bevacizumab targeting diffuse intrinsic pontine glioma ... · bevacizumab targeting diffuse...

Large Molecule Therapeutics

Bevacizumab Targeting Diffuse Intrinsic PontineGlioma:Results of 89Zr-BevacizumabPET Imagingin Brain Tumor ModelsMarc H.A. Jansen1, Tonny Lagerweij2,3, A. Charlotte P. Sewing1,2, Danielle J. Vugts4,Dannis G. van Vuurden1,2, Carla F.M. Molthoff4, Viola Caretti1,2,5, Susanna J.E. Veringa1,2,Naomi Petersen2, Angel M. Carcaboso6, David P. Noske2,3,W. Peter Vandertop3,Pieter Wesseling2,7,8, Guus A.M.S. van Dongen4, Gertjan J.L. Kaspers1, andEsther Hulleman1,2

Abstract

The role of the VEGF inhibitor bevacizumab in the treatment ofdiffuse intrinsic pontine glioma (DIPG) is unclear. We aim tostudy the biodistribution and uptake of zirconium-89 (89Zr)-labeled bevacizumab in DIPG mouse models. Human E98-FM,U251-FM glioma cells, andHSJD-DIPG-007-FLUC primary DIPGcells were injected into the subcutis, pons, or striatum of nudemice. Tumor growth wasmonitored by bioluminescence imaging(BLI) and visualized by MRI. Seventy-two to 96 hours after 89Zr-bevacizumab injections, mice were imaged by positron emissiontomography (PET), and biodistribution was analyzed ex vivo.High VEGF expression in human DIPG was confirmed in apublically available mRNA database, but no significant 89Zr-bevacizumab uptake could be detected in xenografts located inthe pons and striatum at an early or late stage of the disease. E98-

FM, and to a lesser extent the U251-FM and HSJD-DIPG-007subcutaneous tumors, showed high accumulation of 89Zr-beva-cizumab. VEGF expression could not be demonstrated in theintracranial tumors by in situ hybridization (ISH) but was clearlypresent in the perinecrotic regions of subcutaneous E98-FMtumors. The poor uptake of 89Zr-bevacizumab in xenograftslocated in the brain suggests that VEGF targeting with bevaci-zumab has limited efficacy for diffuse infiltrative parts of glialbrain tumors in mice. Translating these results to the clinicwould imply that treatment with bevacizumab in patientswith DIPG is only justified after targeting of VEGF has beendemonstrated by 89Zr-bevacizumab immuno-PET. We aim toconfirm this observation in a clinical PET study with patientswith DIPG. Mol Cancer Ther; 15(9); 2166–74. �2016 AACR.

IntroductionRecent advances in molecular and cellular cancer biology have

resulted in the identification of critical molecular tumor targetsinvolved in the different phases of tumor growth and spreading.

This knowledge has boosted the rational design of novel drugs,especially monoclonal antibodies and tyrosine kinase inhibitors.However, broad application of these targeted therapies to treatbrain tumors has lagged behind in daily clinical practice. By usingmolecular positron emission tomography (PET) imaging, targetexpression and biodistribution can be studied concurrently in arelatively noninvasive manner. It potentially allows the identifi-cation of those patients who may, or may not, benefit fromtargeted therapy.

A disease that may benefit from molecular imaging is diffuseintrinsic pontine glioma (DIPG), a childhoodmalignancy locatedin thebrainstem. In the past 40 years, theoutcomeof patientswithDIPGhas remainedunchanged,with less than 10%of the patientsbeing alive 2 years from diagnosis (1, 2). Given the lack ofgadolinium uptake on MRI in DIPG tumors (3, 4), it is plausiblethat the blood–brain barrier (BBB) in DIPG often remains intactwhich might explain the resistance to systemic chemotherapy inthese patients.

A well-studied drug target in gliomas is VEGF, a signal protein-stimulating angiogenesis and increasing vessel permeability (5).Overexpression of VEGF-A, its receptor VEGFR2, or both, havebeen implicated as poor prognostic markers in various clinicalstudies (5, 6). Bevacizumab is a recombinant humanized mono-clonal antibody that selectively binds with high affinity to allisoforms of human VEGF-A and neutralizes their biologicalactivity (5, 7). Studies in adult patients with recurrent high-grade

1Department of Pediatrics, Pediatric Hematology and Oncology, Can-cer Center, Amsterdam, the Netherlands. 2Neuro-oncology ResearchGroup Cancer Center, Amsterdam, the Netherlands. 3Department ofNeurosurgery VU University Medical Center and Academic MedicalCenter, Amsterdam, the Netherlands. 4Department of Radiology &Nuclear Medicine VU University Medical Center, Amsterdam, theNetherlands. 5Departments of Neurology, Pediatrics and Neurosur-gery, Stanford University School of Medicine, Stanford, California.6Preclinical Therapeutics and Drug Delivery Research Program,Department of Oncology, Hospital Sant Joan de D�eu, Barcelona,Spain. 7Department of Pathology VU University Medical Center.Amsterdam, the Netherlands. 8Department of Pathology, RadboudUniversity Medical Center, Nijmegen, The Netherlands.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

M.H.A. Jansen, T. Lagerweij, and A.C.P. Sewing contributed equally to this work.

Corresponding Author: Esther Hulleman, VU University Medical Center-CancerCenter Amsterdam, De Boelelaan 1117, Amsterdam 1081 HV, the Netherlands.Phone: 312-0444-1779; Fax: 312-0444-2124; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0558

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(9) September 20162166

on May 31, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst June 20, 2016; DOI: 10.1158/1535-7163.MCT-15-0558

http://mct.aacrjournals.org/

gliomas (HGG) reported high radiological response rates withbevacizumab treatment (7–9), but recently 2 large phase IIIrandomized studies showed no improvement in overall sur-vival with bevacizumab treatment in an upfront setting(10–12). Bevacizumab has been studied in a number of non-randomized trials in pediatric brain tumor patients. Efficacy inthese trials has been variable, with a subset of patients showingclear radiological and/or clinical improvement (13, 14). Therole of bevacizumab in the treatment of patients with DIPGis even less clear but is currently studied in several trials (refs.15–18; NCT00890786 and NCT01182350; clinicaltrials.gov;NTR2391 Trialregister.nl).

