Imaging, Diagnosis, Prognosis

Imaging Colon Cancer Response Following Treatment withAZD1152: A Preclinical Analysis of [18F]Fluoro-2-deoxyglucoseand 30-deoxy-30-[18F]Fluorothymidine Imaging

Maxim A. Moroz1, Tatiana Kochetkov1, Shangde Cai2, Jiyuan Wu1, Mikhail Shamis1, Jayasree Nair3,Elisa de Stanchina4, Inna Serganova1, Gary K. Schwartz3, Debabrata Banerjee6,Joseph R. Bertino6, and Ronald G. Blasberg1,4,5

AbstractPurpose: To determine whether treatment response to the Aurora B kinase inhibitor, AZD1152, could

be monitored early in the course of therapy by noninvasive [18F]-labeled fluoro-2-deoxyglucose, [18F]FDG,

and/or 30-deoxy-30-[18F]fluorothymidine, [18F]FLT, PET imaging.

Experimental design: AZD1152-treated and control HCT116 and SW620 xenograft-bearing animals

weremonitored for tumor size and by [18F]FDG, and [18F]FLT PET imaging. Additional studies assessed the

endogenous and exogenous contributions of thymidine synthesis in the two cell lines.

Results: Both xenografts showed a significant volume-reduction to AZD1152. In contrast, [18F]FDG

uptake did not demonstrate a treatment response. [18F]FLT uptake decreased to less than 20% of control

values in AZD1152-treated HCT116 xenografts, whereas [18F]FLT uptake was near background levels in

both treated and untreated SW620 xenografts. The EC50 for AZD1152-HQPA was approximately 10 nmol/

L in both SW620 and HCT116 cells; in contrast, SW620 cells were much more sensitive to methotrexate

(MTX) and 5-Fluorouracil (5FU) than HCT116 cells. Immunoblot analysis demonstrated marginally lower

expression of thymidine kinase in SW620 compared with HCT116 cells. The aforementioned results

suggest that SW620 xenografts have a higher dependency on the de novo pathway of thymidine utilization

than HCT116 xenografts.

Conclusions: AZD1152 treatment showed antitumor efficacy in both colon cancer xenografts. Although

[18F]FDG PET was inadequate in monitoring treatment response, [18F]FLT PET was very effective in

monitoring response in HCT116 xenografts, but not in SW620 xenografts. These observations suggest that

de novo thymidine synthesis could be a limitation and confounding factor for [18F]FLT PET imaging and

quantification of tumor proliferation, and this may apply to some clinical studies as well. Clin Cancer Res;

17(5); 1099–110. �2011 AACR.

Introduction

The objective of this study was to assess whetherresponse to treatment with the novel Aurora Kinase B

inhibitor, AZD1152, could be monitored noninvasivelyby [18F]-labeled fluoro-2-deoxyglucose, [18F]FDG, or 30-deoxy-30-[18F]fluorothymidine, [18F]FLT, PET imaging inan animal xenograft model. These studies were expected toprovide preclinical imaging assessments that would helpguide a decision of whether to proceed with comparablePET imaging studies in clinical trials with AZD1152. Bothradiopharmaceuticals are widely available for clinical PETimaging studies, and the potential exists for direct transla-tion to comparable phase I and II drug treatment andimaging trials.

Aurora kinases are overexpressed in a variety of humantumors, including colorectal tumors, leading to dysregula-tion of mitosis. Over the past 20 years, aurora kinases havebeen extensively studied and are now recognized as pro-mising targets for anticancer drug development (1). AuroraB kinase is the catalytic component of the chromosomalpassenger complex responsible for the accurate segregationof the chromatids, histone modification, and cytokinesis(2). The aurora kinase family comprises 3 related proteins

Authors' Affiliations: 1Department of Neurology; 2Cyclotron and Radio-chemistry Core Facility; 3Department of Medicine, Laboratory of New DrugDevelopment; 4Sloan Kettering Institute Molecular Pharmacology andChemistry Program, and 5Department of Radiology,Memorial Sloan-Ket-tering Cancer Center, New York, New York; and 6Departments of Medicineand Pharmacology, CINJ, RWJMS, UMDNJ, Newark, New Jersey

