization was observedbetween the fluorescent signal, the radioactive signal, and CAIXexpression in tumor tissue. These results, in a system closelyresembling the clinical application, bridge the gap betweenpreclinical research and clinical application of dual-labeledgirentuximab and have led to the initiation of the first targeted

Table 2. Accumulation of dual-labeled antibodies in tissue samples (%ID/g)111In-DOTA-girentuximab-IRDye800CW

Tumor tissue Normal tissue

Experimentnumber

ID (MBq)111In Max Mean (SD) N Max Mean (SD) N Pa

T:N ratiomean(range)

1b — — — 11 — — 2 — —

2 3.4 0.13 0.04 (0.03) 16 0.02 0.01 (0.00) 4 0.118 4 (2–13)3 3.8 0.33 0.15 (0.09) 7 0.04 0.02 (0.01) 9 0.001 8 (4–17)4 3.5 0.31 0.14 (0.09) 12 0.02 0.01 (0.01) 5 0.005 14 (1–31)5 5.1 0.33 0.15 (0.08) 16 0.03 0.01 (0.01) 6 <0.001 8 (1–17)

Control experiments: 131I-girentuximab-IRDye800CW and 125I-IgG-IRDye800CWTumor tissue Normal tissue

131I/125IGirentuximabmean (SD)

Controlmean (SD) N

Girentuximabmean (SD)

Controlmean (SD) N Pa Pc

6 4.6/1.0 0.08 (0.05) 0.01 (0.01) 15 0.01 (0.01) 0.01 (0.01) 5 <0.01 <0.0017 4.1/1.2 0.20 (0.06) 0.02 (0.00) 17 0.02 (0.00) 0.02 (0.00) 9 <0.001 <0.001Abbreviation: N, number of tissue samples.aT test comparing uptake of dual-labeled girentuximab in tumor versus normal tissue.bPerformed without radioactivity.cPaired t test comparing uptake of dual-labeled girentuximab and dual-labeled control antibody in tumor tissue.

Figure 4.Tissue section of tumor thrombus in therenal vein and several small veinsshowing the overlap between thefluorescent and radioactive signals andCAIX expression in tumor tissue.A,H&Estaining. B, fluorescence image(Odyssey).C,CAIX stainingwithDABofCAIX-expressing tumor tissue. D,autoradiography.






dual-modality image-guided surgery study in patients withccRCC (clinicaltrials.gov NCT02497599).

As more complex renal tumors are increasingly treated bypartial nephrectomy these days (32, 33), intraoperative dual-modality imaging can be useful to improve intraoperative tumordetection and improve radical tumor resection. Particularly inmore complex tumors, the percentage of positive surgicalmarginscan be as high as 18% (34). Although no consensus exists aboutthe prognostic impact of positive surgical margins, the uro-onco-logical surgeon should aim for radical tumor resection (34, 35).Targeted dual-modality imaging using dual-labeled girentuximabmay be a valuable imaging technique during surgery of ccRCCpatients. The high tissue penetration depth of gamma radiationfor tumor localization combined with fluorescence imaging forreal-time precise tumor delineation forms a powerful combina-tion to improve surgical outcome. The advantage of the dual-labeling strategy over coinjection of separate radio- and fluores-cently labeled probes is that both imaging signals originate fromthe same tracer molecule (20, 36).

Preclinical studies have already demonstrated the feasibility ofdual-modality imaging using various monoclonal antibodies inseveral animal tumor models, for example, cetuximab (anti-EGFR; ref. 29), panitumumab (anti-HER1; ref. 37), trastuzumab(anti-HER2; refs. 37, 38), TRC105 (anti-CD105; ref. 39), D2B(anti-PSMA; ref. 16), and girentuximab (anti-CAIX; refs. 17, 18,29) in head and neck cancer, breast cancer, prostate cancer, andrenal cell carcinoma, respectively. However, extrapolation ofresults of these studies in mouse-tumor models to the clinicalsetting is cumbersome, because tumor models and the experi-mental setup only partly reflect the clinical situation. To facilitateclinical translation of dual-modality imaging, in this study, an exvivomodel was used to evaluate the in vivo targeting properties ofdual-labeled antibodies (26, 40). This model has several advan-tages compared with the mouse-tumor models. Because humantumor-containing tissue is used, the tumor size, antigenic make-up, vascularization, and growth pattern of the tumor reflect theclinical situation. Furthermore, as the sensitivity of clinical imag-ing systemsdiffers frompreclinical imaging systems,weusedbothtypes of imaging systems in this study (i.e., closed cabinet-typefluorescence imaging systems often used in preclinical researchand a hand-held laparoscopic fluorescence camera system usedfor clinical applications). By selecting an antibody concentrationof 3.4 mg/L (1.2 mg in 350 mL of Ringer's Lactate solution), wemimicked the initial plasma concentration in clinical trials(administration of 5–50 mg girentuximab per patient; refs. 22–25). Clearance of the nonbound antibody from the circulationwas mimicked by washing with Ringer's lactate. However, thisdoes not reflect the complex clearance of antibodies in the humanbody. Nevertheless, we found a maximum accumulation of dual-labeled girentuximab of 0.3 %ID/g in tumor regions, which is inthe same range as the accumulation that was found in clinicalstudies with radiolabeled girentuximab (up to 0.5%ID/g; refs. 22,41). Therefore, our results may be a good predictor for theintensity of the dual-modality signal that can be expected duringsurgery. However, one must realize that real-time intraoperativeimaging involves many challenges that were not encountered inthis imaging model of the lamella, for example, the limitedmovability of a camera during laparoscopy, 3D optical imaging,gamma probing, and an increased noise level because of traceraccumulation in the liver and blood. These aspects have to beevaluated in a clinical trial.