No validated methods are available to identify patients whomay potentially benefit from bevacizumab treatment. Lack ofclinical effect may be due to either poor transport of bevacizumabinto the tumor microenvironment due to an intact BBB, or a lackof VEGF expression. In this study, we analyzed VEGF(R) expres-sion in adult and childhood HGG, including DIPG tumors.Furthermore, we studied bevacizumab distribution in vivo usingmolecular PET imaging with 89Zirconium-labeled bevacizumabin murine DIPG models (19).

Materials and MethodsVEGF-A and VEGFR2 mRNA expression profiles

VEGF-A and VEGFR2 (KDR) mRNA expression in DIPG(n ¼ 27) and pediatric HGG (pHGG; n ¼ 53) were determinedin silico, using publicly available datasets, and compared with adataset of nonmalignant brain tissue (n ¼ 44), low-grade brain-stem glioma (n ¼ 6), and adult HGG (n ¼ 284). These datasetsinclude tumor material from biopsy, resection, and autopsy(DIPG). Differences were analyzed by two-way ANOVA andP < 0.01 was considered significant. As a validation of thesefindings, VEGF-associated gene expression was studied in normalbrain, low-grade brainstem glioma (LG-BSG), DIPG, and glio-blastoma by creating a heatmap using K-means clustering. Allexpression analyseswere performed using R2, a web-basedmicro-array analysis and visualization platform (http://r2.amc.nl).

Immunohistochemistry and in situ hybridizationFormalin-fixed, paraffin-embedded slides were sectioned from

xenograft tumors and brain tissue and subjected to immunohis-tochemical (IHC) staining. Briefly, after deparaffinization andheat-induced antigen retrieval, sections were incubated withprimary mouse anti-Ki67 antibodies (clone MIB-1, DAKO) over-night at 4�C. Thereafter, slides were washed and incubated withHRP-conjugated EnVision (DAKO) and subsequently stained byDAB with hematoxylin counterstaining.

For in situ hybridization (ISH), tumors were cut in 5-mm slicesand incubatedwith VEGF probes against the humanVEGF codingsequence using a previously described protocol (20). Sampleswere evaluated by microscopy with a Zeiss Axioskip microscope(HBO100W/Z) and equipped with a Canon digital camera andimaging software (Canon PowerShot A640, Canon Utilities,ZoomBrowser Ex. 5.7, Canon Inc.).

Labeling and quality control of 89Zr-bevacizumabBevacizumab was labeled with 89Zr using N-succinyl-desfer-

rioxamine (N-suc-Df) as described previously (21). In short, thechelator, desferrioxamine, was succinylated toN-suc-Df. Next, thehydroxamate groups were blocked with iron and the succinyl

group was activated as its TFP-ester (Fe-N-suc-Df-TFP ester).Bevacizumab (6 mg/mL) was reacted with 2 equivalents of Fe-N-suc-Df-TFP ester at pH 9 for 30 minutes at room temperature.Hereafter, iron was removed at pH 4.2 to 4.5 with an excess ofEDTA for 30 minutes at 35�C, and N-suc-Df-bevacizumab waspurifiedby size exclusion chromatography using aPD-10 column.Radiolabeling of N-suc-Df-bevacizumab was performed inHEPES buffer: to 200 mL 89Zr in 1 mol/L oxalic acid 90 mL 2MNa2CO3was added. After 3minutes, 300 mL 0.5mol/L HEPES,N-suc-Df-bevacizumab, and 700 mL 0.5 mol/L HEPES were added.After 60-minute reaction time, 89Zr-N-suc-Df-bevacizumab waspurified by PD10 using 5 mg/mL gentisic acid in 0.9% NaCl (pH4.9–5.4) as the mobile phase.

Radiochemical purity and antibody integrity were deter-mined using instant thin-layer chromatography (iTLC), high-performance liquid chromatography (HPLC), and SDS-PAGEfollowed by phosphor imager analysis. For analysis of immu-noreactivity, an ELISA-based assay was used. iTLC analysis of89Zr-bevacizumab was performed on TEC control chromatog-raphy strips (Biodex). As the mobile phase, citrate buffer (20mmol/L, pH 5.0) containing 10% acetonitrile was used. HPLCanalyses of bevacizumab modification and radiolabeling wereperformed using a Jasco HPLC system equipped with a Super-dex 200 10/30 GL size exclusion column (GE healthcare Lifesciences) using a mixture of 0.05 mol/L sodium phosphate,0.15 mol/L sodium chloride (pH 6.8), and 0.01 mol/L NaN3 asthe eluent at a flow rate of 0.5 mL/min. The radioactivity of theeluate was monitored using an inline NaI(Tl) radiodetector(RaytestSockett).

Cell lines and animal modelsAnimal experiments were performed in accordance with the

European Community Council Directive 2010/63/EU for labo-ratory animal care and theDutch Lawonanimal experimentation.The experimental protocol was validated and approved by thelocal committee on animal experimentation of the VUUniversityMedical Center. Athymic nude-Foxn1nu mice (6 weeks old) werepurchased from Harlan/Envigo and kept under filter top condi-tions and received food and water ad libitum.

The primary HSJD-DIPG-007 cell line was established fromDIPG tumor material obtained at Hospital Sant Joan de Deu(Barcelona, Spain) after autopsy from a 6-year-old patient andwas confirmed to have a H3F3A (K27M) and ACVR1 (R206H)mutation (22). The E98 cell line was obtained from RadboudUniversity Nijmegen Medical Center (Nijmegen, The Nether-lands; ref. 23), the U251 glioma cell line from ATCC. All celllines were transduced in our laboratory to express firefly luciferase(FLUC) and/or mCherry (24). Cell lines were mycoplasma-neg-ative and were authenticated by STR analysis modified from DeWeger and colleagues (25).