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

Corresponding Author: Ronald G. Blasberg, Departments of Neurologyand Radiology, MH (Box 52) Molecular Pharmacology & Chemistry Pro-gram, SKI Memorial Sloan Kettering Cancer Center (MSKCC), 415 E68Street, New York, NY 10065. Phone: 646-888-2211; Fax: 646-422-0408;E-mail: blasberg@neuro1.mskcc.org

doi: 10.1158/1078-0432.CCR-10-1430

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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that have similar homology in their catalytic domains (3).Expression of aurora A and B is linked to the proliferationof many types of cells, whereas aurora C expression seemsto be restricted to normal testicular tissue (4).

AZD1152 is a representative of a new class of small-molecule inhibitors, quinazoline-based compounds, withpotent activity against specific Aurora kinases (5, 6).AZD1152 is a quinazoline prodrug, which is rapidly con-verted in plasma into its active metabolite, AZD1152-hydroxyquinazoline pyrazol anilide (AZD1152-HQPA)with high affinity for aurora B and C kinases. AZD1152-HQPA is reported to have no substantial activity against apanel of other kinases (7). In preclinical models, AZD1152inhibited the growth of human colon, lung, and hemato-logic tumor xenografts (8–10). In a phase I clinical trial,AZD1152 was administered by weekly infusions to patientswith advanced, pretreated solid tumors. Dose limitingtoxicity (DLT) was neutropenia (grade 3), with some non-hematologic toxicity. Pharmacokinetic studies confirmedrapid conversion of AZD1152 into AZD1152-HQPA. Threepatients had stable disease (melanoma, nasopharyngealcarcinoma, and adenoid cystic carcinoma). Follow-up

studies are ongoing, based on preclinical tumor models,which suggested that prolonged drug administration sig-nificantly increased the proapoptotic effect of AZD1152-HQPA. AZD1152 has been in development for the treat-ment of patients with breast, colon, and lung cancer and iscurrently being studied for the treatment of acute myeloidleukemia (11–12).

This study focuses on 2 issues: (i) the assessment ofAZD1152 treatment effcacy using noninvasive PET ima-ging, and (ii) the importance of considering both the denovo and salvage components of thymidine utilization inthe interpretation of [18F]FLT imaging studies. Two humancolon carcinoma cell lines, HCT116 and SW620, andcorresponding subcutaneous xenografts were studied. Itwas recently reported that HCT116 xenografts were highlysensitive to AZD1152 treatment (13). The measured para-meters in this study included tumor volume and the uptakeof [18F]FDG and [18F]FLT. The objective of this study was toassess whether treatment response could be monitored bynoninvasive [18F]FDG and/or [18F]FLT PET imaging. Asecond objective developed during the course of this study,namely, to explain the differences in [18F]FLT uptakeobserved in untreated HCT116 and SW620 xenografts.

Material and Methods

Cell linesHCT116 human colorectal carcinoma cells obtained

from the American Type Culture Collection (ATCC) werecultured in 72-cm2 flasks with McCoy’s 5a (modified)medium plus L-glutamine (1.5 mmol/L), sodium bicarbo-nate (2.2 g/L), FBS (10%), penicillin (100 I.U./mL), andstreptomycin (100 mg/mL). SW620 human colorectal car-cinoma cells were obtained from the ATCC and cultured in72-cm2 flasks with DMEM medium plus FBS (10%), peni-cillin (100 I.U./mL) and streptomycin (100 mg/mL). Bothcell lines weremaintained in a humidified atmosphere (5%CO2/95% air) at 37�C. All cell lines were tested for myco-plasma contamination prior to experimentation using aPCR test (14) and were shown to be negative.

Xenograft modelAll animal studies were performed under a Memorial

Sloan Kettering Cancer Center IACUC-approved protocol.Xenografts were established by subcutaneous injection of 1� 106 wild-type cells in the right flank region. Two groupsof 15 athymic rnu/rnu mice (NCI, MD), each bearingHCT116 or SW620 xenografts, were developed. Nine ani-mals were treated with AZD1152 and 6 animals weremaintained as controls. Three preliminary sets of experi-ments with SW620 (n ¼ 9) and HCT116 (n ¼ 17) xeno-grafts were performed, and very similar results wereobtained.