The kidneys included in this study contained large renal cellcarcinomas, including tumor thrombi. This differs from the smallrenalmasses wherewe envision that intraoperative dual-modalityimaging might be particularly useful. However, inclusion ofspecimens with smaller tumor masses was not possible as inthese cases usually partial nephrectomy is indicated, and partialnephrectomy specimens lack the renal artery to perform theperfusion. However, the added value of dual-modality imagingfor tumor detection and resection was elegantly illustrated afterperfusion of specimen #5. In this kidney lamella, a small satelliteccRCC lesion was identified by fluorescence imaging (see Fig. 3B)that had not been recognized on primarymacroscopic inspection.Histopathologic analysis confirmed that this was a ccRCC lesion.According to literature, satellite lesions are found in approximate-ly 7% of patients, depending on tumor stage (42).

A challenge for the clinical application of targeted imagingtechniques will be intratumoral heterogeneity. Large variation inantibody accumulation was seen within tumors. This might beattributable to differences in CAIX expression, tumor viability,vascularization, and/or perfusion (41). We found an excellentspatial overlap between CAIX expression and the fluorescent andradioactive signal of dual-labeled girentuximab. Furthermore,antibody accumulation was found to be high at the tumorborders, which are clinically the most relevant regions. Anotherconcern was that the accumulation of girentuximab could bepartly due to the enhanced permeability and retention (EPR)effect, because of the enhanced vascular permeability in tumortissue (43). Therefore, we performed adual-isotope (131I and 125I)control experiment in two resected tumorous kidneys. Becauseaccumulation of the control IgG in tumor tissue was much lowerthan accumulation of girentuximab (Table 2), we conclude thatthe girentuximab accumulation is mainly due to specific interac-tion of girentuximab with the CAIX antigen in the tumor tissue.Because the concentration of the dual-labeled antibody is similarto the plasma concentration and because the concentration intumor tissue ex vivo and in vivo is in the same order of magnitude,the accumulation of the antibody in the tumor due to the EPReffect is expected to be similar ex vivo and in vivo. This also impliesthat targeted imaging using girentuximab can only be used inCAIX-expressing tumors. However, with a suitable tumor-target-ing agent, dual-modality imaging canbeused in any typeof cancersurgery.

The first clinical trials using fluorescently labeled tumor-targeting probes [folate-FITC (11), cetuximab-IRDye800CW(44), and GE-137 (45)] have clearly demonstrated the potentialof targeted intraoperative fluorescence imaging for improvedintraoperative or endoscopic tumor visualization, which maylead to improved surgical outcome in clinical oncology. Fur-thermore, improved delineation of tumor margins may improvelocoregional control, shorten duration of surgery, and reducemorbidity because a more limited resection may be performed(44). Fluorescence imaging is particularly useful for superficiallylocated tumors, because the penetration depth of the fluorescentsignal is limited (28). Deeper located tumor lesions, for exampleintraparenchymal tumors or metastatic lymph nodes coveredwith fat, may be missed by fluorescence imaging. For theseapplications, the addition of a radiolabel to the imaging probecan be valuable. When the tumor has been localized intraopera-tively using the signal emitted by the radionuclide, resection canbe performed using fluorescence-guided surgery (12). Finally,the surgical cavity can be examined for remnant disease with

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dual-modality imaging. Another advantage of dual-modalityimaging is that a PET/CT or SPECT/CT can be obtained preop-eratively. This gives the surgeon an indication of the location ofthe primary tumor and/or metastases. Hence, preoperative andintraoperative imaging can be performed with the same imagingprobe. Thus, targeted dual-modality imaging could revolution-ize surgical oncology.