E98-FM cells were injected subcutaneously in female athymicnudemice (7–9weeks of age) to expand the number of cells (19).When the subcutaneous tumor reached a diameter of 1 cm, thetumorwas removed, and a single-cell suspensionwas prepared bymechanical disruption through a 100-mm nylon cell strainer.HSJD-DIPG-007-FLUC was cultured in tumor stem medium(TSM; ref. 26), U251-FM in DMEM supplemented with 10%FCS andpenicillin/streptomycin. Shortly before stereotactic injec-tion, cells werewashedoncewith PBS and concentrated to 1�105

cells per mL. Mice were stereotactically injected with 5 � 105 cellsin a final volume of 5 mL into either the pons or striatum

Bevacizumab Distribution in Brainstem Glioma Xenografts

www.aacrjournals.org Mol Cancer Ther; 15(9) September 2016 2167




or injected subcutaneously with 3� 106 cells in a final volume of100 mL/flank. Coordinates used for intracranial injections were1.0 mm X, �0.8 mm Y, 4,5 mm Z from the lambda for pontinetumors and 0.5 mm X, 2 mm Y, �2 mm Z from the bregma forthe striatum tumors. Coordinates were previously validated andon the basis of "The mouse brain in stereotaxic coordinates" byFranklin and Paxinos (27). Tumor engraftment wasmonitored bybioluminescence measurement of the Fluc signal. For E98-FM,early-stage tumors in the pons (n ¼ 8), striatum (n ¼ 7), andsubcutaneous xenografts (n¼ 3)were allowed to grow for 18 daysand late-stage tumors (n ¼ 9, striatum; n ¼ 3, subcutaneous) for35 days after xenograft injection. E98 xenograft tumors in theponswere not available 35 days postinjection because injection ofE98FM cells in the pons would result in death of the mice due totumor growth within 3 weeks. For HSJD-DIPG-007-FLUC andU251-FM, tumors (pons, striatum, subcutaneous) were evaluat-ed, at days 78 and 22, respectively (Fig. 2A).

Two to 3 days before the endpoint of the study, mice wereinjected intraperitoneally with 89Zr-labeled bevacizumab (40 mg,5 MBq for PET analysis and ex vivo biodistribution or 185 kBq forex vivo biodistribution only).

PET imaging and ex vivo analysisThedistributionof 89Zr-bevacizumabwas determined72hours

(ex vivo only) or 96 hours (PET followed by ex vivo analysis) afteradministration, as a minimum of 72-hour interval betweeninjection and scanning has previously been shown to achieveoptimal tumor-to-nontumor ratios (28). Scanning of E98-FMtumors was performed using a LSO/LYSO double-layer ECATHigh-Resolution Research Tomograph (HRRT, CTI/Siemens): asmall-animal and human brain 3-dimensional (3D) scanner withhigh spatial resolution (2.3–3.4mm full width at halfmaximum)and high sensitivity (29). Mice were anesthetized by isofluraneinhalation anesthesia (1.5 L O2/min and 2.5% isoflurane) beforepositioning in the HRRT. A static transmission scan (6 minutes)using a rotating 740 MBq 137Cs point source was performed.Before positioning the mice in the HRRT, a canula was placedintraperitoneally to enable later injection of 18F(�). Static imagesof 60-minute acquisition time were obtained. Immediately there-after, 18F(�) was injected intraperitoneally (10 MBq/mouse) forcolocalization of bone structures (static images of 60-minuteacquisition). Body temperature was controlled with a heatedplatform (kept at 37�C).

Figure 2.

A, schematic overview of theexperiment. Cells are injected at day 0in the pons, striatum, or subcutis(early-stage tumors) or the striatumand subcutis (late-stage E98-FMtumors). Tumor progression wasmonitored by bioluminescentimaging. The time point for 89Zr-bevacizumab injectionwas dependenton the growth speed of used celllines. This injection was done 72 to96 hours before PET scanning. MRI,PET, or PET/CT imaging was followedby ex vivo measurement of 89Zr-bevacizumab accumulation in tissues(biodistribution). B, charged coupledevice (CCD) camera images of micebearing E98-FM tumors. BLI imageswere obtained at the study endpoint,day 18 (early stage, top) and day35 (late stage, bottom).

Figure 1.

Expression profiles of VEGF-A andVEGFR2. A, box plots representingrelative median mRNA expression inDIPG (n ¼ 27) and pHGG (n ¼ 53)versus datasets of normal brain tissue(n ¼ 44, dark grey), low-gradebrainstem glioma (n ¼ 6), and adultHGG (n ¼ 284). VEGF-Aoverexpression is shown in DIPGcompared with normal brain andcompared with adult glioma. B,VEGFR2 is not overexpressed in DIPGand pediatric glioma compared withnormal brain. � , P < 0.01, ANOVA.

Jansen et al.

Mol Cancer Ther; 15(9) September 2016 Molecular Cancer Therapeutics2168




Mice with HSJD-DIPG-007-FLUC or U251-FM tumors andwhere tumor growth was confirmed by increase of BLI signalwere imaged using another, preclinical, PET system, (NanoscanPET-CT, MEDISO), 72 hours after 89Zr-bevacizumab injection.PET images were analyzed using AMIDE software (Amide's aMedical Image Data Examiner, version 1.0.1; ref. 30).

MRIMice with representative HSJD-DIPG-007-FLUC or U251-FM

tumors were selected for MRI on the basis of the intensity of thebioluminescence signal. Gadolinium (750 mmol, Dotarem) wasadministered intravenously immediately before imaging. Micewere anesthetized by isoflurane inhalation anesthesia (1.5 L O2

/min and 2.5% isoflurane), placed in a preclinical PET-MRIsystem (Nanoscan system, MEDISO), and T1- and T2-weighedimages with gadolinium contrast were acquired. MR images wereanalyzed usingMIPAV software (Medical Image Processing, Anal-ysis, and Visualization, version 7.2.0).