Treatment with AZD1152 was initiated when tumorvolumes were approximately 250 mm3. HCT116 xeno-grafts have reached this volume at day 4 post implantationand SW620 xenografts at day 14, AZD1152 was providedby Astra Zeneca. The treatment protocol involved 3 weekly

Translational Relevance

Early assessment of treatment response using [18F]-labeled fluoro-2-deoxyglucose, [18F]FDG, and 30-deoxy-30-[18F]fluorothymidine, [18F]FLT, PET imaging hasbeen reported in many clinical as well as preclinicalstudies. In this study, we tested whether [18F]FDG and[18F]FLT PET imaging, early during the course of treat-ment with an Aurora Kinase B inhibitor, AZD1152,could be used to monitor treatment-response in twodifferent colon cancer xenografts models. Both imagingprobes demonstrated a response to AZD1152 therapy,but in quite different ways. These differences are likely tobe important considerations in the assessment treat-ment response, using [18F]FDG and [18F]FLT PETimaging in clinical studies as well. For example, max-imum-voxel values are a commonly used measure oftumor metabolic activity to reduce partial volumeerrors, whereas total tumor radioactivity/metabolismalso takes into account changes in tumor volume. Therewas a significant difference between these two measureswith [18F]FDG in both HCT116 and SW620 xenografts.Although [18F]FLT imaging showed a robust response inAZD1152-treated HCT116 xenografts, this was not seenin AZD1152-treated SW620 xenografts, despite promi-nent Ki67 staining of the tumor cells. This study presentsadditional data that suggests thymidine incorporationinto the DNA of SW620 cells occurs predominantlythrough the de novo pathway of thymidine synthesisand highlights the importance of recognizing the rela-tive contributions of both the de novo and salvage path-ways of thymidine utilization when [18F]FLT, or[11C]thymidine, PET imaging is used to image tumorproliferation or response to therapy.

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cycles. Each cycle included 2 consecutive days of AZD1152treatment by intraperitoneal injections (100 mg/kg). Toassess drug effect on xenografts growth, caliper measure-ments of tumors were performed 3 times a week. Xenograftvolume was calculated using the following equation:W2 �L � 3.14/6 (W ¼ width; L ¼ length). The mean tumorvolume profiles were plotted and fitted to a single expo-nential equation to estimate xenograft doubling time.

Radiotracers synthesis[18F]FDG and [99mTc]DTPA were provided by IBA Mole-

cular via the MSKCC nuclear pharmacy. For [18F]FDGaverage purity was 99%, concentration of radioactivity68 to 357 mCi/mL and specific activity was no less than11 Ci/mmol at the end of bombardment. The averagepurity of [99mTc]DTPA was 99%; the concentration ofradioactivity was approximately 60 mCi/mL and specificactivity was no less than 100 mCi/mmol. [18F]FLT wasprovided by the MSKCC radiochemistry and cyclotronfacility core; purity was approximately 95%, and specificactivity approximately 5,000 Ci/mmol.

PET imaging studiesControl and AZD1152-treated mice were imaged con-

currently on a R4 MicroPET dedicated small-animal PETscanner (Concorde Microsystems Inc.) following intrave-nous injections of 200 mCi [18F]FDG or [18F]FLT. A short-acting isoflurane anesthesia (2% isoflurane, 98% air mix)was used throughout these studies. All animals wereallowed to regain consciousness after radiotracer adminis-tration andmove freely in their cages before repeat anesthe-sia and imaging.Imaging studies were performed weekly and sequentially