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

Authors' ContributionsConception and design:M.C.H. Hekman, O.C. Boerman, E. Oosterwijk, P.F.A.Mulders, M. RijpkemaDevelopment ofmethodology:M.C.H.Hekman,O.C. Boerman,M. deWeijert,E. Oosterwijk, P.F.A. Mulders, M. RijpkemaAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): M.C.H. Hekman, D.L. Bos, P.F.A. Mulders,M. RijpkemaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.C.H. Hekman, E. Oosterwijk, H.F. Langenhuijsen,P.F.A. Mulders, M. Rijpkema

Writing, review, and/or revision of the manuscript: M.C.H. Hekman,O.C. Boerman, D.L. Bos, E. Oosterwijk, H.F. Langenhuijsen, P.F.A. Mulders,M. RijpkemaAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases):M.C.H. Hekman, M. de Weijert, D.L. Bos,E. OosterwijkStudy supervision: M.C.H. Hekman, O.C. Boerman, E. Oosterwijk,H.F. Langenhuijsen, P.F.A. Mulders, M. Rijpkema

AcknowledgmentsThe authors thank Anja van Wincoop for her assistance in logistics on the

operation room to obtain the radical nephrectomy specimens. They also thankArieMaat for the preparation of the kidney lamellae and Tim vanOostenbruggefor his support during the experiments.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 9, 2015; revised April 12, 2016; accepted April 12, 2016;published OnlineFirst April 21, 2016.

References1. Borghesi M, Brunocilla E, Schiavina R, Martorana G. Positive surgical

margins after nephron-sparing surgery for renal cell carcinoma: Inci-dence, clinical impact, and management. Clin Genitourin Cancer 2013;11:5–9.

2. Swindle P, Eastham JA, Ohori M, Kattan MW, Wheeler T, Maru N, et al. Domargins matter? The prognostic significance of positive surgical margins inradical prostatectomy specimens. J Urol 2008;179(5 Suppl):S47–51.

3. Meric F, Mirza NQ, Vlastos G, Buchholz TA, Kuerer HM, Babiera GV, et al.Positive surgical margins and ipsilateral breast tumor recurrence predictdisease-specific survival after breast-conserving therapy. Cancer 2003;97:926–33.

4. Singletary SE.Surgical margins in patients with early-stage breast cancertreated with breast conservation therapy. Am J Surg 2002;184:383–93.

5. Dotan ZA, Kavanagh K, Yossepowitch O, Kaag M, Olgac S, Donat M, et al.Positive surgical margins in soft tissue following radical cystectomy forbladder cancer and cancer specific survival. J Urol 2007;178:2308–12;discussion 13.

6. Haque R, Contreras R, McNicoll MP, Eckberg EC, Petitti DB. Surgicalmargins and survival after head and neck cancer surgery. BMC Ear NoseThroat Disord 2006;6:2.

7. Cote V, Kost K, Payne RJ, Hier MP. Sentinel lymph node biopsy insquamous cell carcinoma of the head and neck: Where we stand now,and where we are going. J Otolaryngol 2007;36:344–9.

8. Tangoku A, Seike J, Nakano K, Nagao T, Honda J, Yoshida T, et al. Currentstatus of sentinel lymph node navigation surgery in breast and gastroin-testinal tract. J Med Invest 2007;54:1–18.

9. Somasundaram SK, Chicken DW, Keshtgar MR. Detection of the sentinellymph node in breast cancer. Br Med Bull 2007;84:117–31.

10. Luker GD, Luker KE. Optical imaging: Current applications and futuredirections. J Nucl Med 2008;49:1–4.

11. van Dam GM, Themelis G, Crane LM, Harlaar NJ, Pleijhuis RG, Kelder W,et al. Intraoperative tumor-specific fluorescence imaging in ovarian cancerby folate receptor-alpha targeting: First in-human results. Nat Med2011;17:1315–9.

12. Vidal-Sicart S, van Leeuwen FW, van den Berg NS, Valdes Olmos RA.Fluorescent radiocolloids: Are hybrid tracers the future for lymphaticmapping? Eur J Nucl Med Mol Imaging 2015;42:1627–30.