Ex vivo analysisImmediately after PET imaging, animals were sacrificed for ex

vivo tissue distribution analysis. Blood, urine, tumor, and varioustissues were excised, rinsed in PBS to remove residual blood, andweighed. Radioactivity in blood and tissues (in percentageinjected dose per gram of tissue: % ID/g) was determined usingan LKB 1282 gamma-counter (Compugamma, LKB Wallac).Differences in the amount of radioactivity inhealthy brain regionsversus subcutaneous-, pontine-, and striatal tumorswere analyzedby Kruskal–Wallis test with Dunn multiple comparison posthoctesting. P < 0.05 was considered statistically significant.

ResultsVEGF-A-, VEGFR2-, and VEGF-associated gene expression inDIPG

A search in a publically available mRNA expression databasecurated by the Amsterdam Medical Center (R2.amc.nl) revealedthat VEGF-A mRNA is overexpressed in DIPG compared with

normal brain and compared with low-grade brainstem glioma(LG-BSG) and non-pontine adult and pediatric HGG (Fig. 1A; P <0.01, ANOVA). VEGFR2 (KDR)mRNAexpressionwas low inbothpediatric and adult glioma, compared with normal brain (Fig. 1B;P<0.01, ANOVA). Aheatmap generated usingK-means clusteringof expression of VEGF-A–associated genes confirmed an aberrantVEGF-A pathway to be more prominent in DIPG compared withLG-BSG. Most DIPG (red) samples clustered together with pedi-atricGBM(green) and LG-BSG (blue) clusteredwith normal braintissue (purple) (Supplementary Fig. S1).

Biodistribution of 89Zr-bevacizumabAfter tumor growth was confirmed by an increase in BLI signal,

89Zr-bevacizumab was injected in these animals. Seventy-two to96 hours after intraperitoneal injection of 89Zr-bevacizumab,animals were imaged by PET; a schematic overview of the exper-iment is given in Fig. 2, with representative BLI figures of E98-FM–

engrafted mice shown in Fig. 2B. 89Zr-bevacizumab uptake wasnot visible in the E98 gliomas located in the pons (only earlystage, Fig. 3A) or in the striatum at early or late stages. The rest ofthe brain showed no uptake of 89Zr-bevacizumab either, inde-pendently of the presence of a tumor. However, subcutaneousE98 tumors showed high accumulation of 89Zr-bevacizumabindicated by a red hotspot (Fig. 3A). Ex vivo tissue distributionmeasurements confirmed that no significant 89Zr-bevacizumabuptake was detected in the brain or brain tumor at any stage ofdisease,whereas therewashighuptake in the subcutaneous tumor(P < 0.01; Fig. 4A). Besides accumulation in the subcutaneoustumor with an average level of 50% ID/g, 89Zr-bevacizumabwas observed in all animals (to a lesser extent) in the blood pooland in well-perfused organs, such as the liver, spleen, and lungs(Fig. 5). Experiments using the 2 other cell lines (U251FM andHSJD-DIPG-007-FLUC) showed comparable results. Using thesecell lines, no uptake of 89Zr-bevacizumab was visualized on PETin any of the xenografts (Fig. 3B). Ex vivo biodistribution analysisshowed higher 89Zr-bevacizumab uptake in subcutaneous HSJD-DIPG-007-FLUC tumors (P < 0.05) as compared with brain(areas) without tumor (Fig. 4B). The subcutaneous U251FM

Figure 3.

A, 89Zr-PET combined with 18F(-)imaging of mice with an E98 tumorlocated in the pons (top) orsubcutaneous (bottom). White circlesindicate expected location ofintracranial tumors.While nouptake of89Zr-bevacizumab is observed in thepontine tumor, the subcutaneoustumor shows a PET hotspot reflecting89Zr-bevacizumabaccumulation in thetumor (arrow). In all animals, highintensity (visualized by red color code)is seen in the heart and liver of themice, reflecting 89Zr-bevacizumab inthe blood pool. Images acquired withSiemens HRRT. B, immuno-PETscanning of mice with HSJD-DIPG-007-Fluc (top and middle) or U251FMtumors (bottom) confirmed the lack ofbevacizumab uptake in intracranialtumors. Image acquired with MEDISONanoscan PET-CT system. Whitecircles indicate expected location ofintracranial tumors.






tumors showed no significant increase in 89Zr-bevacizumabuptake (Fig. 4C) as compared with non-tumor brain. Of note,both HSJD-DIPG-007-FLUC and U251FM cells formed only verysmall subcutaneous tumors during the time window in whichmice baring intracranial tumors had reached human endpoints(78 vs. 22 days after tumor injection).

ISH of VEGFTo determine whether the differences in 89Zr-bevacizumab

uptake in the subcutaneous and the intracranial tumors were due

to an impaired distribution of 89Zr-bevacizumab into the brain orto a differential expression of its target (or both), VEGF expressionwas analyzed in the different xenografts. ISH confirmed expres-sion of VEGF in the subcutaneous E98FM tumor (Fig. 6A),whereas VEGF expression was absent in E98FM brain tumors(Fig. 6B) and brain tissue without a xenograft. Of note, in thesubcutaneous tumors, VEGF was preferentially expressed in peri-necrotic areas, whereas necrosis was absent in all of the striatal andpontine tumors. A Ki67 (Mib-1) staining was performed toconfirm presence of proliferating tumor tissue (Fig. 6C and D).

Figure 4.89Zr-bevacizumabmeasured ex vivo by a gamma-counter and normalized to counts found in healthy brain tissue (brain tissue of animals without xenografted braintumors). A, uptake is significantly higher in the subcutaneous E98-FM tumors (�� , P < 0.01), but there is no significant difference in uptake in pontine orstriatal xenografts at any stage of the disease comparedwith normal brain.B, uptake in subcutaneous DIPG7-Fluc tumors is higher than in normal brain (� , P < 0.05).C, no significant differences between U251-FM tumors (subcutaneous or intracranial) versus normal brain.

Figure 5.89Zr-bevacizumab uptake measuredex vivo by radioactivity of the specificorgans and the tumor after dissection,expressed as percentage of theinjected dose per gram tissue in E98-FM tumor–bearing animals. Note thehigh uptake in subcutaneous tumors,compared with the negligible uptakein the intracerebral tumors.