with [18F]FDG and [18F]FLT imaging performed 24 hoursapart. [18F]FDG imaging (10 minute acquisition) was per-formed 1 hour after i.v. injection; [18F]FLT imaging (10minute acquisition) was performed 2 hours after i.v. injec-tion. In preliminary [18F]FLT imaging studies, the dynamicprofile of FLT accumulation in SW620 xenografts showedno evidence of uptake followed by washout, and back-ground radioactivity was lower at 2 hours compared with 1hour. The image data were corrected for nonuniformity ofscanner response, dead time count losses, and physicaldecay to the time of injection but no attenuation, scatter,or partial-volume averaging. The measured spatial resolu-tion of the R4 is approximately 2.2 mm full-width halfmaximum (FWHM) at the center of the field of view ofreconstructed images. PET-measured tissue radioactivitywas expressed as percentage of the injected dose per gramof tissue (%ID/g). Data analysis was performed on allimages using ASIPro software with 2D regions of interest(ROI). ROI’s were drawn around the tumor on 2Dslices through the tumor and the maximum-voxel value(% injected dose/cc) was measured and used in the calcu-lations. For the assessment of tumor radioactivity (uCi/tumor), the mean tumor radioactivity (uCi/cc) � tumorvolume (cc) product was calculated as previously described(15). Briefly, amanual ROI was drawn around the tumor in

each slice where tumor was visible. Themean accumulation(mCi/cc) and volume (cc) of each ROI was determined andsummed.

Ki-67 and hematoxylin and eosinimmunohistochemical staining procedure

Tissue sample preparation and staining procedure toassess the effect of AZ1152-HQPA on cells proliferationwas performed as previously described (16, 17).

In vitro studiesAll experiments were performed in triplicate or more.Studies with AZD1152-HQPA. AZD1152-HQPA is the

active component of AZD1152 and was provided byAstraZeneca. The compound was diluted in 100%dimethyl sulfoxide (DMSO) to 10 mmol/L stock solutionand stored frozen (�20� C) until use. The concentrationof AZD1152-HQPA in culture medium was 0, 100, and500 nmol/L; the corresponding concentrations of DMSOwere 0, 0.064, and 0.32 mmol/L per mL were used in thecontrol cultures, respectively. Cell monolayers wereexposed to drug containing medium for 1 or 24 hoursin 6-well plates before the [18F]FDG uptake experimentswere performed. The AZ1152-HQPA–containing mediumwas removed and replaced with 2 mL of DMEM highglucose medium containing [18F]FDG (0.5 mCi/mL/well)containing media, and a 60-minute incubation was per-formed as described previously (18). A clonogenic assayto estimate the sensitivity of cells to AZ1152-HQPA wasperformed as previously described (19).

EC50 cell viability studies with methotrexate and 5-fluor-ouracil. These studies were performed to obtain a mea-sure which reflects cell utilization of the de novo pathway ofthymidine (TdR) synthesis and incorporation of endogen-ous TdR in DNA synthesis (20); the WST-1 assay was usedas previously described (21). Briefly, cells were seeded on96-well micro plates (4,000 cells/well; 8 wells/treatmentdose), and incubated with methotrexate (MTX) over a widedose range for 72 hours. WST-1 (cell viability reagent) wasadded to each well, and after a 4-hour reaction period, theoptical absorbance was determined at a wavelength of562 nm with an ELISA reader. The optical absorbance ofcontrol (untreated) cells was taken as the 100% value; EC50

values were calculated using PRISM 5 software.Thymidine and fluorothymidine uptake studies. [3H]FLT

and [14C]TdR were obtained from Moravek Biochemicals[purity >95%, specific activity 3.4 Ci/mmol (for FLT) and19.4 Ci/mmol (for TdR)]. HCT116 and SW620 wereplated in 6-well plates and after 12 hours of incubationin 37�C and 5% CO2. The average confluence of cells onthe plates was approximately 50%. The cell incubationmedium was replaced with preheated medium containing[3H]FLT (0.5 mCi/mL), [99mTc]DTPA (0.5 mCi/mL), and[14C]TdR (0.05 mCi/mL), and uptake was measured after5, 15, 60, and 120 minutes of incubation. The incubationmedium was removed and centrifuged at 14,000 rpm toremove any cells in the medium. Cells were washed withPBS preheated to 37�C once and lysated by adding RIPA

Imaging Response to AZD1152 with FDG and FLT-PET

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buffer 1� with Halttm protease inhibitor single use cock-tail (both by Thermo Scientific). [99mTc]DTPA provided ameasure of adhered medium that was not removed by thewashing procedure. An aliquot of medium and lysatesamples have been used for counting of radioactivity.The remaining lysate was centrifuged at 14,000 rpm for10 minutes and frozen at �20�C and assayed later for thedetermination of protein concentration using a BCAprotein assay kit (Thermo Scientific).