13. Verbeek FP, Tummers QR, RietbergenDD, Peters AA, Schaafsma BE, van deVelde CJ, et al. Sentinel lymph node biopsy in vulvar cancer usingcombined radioactive and fluorescence guidance. Int J Gynecol Cancer2015;25:1086–93.

14. van den BergNS, SimonH, KleinjanGH, Engelen T, Bunschoten A,WellingMM, et al. First-in-human evaluation of a hybrid modality that allows

combined radio- and (near-infrared) fluorescence tracing during surgery.Eur J Nucl Med Mol Imaging 2015;42:1639–47.

15. Brouwer OR, van den Berg NS, Matheron HM, van der Poel HG, van RhijnBW, Bex A, et al. A hybrid radioactive and fluorescent tracer for sentinelnode biopsy in penile carcinoma as a potential replacement for blue dye.Eur Urol 2014;65:600–9.

16. Lutje S, Rijpkema M, Franssen GM, Fracasso G, Helfrich W, Eek A, et al.Dual-modality image-guided surgery of prostate cancer with a radiola-beled fluorescent anti-PSMA monoclonal antibody. J Nucl Med 2014;55:995–1001.

17. Muselaers CH, Rijpkema M, Bos DL, Langenhuijsen JF, Oyen WJ, MuldersPF, et al. Radionuclide and fluorescence imaging of clear cell renal cellcarcinoma using dual labeled anti-carbonic anhydrase IX antibody G250.J Urol 2015;194:532–8.

18. Muselaers CH, Stillebroer AB, Rijpkema M, Franssen GM, Oosterwijk E,Mulders PF, et al. Optical Imaging of renal cell carcinoma with anti-carbonic anhydrase IX monoclonal antibody girentuximab. J Nucl Med2014;55:1035–40.

19. Rijpkema M, Oyen WJ, Bos D, Franssen GM, Goldenberg DM, BoermanOC. SPECT- and fluorescence image-guided surgery using a dual-labeledcarcinoembryonic antigen-targeting antibody. J Nucl Med 2014;55:1519–24.

20. Lutje S, Rijpkema M, Helfrich W, Oyen WJ, Boerman OC. Targetedradionuclide andfluorescencedual-modality imaging of cancer: Preclinicaladvances and clinical translation. Mol Imaging Biol 2014;16:747–55.

21. Oosterwijk E, Ruiter DJ, Hoedemaeker PJ, Pauwels EK, Jonas U, Zwarten-dijk J, et al. Monoclonal antibody G 250 recognizes a determinant presentin renal-cell carcinoma and absent from normal kidney. Int J Cancer1986;38:489–94.

22. Steffens MG, Boerman OC, Oosterwijk-Wakka JC, Oosterhof GO, WitjesJA, Koenders EB, et al. Targeting of renal cell carcinoma with iodine-131-labeled chimeric monoclonal antibody G250. J Clin Oncol 1997;15:1529–37.

23. Muselaers CH, Boerman OC, Oosterwijk E, Langenhuijsen JF, Oyen WJ,Mulders PF. Indium-111-labeled girentuximab immunoSPECT as a diag-nostic tool in clear cell renal cell carcinoma. Eur Urol 2013;63:1101–6.

24. Divgi CR, Pandit-Taskar N, Jungbluth AA, Reuter VE, Gonen M, RuanS, et al. Preoperative characterisation of clear-cell renal carcinomausing iodine-124-labelled antibody chimeric G250 (124I-cG250) andPET in patients with renal masses: A phase I trial. Lancet Oncol2007;8:304–10.

25. Divgi CR, Uzzo RG, Gatsonis C, Bartz R, Treutner S, Yu JQ, et al. Positronemission tomography/computed tomography identification of clear cell






renal cell carcinoma: results from the REDECT trial. J Clin Oncol 2013;31:187–94.

26. van Dijk J, Oosterwijk E, van Kroonenburgh MJ, Jonas U, Fleuren GJ,Pauwels EK, et al. Perfusion of tumor-bearing kidneys as a model forscintigraphic screening of monoclonal antibodies. J Nucl Med 1988;29:1078–82.

27. Cohen R, Stammes MA, de Roos IH, Stigter-van Walsum M, Visser GW,van Dongen GA. Inert coupling of IRDye800CW to monoclonal anti-bodies for clinical optical imaging of tumor targets. EJNMMI Res 2011;1:31.

28. Frangioni JV. In vivo near-infrared fluorescence imaging. Curr Opin ChemBiol 2003;7:626–34.

29. Rijpkema M, Bos DL, Cornelissen AS, Franssen GM, GoldenbergDM, Oyen WJ, et al. Optimization of dual-labeled antibodies fortargeted intraoperative imaging of tumors. Mol Imaging 2015;14:15–25.

30. Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PAJr. Determination of theimmunoreactive fraction of radiolabeled monoclonal antibodies by linearextrapolation to binding at infinite antigen excess. J Immunol Methods1984;72:77–89.

31. Muselaers CH, Stillebroer AB, Rijpkema M, Franssen GM, Oosterwijk E,Mulders PF, et al. Optical imaging of renal cell carcinoma with anti-carbonic anhydrase IX monoclonal antibody girentuximab. J Nucl Med2014;55:1035–40.

32. Thorstenson A, Harmenberg U, Lindblad P, Ljungberg B, LundstamS. Impact of quality indicators on adherence to National andEuropean guidelines for renal cell carcinoma. Scand J Urol 2015:1–7.

33. Alanee S, Nutt M, Moore A, Holland B, Dynda D, Wilber A, et al. Partialnephrectomy for T2 renal masses: Contemporary trends and oncologicefficacy. Int Urol Nephrol 2015;47:945–50.

34. Marszalek M, Carini M, Chlosta P, Jeschke K, Kirkali Z, Knuchel R, et al.Positive surgical margins after nephron-sparing surgery. Eur Urol 2012;61:757–63.

35. Bensalah K, Pantuck AJ, Rioux-Leclercq N, Thuret R, Montorsi F, Karakie-wicz PI, et al. Positive surgical margin appears to have negligible impact onsurvival of renal cell carcinomas treated by nephron-sparing surgery. EurUrol 2010;57:466–71.

36. Terwisscha van Scheltinga AG, van DamGM, Nagengast WB, NtziachristosV, Hollema H, Herek JL, et al. Intraoperative near-infrared fluorescencetumor imaging with vascular endothelial growth factor and human epi-dermal growth factor receptor 2 targeting antibodies. J Nucl Med2011;52:1778–85.

37. Ogawa M, Regino CA, Seidel J, Green MV, Xi W, Williams M, et al. Dual-modality molecular imaging using antibodies labeled with activatablefluorescence and a radionuclide for specific and quantitative targetedcancer detection. Bioconjug Chem 2009;20:2177–84.

38. Sampath L, Kwon S, Hall MA, Price RE, Sevick-Muraca EM. Detection ofcancer metastases with a dual-labeled near-infrared/positron emissiontomography imaging agent. Transl Oncol 2010;3:307–217.

39. Hong H, Zhang Y, Severin GW, Yang Y, Engle JW, Niu G, et al. Multi-modality imaging of breast cancer experimental lung metastasis withbioluminescence and a monoclonal antibody dual-labeled with 89Zr andIRDye 800CW. Mol Pharm 2012;9:2339–49.

40. Costello B, Li C, Duff S, Butterworth D, Khan A, Perkins M, et al. Perfusionof 99Tcm-labeled CD105 Mab into kidneys from patients with renalcarcinoma suggests that CD105 is a promising vascular target. Int J Cancer2004;109:436–41.

41. Steffens MG, Oosterwijk E, Zegwaart-Hagemeier NE, van't Hof MA, Deb-ruyne FM, Corstens FH, et al. Immunohistochemical analysis of intratu-moral heterogeneity of [131I]cG250 antibody uptake in primary renal cellcarcinomas. Br J Cancer 1998;78:1208–13.

42. Oya M, Nakamura K, Baba S, Hata J, Tazaki H. Intrarenal satellites of renalcell carcinoma: Histopathologic manifestation and clinical implication.Urology 1995;46:161–4.

43. Maeda H. Toward a full understanding of the EPR effect in primary andmetastatic tumors as well as issues related to its heterogeneity. Adv DrugDeliv Rev 2015;91:3–6.

44. Rosenthal EL, Warram JM, de Boer E, Chung TK, Korb ML, Brandwein-Gensler M, et al. Safety and tumor specificity of cetuximab-IRDye800 forsurgical navigation in head and neck cancer. Clin Cancer Res . 2015;21:3658–66.

45. Burggraaf J, Kamerling IM, Gordon PB, Schrier L, de Kam ML, Kales AJ,et al. Detection of colorectal polyps in humans using an intravenouslyadministered fluorescent peptide targeted against c-Met. Nat Med 2015;21:955–61.


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2016;22:4634-4642. Published OnlineFirst April 21, 2016.Clin Cancer Res Marlène C.H. Hekman, Otto C. Boerman, Mirjam de Weijert, et al.

Kidney Perfusion StudyVivoExTargeted Dual-Modality Imaging in Renal Cell Carcinoma: An

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