Jansen et al.





In HSJD-DIPG-007-FLUC and U251FM tumors, VEGF mRNAexpression was not detectable in striatal and pontine gliomas.The subcutaneous tumors were too small to adequately performVEGF ISH and therefore no conclusions could be drawn regardingVEGF expression in these subcutaneous tumors.

MRITo visualize disruption of the BBB, mice with HSJD-DIPG-007-

FLUCandU251-FM intracranial tumorswere imagedbyMRI afterintravenous administration of gadolinium. In the diffusely grow-ing HSJD-DIPG-007-FLUC tumor, gadolinium enhancement onT1-weighed images was limited (Fig. 7A, arrow), whereas gado-linium enhancement was clearly visible in the U251-FM tumor(Fig. 7C, arrow). On T2-weighed images, tumors were not clearlyvisible (Fig. 7B and D; arrow).

DiscussionThe potential benefit of bevacizumab in the treatment of DIPG

is unclear, as efficacy depends on expression of VEGF-A as well asappropriate drug distribution (31). We used molecular PETimaging to study the influence of location and stage of diseaseon biodistribution of 89Zr-bevacizumab in 3 glioma mousemodels (pontine, striatal, subcutaneous) using 3 different celllines. The E98-FM pontine and striatal and HSJD-DIPG-007pontine xenograft models have previously been described toresemble the diffuse phenotype of humanDIPG and other diffuseHGGs (19, 32, 33). U251 has been described as an intracranialmurine tumor model that recapitulates most of the key figures ofadult glioblastoma (34). We found no significant uptake of 89Zr-bevacizumab in the intracranial tumor models at any stage of thedisease, nor in the normal/nonneoplastic surrounding brain. Incontrast, high accumulation of 89Zr-bevacizumabwas observed in

the subcutaneous E98-xenograft and moderate uptake in thesubcutaneous HSJD-DIPG-007-FLUC.

We initially hypothesized that lack of 89Zr-bevacizumabuptakecould be explained solely by poor distribution into the brain, aslarge molecules like monoclonal antibodies may not be able topass the BBB. This hypothesis is supported by the absence ofenhancement of the tumor on MRI after administration of gad-olinium in animals with HSJD-DIPG-007-FLUC pontine tumors.However, MRI analysis of U251-FM tumors in the brainstem ofmice showed clear gadolinium enhancement, which is indicativeof "leaky" blood vessels in the tumor. Furthermore, VEGF expres-sion of the E98FM glioma cells—analyzed by ISH—also differedbetween tumor locations: E98FM gliomas in both striatum andpons appeared VEGF-negative, whereas the subcutaneous E98FMtumorswerepartly VEGF-positive. Thedifferences inVEGFexpres-sion of the tumors in distinct locations originating from the samecell line confirm that the orthotopic microenvironment and theresulting growth pattern significantly influence gene expression inglioma cells, a phenomenon that has been described previously(19, 20).Moreover, it has been shown that in glioblastoma, VEGFis predominantly overexpressed in hypoxic, perinecrotic cells(35, 36). Indeed, in our study, the VEGF expression in subcuta-neous E98FM tumors was especially present around areas ofnecrosis, whereas in intracranial E98FM tumors, necrosis andVEGF expression were lacking and this also coincided with lackof bevacizumab uptake studied ex vivo and by PET. Of note,bevacizumab does not bind to murine VEGF-A (37), but astypically, the neoplastic cells are upregulating VEGF expressionin tumor angiogenesis, we consider it unlikely that stromal cell–derived mouse VEGF-A plays an important role in this particularxenograft model (33).

In contrast to our preclinical findings, the in silico analysis thatwe performed in this study indicates that human DIPG tumors

Figure 6.

VEGF expression in DIPG tumors asassessed by VEGF ISH. A, positiveVEGF staining in subcutaneousE98-FM tumor cells (magnification,20�, insert 2.5�). The tumor showsextensive necrosis surrounded byVEGF-positive cells. B, no VEGFmRNA is detected in the E98-FMtumor tissue in the striatum(magnification, 20�, insert 1.25�).C, Ki67 staining shows the presenceof proliferating tumor cells in thesubcutaneous tumor tissue(magnification 20�, insert 2.5�).D, Ki67-positive tumor cells in thestriatal E98-FM tumor (magnification20�, insert 10�).






have relatively high expression levels of VEGF mRNA. However,the majority of tumors (23 of 27) used for the microarrayexperiments were collected post-mortem (38, 39) and thereforethese samples represent the end stage of the disease and are post-radiation therapy. In the end stage of the disease, DIPG is knownto have necrotic areas with microvascular proliferations and BBBdisruption, compatible with the histology of a glioblastoma,which is associated with high VEGF expression. Although thenumbers are low, it is important to point out that VEGF-Aexpression levels in samples obtained during pretreatment werelow compared with expression in post-mortem/end-stage sam-ples (Supplementary Fig. S2). In addition, biopsy samples thatwere included in the analysis are frequently directed at contrast-enhancing regions, and this "biased sampling" may well lead tooverestimation of the role of VEGF in the tumor as awhole.We arecurrently studying the differences in VEGF-A expression in autop-sy-derived DIPG tissue between the perinecrotic areas and themore diffusely growing tumor parts without necrosis.

Experimental and clinical research in both adult and pHGGand DIPG has suggested that there is a complex relationbetween a histologically diffuse growth pattern of brain tu-mors, VEGF expression, and availability and BBB integrity (20,40–44). Traditionally, VEGF is viewed as the main cause of in-creased BBB permeability in central nervous system (CNS)tumors as represented by contrast-enhancing lesions on MRI(45). More recently, anti-VEGF therapy is thought to potenti-ally induce a more diffuse and distant spread of tumor cells(41, 42). In contrast to adult HGGs, gadolinium contrastenhancement on MRI in DIPGs at diagnosis is generally lim-ited, with 50% of the patients showing no enhancement at all(3). The lack of gadolinium enhancement suggests an intactBBB, at least for large molecules, in a substantial percentage of

the patients, coinciding with low VEGF expression and inabi-lity of bevacizumab to target the tumor (46).