Radioactivity in the cells and medium was measuredusing both a g-counter (MINAXI Auto-Gamma 5000Series Gamma Spectrometer, United Technologies Pack-ard), followed by liquid scintillation b-counting (Packard1600 TR Beta Spectrometer, United Technologies Pack-ard) after 99mTc had decayed away. Radioactivity uptakeresults were expressed as a cell/medium uptake ratio:dpm/mg cell protein/dpm/mL of medium (mL medium/mg cell protein).

Cell proliferation and doubling time studies. These stu-dies were performed as previously described (22). Briefly,the experimental cell lines HCT116 and SW620 wereplaced on 75cm plates, 100,000 viable cells per plate.Twelve, 24, 48, 72, and 96 hours after the placement, cellswere removed by brief incubation with 0.05% Trypsinand counted. The cell number versus time profiles wereplotted and fitted to a single exponential equation toestimate doubling time. Immunoblots using antibodiesagainst human thymidine kinase (TK) and thymidylatesynthetase (TS) obtained from Santa Cruz Biotechnologywere performed as previously described (23).

Statistical analysisMean values and SDs were calculated using theMSOffice

2003 Excel 11.0 statistical package (Microsoft). Statisticalsignificance of differences between mean values was esti-mated using the independent t test for unequal variances. Pvalues of less than 0.05 were considered to be statisticallysignificant.

Results

[18F]FDG and [18F]FLT microPET imagingSequential [18F]FDG and [18F]FLT micro PET imaging of

animals bearing s.c. HCT116 or SW620 xenografts wasperformed at weekly intervals (Figs. 1 and 2). The max-imum-voxel FDG value (%dose/cc) and total tumor FDGradioactivity (mCi/tumor; %dose) values were determinedfor each of the xenografts. The maximum-voxel FDG activ-ity was somewhat variable in both AZD1152-treated andnon-treated xenografts (Figs. 3A and 4A). The normalizedtumor-to-adjacent tissue FDG ratio showed considerablyless variation and did not change significantly over time,nor were there any significant differences between treatedand nontreated animals (Supplementary Fig. S1A andS1B). Measuring the total accumulation of FDG in eachtumor yielded a different profile. There was a profoundeffect of AZD1152 treatment on total FDG accumulation inHCT116 xenografts (Fig. 3B); this profile change was alsosignificant for SW620 xenografts, but it was less striking(Fig. 4B). The amount of FDG accumulated in both SW620and HCT116 was much lower in AZD1152-treated animals

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Figure 1. Sequential imagingof representative HCT116 flankxenografts (dotted circle).[18F]FDG (A) and [18F]FLT (B)microPET 2D transaxial imagesthrough a HCT116 xenograftfrom the same control animaland from the same AZD1152-treated animal are shown. *,The first [18F]FDG study in thetreatment group (day 7) wasperformed under anesthesiaduring the entire study; in allother studies the animals wereawake after radiotracer injectionuntil time of imaging. Note thecentral area of necrosis (lowtracer uptake) as the xenograftsget larger.

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than that in controls, which largely reflects differences intumor volume (Figs. 3E and 4E). After the end of thetreatment, both xenografts showed a rebound effect.The pattern of tumor radiotracer uptake with [18F]FLT

imaging in HCT116 and SW620 xenografts was somewhatdifferent than that observed with [18F]FDG. Themaximum-voxel FLT activity (%ID/cc) in untreated HCT116 xeno-grafts was approximately 10-fold higher than that inuntreated SW620 xenografts (P < 0.001; Figs. 4C and5C), and a similar pattern was observed after calculatingtotal FLT accumulation per tumor. Radioactivity levels inboth AZD1152-treated and nontreated SW620 xenograftswere at or near background levels (Fig. 4C) and the SW620xenograft-to-surrounding tissue ratio was very low,approximately 1.5 (Supplementary Fig. S1D). If back-ground radioactivity is subtracted from that measured inthe xenografts, the difference between HCT116 and SW620xenografts is 37-fold (1.01 � 0.09%ID/g vs. 0.028 �0.005%ID/g, respectively). FLT accumulation in HCT116xenografts was high in nontreated control animals and wassignificantly less in AZD1152-treated animals (P < 0.05;Fig. 3C and D). During the latter posttreatment period, FLTactivity in HCT116 xenografts showed a marked rebound,reaching a significantly higher value (P < 0.05) than thatmeasured in nontreated xenografts (Fig. 3C). The normal-ized tumor-to-adjacent tissue FLT ratio demonstrates asimilar profile (Supplementary Fig. S1C).

AZD1152 treatment effect on HCT116 and SW620xenograft growth profilesNontreated HCT116 and SW620 s.c. xenografts showed

an exponential increase in tumor volume during the studyperiod with estimated doubling times of 3.6 � 0.5 and 8.1

� 0.8 days, respectively (Figs. 4C and 5A). A clear responseto weekly treatment with AZD1152 (100 mg/kg i.p. injec-tions on 2 consecutive days per week over a 3-week inter-val) was observed in both HCT116 and SW620 xenografts.However, the xenograft growth profiles were different.HCT116 xenografts stopped growing immediately afterthe first treatment and decreased in size during the 3treatment cycles. In contrast, SW620 xenografts continuedto grow during AZD1152 treatment, but showed a decreasein growth rate during the 3 cycles of treatment. Followingthe cessation of treatment, there was a fairly rapid resump-tion of exponential growth of HCT116 xenografts with adoubling time of 5.1 � 0.7 days. In contrast, SW620xenografts showed an approximately 10-day delay in theresumption of exponential growth with a doubling time of9.4 � 1.5 days.

Histology and Ki67 ImmunohistochemistryAZD1152 treatment of experimental animals, bearing

HCT116 xenografts induced the formation of large multi-nucleated-giant tumor cells, with a marked decline inKi67 staining over the treatment period (Fig. 5). Samplescollected from untreated animals showed normal tumormorphology with a robust pattern of Ki67 staining. Incontrast, SW620 xenografts treated with AZD1152 did notdemonstrate comparable changes in morphology or Ki67expression, suggesting little or no change in proliferation(Fig. 5).

AZD1152-HQPA, MTX, and 5-fluorouracil treatment,in vitro

EC50 values for AZD1152-HQPA treatment of HCT116and SW620 cells were 10 � 2.1 and 11 � 3.3 nmol/L,

Figure 2. Sequential imaging ofrepresentative SW620 flankxenografts (dotted circle).[18F]FDG uptake (A) and [18F]FLT(B) microPET 2D transaxialimages through a SW620xenograft from the same controlanimal and from the sameAZD1152-treated animal areshown. Note the central area ofnecrosis (low tracer uptake) as thexenografts get larger.

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Imaging Response to AZD1152 with FDG and FLT-PET

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respectively, as determined by a clonogenic assay (Fig. 6A).However, no significant effect of AZD1152-HQPA treat-ment was observed on [18F]FDG uptake, including expo-sures to 100 and 500 nmol/L of AZD1152-HQPA for 1, 24,and 48 hours (Supplementary Table S1). We also tested thesensitivity of the HCT116 and SW620 cell lines to MTX and5-fluorouracil (5FU). In contrast to AZD1152-HQPA,MTX,and 5FU treatment yielded markedly different sensitivity

results for the 2 cell lines (Fig. 6A). SW620 cells weresensitive to MTX and 5FU, whereas HCT116 cells were not.

[3H]FLT and [14C]thymidine uptake and immunoblotsin HCT116 and SW620 cells

To assess whether the different patterns of FLT uptakeobserved in HCT116 and SW620 xenografts were alsoreflected in cell culture uptake studies, the accumulation

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Figure 3. Measurements of[18F]FDG and [18F]FLT uptake inHCT116 xenografts. Units are%ID/cc of the maximum-voxelvalue (A, C), and mCi of total tumorradioactivity (B, D). Hatched barrepresents nontreated, controlanimals; solid bar representsAZD1152-treated animals. Growthprofiles of HCT116 s.c. xenografts(E). Open circles represent themean tumor volume of untreated(control) animals. Closed circlesrepresent the mean tumor volumeof AZD1152-treated animals.Arrows show the beginning ofeach 2-day AZD1152 treatmentcycle; the day post implantation isshown in the abcissa. *,The first[18F]FDG study in the treatmentgroup (day 7) was performedunder anesthesia during the entirestudy; in all other studies theanimals were awake afterradiotracer injection until time ofimaging.