Results from our preclinical PET, MRI with gadoliniumcontrast, and ISH studies suggest that this relation is not sostraightforward, pressing the need for studying VEGF targetingin patients treated with anti-VEGF therapy. One could howeverargue whether high local tumor accumulation of bevacizumabis at all needed to obtain potential therapeutic effects in DIPG.Bevacizumab is capable of decreasing VEGF levels in blood toundetectable range in less than 20 days in a large cohort ofadult cancer patients (47), but only a subgroup of patientsresponded to anti-VEGF therapy. Also in DIPG, decreasedphosphoVEGR2 levels in peripheral blood mononuclear cells(PBMC) did not correlate with treatment response (15). In theE98 xenograft model used in this study, treatment with bev-acizumab did not increase survival, nor did it influence thegrowth pattern in the diffusely growing parts of the tumor (36).This suggest simply decreasing VEGF in the blood pool is, atleast for the tumor types studied and our xenograft model,often insufficient for adequate tumor targeting and instead,local bevacizumab accumulation seems needed (47–49).

The results of immuno-PET imaging and VEGF-ISH in theseDIPGmodels are in line with the poor clinical response rates thusfar obtained with bevacizumab in children with DIPG (15). Thedata presented suggest that no adequate uptake of bevacizumabwill occur in diffusely growing gliomas, which present with BBBdisruption but without drastically increased VEGF expression.Therefore, we suggest that bevacizumab treatment is only justifiedif targeting of VEGF by bevacizumab has been visualized byimmuno-PET scan.We aim to confirm this hypothesis in a clinicalPET study with patients with DIPG.

Future directionsThis study underlies the importance of using strong biological

and biodistributional rationale before using any therapy in anypatient. Following the results of this study, we developed amolecular drug imaging trial with 89Zr-bevacizumab in childrenwith DIPG (study number NTR3518 www.trialregister.nl). Thistechnique aims to further unravel the role of bevacizumab treat-ment in DIPG. Ideally, such molecular imaging is combined withVEGF-A and VEGFR2 expression analysis on tumor tissue origi-nating from biopsies taken from several (contrast-enhancing andnonenhancing) parts of the tumor. BecauseDIPG can generally bediagnosed on the basis of its typical radiological presentation andthe delicate nature of the brain involved, taking biopsies fromDIPGs is still no common practice and sampling of multipleregions is even more cumbersome. In general, integrating molec-ular imaging with radiolabeled drugs (classic cytostatic agents,small molecules, other monoclonal antibodies) in the treatmentof childhood brain cancer provides an insight in drug targetingand might help personalize treatment and thereby to avoidunnecessary side effects of drugs that do not reach the tumor.

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

Authors' ContributionsConception and design: M.H.A. Jansen, T. Lagerweij, A.C.P. Sewing, D.G. vanVuurden, G.A.M.S. van Dongen, G.J.L. Kaspers, E. HullemanDevelopment of methodology: M.H.A. Jansen, T. Lagerweij, A.C.P. Sewing,V. Caretti, G.A.M.S. van Dongen

Figure 7.

MRI scans of pontine tumors. The 89Zr-bevacizumab uptake in the tumors ofthese 2mice is presented as%ID/g.A, limited gadolinium contrast enhancementin the tumor area (arrow) on T1 scan. B, HSJD-DIPG-007-FLUC tumor is poorlyvisible on T2-weighed MRI image (arrow). C, clear gadolinium contrastenhancement in the U251-FM tumor area (T1-weighed MRI scan) indicatesdisruption of the BBB. D, U251-FM tumor is visible on T2-weighed MRI scan.

Jansen et al.





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M.H.A. Jansen, T. Lagerweij, A.C.P. Sewing, D.J. Vugts,C.F.M. Molthoff, V. Caretti, N. Petersen, A.M. Carcaboso, D.P. Noske,P. WesselingAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.H.A. Jansen, T. Lagerweij, A.C.P. Sewing,D.J. Vugts, C.F.M. Molthoff, S.J.E. Veringa, P. Wesseling, G.A.M.S. van DongenWriting, review, and/or revision of the manuscript: T. Lagerweij, A.C.P.Sewing, D.J. Vugts, D.G. van Vuurden, C.F.M. Molthoff, A.M. Carcaboso,D.P. Noske,W.P. Vandertop, P.Wesseling, G.A.M.S. vanDongen, G.J.L. Kaspers,E. HullemanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.H.A. Jansen, T. LagerweijStudy supervision: G.J.L. Kaspers, E. Hulleman

AcknowledgmentsWe are thankful to Ricardo Vos, Iris Mes and Alex Poot (Department of

Radiology & Nuclear Medicine, VUmc) for technical support with labeling of

PET tracers and PET imaging and PiotrWaranecki and Yanyan Veldman (Neuro-oncology ResearchGroup, VUmc) for technical assistance and animal handling.DIPG research at the VUmchas beenmade possible by the invaluable support ofthe Semmy Foundation, Stichting Egbers and of Stichting Kika (Children-Cancer-free, project 69).

Grant SupportE. Hulleman received funding from Semmy Foundation and Stichting Kika

(Children-Cancer-free, project 69).M.H. Jansen received funding fromStichtingEgbers. A.M. Carcaboso acknowledges funding from ISCIII-FEDER (CP13/00189).

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 July 8, 2015; revised May 12, 2016; accepted May 26, 2016;published OnlineFirst June 20, 2016.

References1. Hargrave D, Bartels U, Bouffet E. Diffuse brainstem glioma in children:

critical review of clinical trials. Lancet Oncol 2006;7:241–8.2. Jansen MHA, van Vuurden DG, Vandertop WP, Kaspers GJL. Diffuse

intrinsic pontine gliomas: a systematic update on clinical trials andbiology.Cancer Treat Rev 2012;38:27–35.

3. Hargrave D, Chuang N, Bouffet E. Conventional MRI cannot predictsurvival in childhood diffuse intrinsic pontine glioma. J Neurooncol2008;86:313–9.