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of [3H]FLT, [14C]thymidine ([14C]TdR), and[99mTc]DTPA (extracellular fluid reference) in HCT116and SW620 cells was compared (Fig. 6C). The accumula-tion rate of [3H]FLT and [14C]TdR radioactivity was 8-foldmore rapid in HCT116 cells than that in SW620 cells: 1.4� 0.3 and 4.3 �0.5 mL/mg/minute, respectively, for

HCT116 versus 0.18 � 0.15 and 0.55 �0.23 mL /mg/minute for SW620. The corresponding in vitro growthprofiles of HCT116 and SW620 cells were obtained(Fig. 6D), and the calculated exponential growth dou-bling time was 14.1 � 0.7 and 20.9 � 0.4 hours, respec-tively. Immunoblots for TK and TS levels in HCT116 cells

Figure 4. Measurements of[18F]FDG and [18F]FLT uptake(%dose/g) in SW620 xenografts.Units are%ID/cc of themaximum-voxel value (A, C), and mCi of totaltumor radioactivity (B, D).Normalized uptake ratios ofxenograft-to-surrounding tissue(B, D). Hatched bar representsnontreated, control animals; solidbar represents AZD1152-treatedanimals. E, growth profiles ofSW620 s.c. xenografts. Opencircles represent the mean tumorvolume of untreated (control)animals. Closed circles representthe mean tumor volume ofAZD1152-treated animals. Arrowsshows the beginning of each2-day AZD1152 treatment cycle;the day post implantation isshown in the abcissa.

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during rapid exponential growth showed slightly higherexpression than that in SW620 cells Fig. 6B). Conversely,SW620 cells showed slightly higher levels of TS thanHCT116 cells (Fig. 6B). These differences are not large,

but consistent with HCT 116 cells being better able tosalvage thymidine in comparison to SW620 cells. How-ever, impairment of thymidine uptake in SW620 cellscannot be excluded and needs to be investigated further.

Figure 5. Immunohistochemistry.Hematoxylin and eosin and Ki67staining of tumor samplesacquired from experimentalanimals bearing HCT116 andSW620 xenografts at the differenttime points of the study.

Figure 6. In vitro assessments. A, EC50 estimates for AZD1152-HQPA, MTX, and 5FU in HCT116 and SW620 cell lines. B, immunoblots for TK, TS, and atubulin in HCT116 and SW620 cells. C, [3H]FLT, [14C]TdR, and [99mTc]DTPA accumulation in HCT116 and SW620 cells; data are plotted as radioactivity–timeprofiles for each tracer; tracer uptake is expressed as (mL medium/ mg total cell protein). D, proliferation-time profiles for HCT116 and SW620 cells.

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Discussion

AZD1152 is effective in inhibiting the growth of 2human colorectal/colonic cancer cell lines (HCT116 andSW620) in culture and xenografts in nude mice (9, 13, 23;Figs. 3E and 4E). Both cell lines were sensitive to AZD1152-HQPA in the low nanomolar range. A robust treatmentresponse based on tumor volume measurements wasclearly obtained in HCT116 xenografts, using a weeklyschedule of i.p. AZD1152 administration (100 mg/kgi.p. injections daily on 2 consecutive days per week), adose schedule previously reported to be effective in mice(9). The treatment response of SW620 xenografts wassomewhat delayed and less robust compared with theHCT116 xenograft response.One question we asked was whether [18F]FDG or