4. Jansen MH, Veldhuijzen van Zanten SE, Sanchez Aliaga E, Heymans MW,Warmuth-Metz M, Hargrave D, et al. Survival predictionmodel of childrenwith diffuse intrinsic pontine glioma based on clinical and radiologicalcriteria. Neuro Oncol 2015;17:160–6.

5. Ferrara N. Vascular endothelial growth factor: basic science and clinicalprogress. Endocr Rev 2004;25:581–611.

6. Shibuya M. Differential roles of vascular endothelial growth factorreceptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol 2006;39:469–78.

7. Friedman HS, Prados MD, Wen PY, Mikkelsen T, Schiff D, Abrey LE, et al.Bevacizumab alone and in combination with irinotecan in recurrentglioblastoma. J Clin Oncol 2009;27:4733–40.

8. Vredenburgh JJ, Desjardins A, Herndon JE, Dowell JM, ReardonDA,QuinnJA, et al. Phase II trial of bevacizumab and irinotecan in recurrentmalignantglioma. Clin Cancer Res 2007;13:1253–9.

9. Vredenburgh JJ, Desjardins A, Herndon JE, Marcello J, Reardon DA, QuinnJA, et al. Bevacizumab plus irinotecan in recurrent glioblastoma multi-forme. J Clin Oncol 2007;25:4722–9.

10. Chinot OL, Wick W, Mason W, Henriksson R, Saran F, Nishikawa R, et al.Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glio-blastoma. N Engl J Med 2014;370:709–22.

11. Gilbert MR, Dignam JJ, Armstrong TS, Wefel JS, Blumenthal DT, Vogel-baum MA, et al. A randomized trial of bevacizumab for newly diagnosedglioblastoma. N Engl J Med 2014;370:699–708.

12. Khasraw M, Ameratunga MS, Grant R, Wheeler H, Pavlakis N. Antiangio-genic therapy for high-grade glioma. Cochrane Database Syst Rev 2014;9:CD008218.

13. Narayana A, Kunnakkat S, Chacko-Mathew J, Gardner S, Karajannis M,Raza S, et al. Bevacizumab in recurrent high-grade pediatric gliomas. NeuroOncol 2010;12:985–90.

14. Parekh C, Jubran R, Erdreich-Epstein A, Panigrahy A, Bluml S,Finlay J, et al. Treatment of children with recurrent high gradegliomas with a bevacizumab containing regimen. J Neurooncol2011;103:673–80.

15. Gururangan S, Chi SN, Young Poussaint T, Onar-Thomas A, Gilbert-son RJ, Vajapeyam S, et al. Lack of efficacy of bevacizumab plusirinotecan in children with recurrent malignant glioma and diffusebrainstem glioma: a pediatric brain tumor consortium study. J ClinOncol 2010;28:3069–75.

16. Aguilera DG, Mazewski C, Hayes L, Jordan C, Esiashivilli N, Janns A, et al.Prolonged survival after treatment of diffuse intrinsic pontine glioma with

radiation, temozolamide, and bevacizumab: report of 2 cases. J PediatrHematol Oncol 2013;35:e42–6.

17. Zaky W, Wellner M, Brown RJ, Bl€uml S, Finlay JL, Dhall G.Treatment of children with diffuse intrinsic pontine gliomas withchemoradiotherapy followed by a combination of temozolomide,irinotecan, and bevacizumab. Pediatr Hematol Oncol 2013;30:623–32.

18. Hummel TR, Salloum R, Drissi R, Kumar S, Sobo M, Goldman S, et al. Apilot study of bevacizumab-based therapy in patients with newly diag-nosed high-grade gliomas and diffuse intrinsic pontine gliomas. J Neu-rooncol 2016;127:53–61.

19. Caretti V, Zondervan I, Meijer DH, Idema S, Vos W, Hamans B, et al.Monitoring of tumor growth and post-irradiation recurrence in adiffuse intrinsic pontine glioma mouse model. Brain Pathol 2011;21:441–51.

20. Roodink I, van der Laak J, Kusters B,Wesseling P, Verrijp K, deWaal R, et al.Development of the tumor vascular bed in response to hypoxia-inducedVEGF-A differs from that in tumors with constitutive VEGF-A expression.Int J Cancer 2006;119:2054–62.

21. Verel I, Visser GWM,Boellaard R, Stigter-van Walsum M, Snow GB, vanDongen GAMS. 89Zr immuno-PET: comprehensive procedures for theproduction of 89Zr-labeled monoclonal antibodies. J Nucl Med 2003;44:1271–81.

22. Taylor KR, Mackay A, Truffaux N, Butterfield YS, Morozova O, Philippe C,et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontineglioma. Nat Genet 2014;46:457–61.

23. BernsenHJ, RijkenPF, PetersH, BakkerH, vander Kogel AJ. The effect of theanti-angiogenic agent TNP-470 on tumor growth and vascularity in lowpassaged xenografts of human gliomas in nude mice. J Neurooncol1998;38:51–7.

24. Mir SE, De Witt Hamer PC, Krawczyk PM, Balaj L, Claes A, Niers JM,et al. In silico analysis of kinase expression identifies WEE1 as agatekeeper against mitotic catastrophe in glioblastoma. Cancer Cell2010;18:244–57.

25. de Weger RA, Tilanus MG, Scheidel KC, van den Tweel JG, Verdonck LF.Monitoring of residual disease and guided donor leucocyte infusion afterallogeneic bonemarrow transplantation by chimaerism analysiswith shorttandem repeats. Br J Haematol 2000;110:647–53.

26. Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily M-A, et al.Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.Nat Med 2015;21:555–9.

27. Paxinos GKF. The mouse brain in stereotaxic coordinates, compact, 3rdEdition | George Paxinos, Keith Franklin | ISBN 9780123742445[Internet]; 2008. Available from: http://store.elsevier.com/product.jsp?isbn¼9780123742445&pagename¼search.

28. Verel I, Visser GWM, Boellaard R, Boerman OC, van Eerd J, Snow GB, et al.Quantitative 89Zr immuno-PET for in vivo scouting of 90Y-labeled mono-clonal antibodies in xenograft-bearing nude mice. J Nucl Med 2003;44:1663–70.






29. de Jong HWAM, van Velden FHP, Kloet RW, Buijs FL, Boellaard R,Lammertsma AA. Performance evaluation of the ECAT HRRT: an LSO-LYSO double layer high resolution, high sensitivity scanner. PhysMed Biol2007;52:1505–26.

30. Loening AM, Gambhir SS. AMIDE: a free software tool for multimod-ality medical image analysis. Mol Imaging 2003;2:131–7.

31. Papadopoulos N, Martin J, Ruan Q, Rafique A, Rosconi MP, Shi E, et al.Binding and neutralization of vascular endothelial growth factor (VEGF)and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angio-genesis 2012;15:171–85.

32. Boult JKR, Taylor KR, Vinci M, Popov S, Jury A, Molinari V, et al. Abstract3271: Novel orthotopic pediatric high grade glioma xenografts evaluatedwith magnetic resonance imaging mimic human disease. Cancer Res2015;75:3271–3271.

33. Claes A, Schuuring J, Boots-Sprenger S, Hendriks-Cornelissen S,Dekkers M, van der Kogel AJ, et al. Phenotypic and genotypiccharacterization of orthotopic human glioma models and its rele-vance for the study of anti-glioma therapy. Brain Pathol 2008;18:423–33.

34. Jacobs VL, Valdes PA, Hickey WF, De Leo JA. Current review of in vivoGBMrodentmodels: emphasis on theCNS-1 tumourmodel. ASNNeuro2011;3:e00063.

35. JohanssonM, Br€annstr€omT, BergenheimAT, Henriksson R. Spatial expres-sion of VEGF-A in human glioma. J Neurooncol 2002;59:1–6.

36. Navis AC, Hamans BC, Claes A, Heerschap A, Jeuken JWM, Wesseling P,et al. Effects of targeting the VEGF and PDGF pathways in diffuse ortho-topic glioma models. J Pathol 2011;223:626–34.

37. Yu L, Wu X, Cheng Z, Lee CV, LeCouter J, Campa C, et al. Interactionbetween bevacizumab and murine VEGF-A: a reassessment. InvestOphthalmol Vis Sci 2008;49:522–7.

38. Paugh BS, Broniscer A, Qu C, Miller CP, Zhang J, Tatevossian RG, et al.Genome-wide analyses identify recurrent amplifications of receptor tyro-sine kinases and cell-cycle regulatory genes in diffuse intrinsic pontineglioma. J Clin Oncol 2011;29:3999–4006.

39. Paugh BS, Qu C, Jones C, Liu Z, Adamowicz-Brice M, Zhang J, et al.Integrated molecular genetic profiling of pediatric high-grade gliomasreveals key differences with the adult disease. J Clin Oncol 2010;28:3061–8.

40. Zhao L, Yang Z, Liu Y, YingH, ZhangH, Xue Y. Vascular endothelial growthfactor increases permeability of the blood-tumor barrier via caveolae-mediated transcellular pathway. J Mol Neurosci 2011;44:122–9.

41. P�aez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Vi~nals F, et al.Antiangiogenic therapy elicits malignant progression of tumors toincreased local invasion and distant metastasis. Cancer Cell 2009;15:220–31.

42. Salloum R, DeWire M, Lane A, Goldman S, Hummel T, Chow L, et al.Patterns of progression in pediatric patients with high-grade glioma ordiffuse intrinsic pontine glioma treated with Bevacizumab-based therapyat diagnosis. J Neurooncol 2015;121:591–8.

43. Gomez-Manzano C, Holash J, Fueyo J, Xu J, Conrad CA, Aldape KD, et al.VEGF Trap induces antiglioma effect at different stages of disease. NeuroOncol 2008;10:940–5.

44. Wesseling P, Ruiter DJ, Burger PC. Angiogenesis in brain tumors; patho-biological and clinical aspects. J Neurooncol 1997;32:253–65.

45. Gagner J-P, Law M, Fischer I, Newcomb EW, Zagzag D. Angiogenesis ingliomas: imaging and experimental therapeutics. Brain Pathol 2005;15:342–63.

46. Jansen MHA, Kaspers GJ. A new era for children with diffuse intrinsicpontine glioma: hope for cure? Expert Rev Anticancer Ther 2012;12:1109–12.

47. Jubb AM, Harris AL. Biomarkers to predict the clinical efficacy of bevaci-zumab in cancer. Lancet Oncol 2010;11:1172–83.

48. Jubb AM. Impact of vascular endothelial growth factor-A expression,thrombospondin-2 expression, and microvessel density on the treatmenteffect of bevacizumab in metastatic colorectal cancer. J Clin Oncol2005;24:217–27.

49. Shih T, Lindley C. Bevacizumab: an angiogenesis inhibitor for the treat-ment of solid malignancies. Clin Ther 2006;28:1779–802.


Jansen et al.




2016;15:2166-2174. Published OnlineFirst June 20, 2016.Mol Cancer Ther Marc H.A. Jansen, Tonny Lagerweij, A. Charlotte P. Sewing, et al.

Zr-Bevacizumab PET Imaging in Brain Tumor Models89Bevacizumab Targeting Diffuse Intrinsic Pontine Glioma: Results of

Updated version

10.1158/1535-7163.MCT-15-0558doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2016/06/18/1535-7163.MCT-15-0558.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/15/9/2166.full#ref-list-1

This article cites 48 articles, 9 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/15/9/2166.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/15/9/2166To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0558

http://mct.aacrjournals.org/content/suppl/2016/06/18/1535-7163.MCT-15-0558.DC1

http://mct.aacrjournals.org/content/15/9/2166.full#ref-list-1

http://mct.aacrjournals.org/content/15/9/2166.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/15/9/2166


bevacizumab targeting diffuse intrinsic pontine glioma ... · bevacizumab targeting diffuse...

Documents