[18F]FLT PET imaging could predict treatment response"early", within several days or weeks of initiating treatment.Because hematologic toxicity is observed with prolongedadministration of AZD1152, early assessment of treatmentresponse could be important in at least 2 respects. First, if itis possible to identify a nonresponding tumor "early" by[18F]FDG or [18F]FLT PET imaging, would the imagingresults be sufficient criteria for discontinuing AZD1152treatment before hematologic toxicity develops? Second,could PET imaging be used to establish the minimumeffective dose of AZD1152 in individual patients andthereby reduce hematologic toxicity during prolongedadministration of the drug?The [18F]FDG PET imaging results were surprising, given

the tumor volume treatment responses that were observedin both xenograft models. Sequential FDG PET imagingresults showed little or no difference in FDG uptakebetween AZD1152-treated and nontreated xenografts,when the radioactivity data are expressed as maximum-voxel values. (Measures based on maximum-voxel valuesare used to avoid partial volume imaging effects for smalltumors; measures based on region of interest (ROI) mea-surements are usually used for larger tumors.) These resultssuggested that glucose utilization by HCT116 and SW620tumor cells is not significantly influenced by AZD1152treatment, although treatment does have a significant effecton tumor growth and volume. However, this finding wasinconsistent with our understanding of the mechanism ofAZD1152 antitumor effects (9). The major regulatory path-ways of glucose metabolism are largely mediated throughinsulin receptor (IR) signaling and the AMP-activated pro-tein kinase signaling pathways (24–27). Because the IR andthe AMP signaling pathways are activated by AuroraKinases, selective inhibition of Aurora Kinases (and AuroraKinase B, in particular) was expected to reduce tumorglucose utilization.When the FDG results are expressed as total tumor

metabolism (or radioactivity) as has previously been sug-gested (15), a highly significant AZD1152 treatment effectwas observed in both HCT116 and SW620 xenografts.However, the total tumor metabolic response observedin this study largely reflects differences in tumor volume

between treated and nontreated tumors, not a change inglucose metabolism of tumor cells within an imagingvoxel. The absence of a drug effect on maximum-voxelFDG uptake is more clearly appreciated when the xenograftvalues were normalized to that of the surrounding, non-tumor tissue; this normalization accounts in part for inter-animal variations and FDG input function differences.Thus, FDG PET may not be a useful paradigm for non-invasive monitoring of AZD1152 treatment response, atleast as reflected in these two animal xenograft models.

The [18F]FLT PET imaging studies also yielded surprisingresults. SW620 xenografts showed little or no accumulationof [18F]FLT above background levels, and there was nodifference in [18F]FLT accumulation between AZD1152-treated and nontreated SW620 xenografts. These resultsconflicted with the robust Ki67 staining pattern observed inuntreated SW620 xenografts. In contrast, [18F]FLT uptakein untreated HCT116 xenografts was 10-fold higher thanthat in untreated SW620 xenografts, and this difference was37-fold when the values are corrected for backgroundradioactivity. The [18F]FLT imaging response pattern, fol-lowing AZD1152 treatment of HCT116 xenografts was alsoquite different. [18F]FLT uptake decreased to 20% or less ofthat measured in nontreatedHCT116 xenografts over the 3-week treatment period, and these imaging results wereconsistent with the decrease in Ki67 staining followingAZD1152 treatment.

[18F]FLT, as well as [11C]thymidine ([11C]TdR) tumor-proliferation imaging depends on 2 major components: (i)transport of pyrimidine nucleotides across cell membranesand (ii) the activity of thymidine kinase. Both componentsare highly regulated and the expression levels of bothtransporter and kinase depend on the cell cycle and rateof cell proliferation (28–30). Another determinant, which issometimes ignored in [18F]FLT and [11C]TdR PET imagingstudies, is an assessment of whether the endogenous path-way of thymidine synthesis is active in the cell or whetherexogenous thymidine is the dominant source of the nucleo-tide for DNA synthesis via the "salvage pathway" (31).

To further test whether the de novo pathway of thymidinesynthesis was dominant in SW620 cells, but not in HCT116cells, we performed 4 different in vitro assays. First, wefound an 8-fold difference in the rates [14C]thymidine and[3H]FLT accumulation in HCT116 and SW620 cells inculture, which demonstrated an impairment of traceruptake (the combination of transport and phosphoryla-tion) of these compounds in SW620 cells. This suggests thatutilization of exogenous thymidine as a salvagemechanismmay be less predominant in the SW620 cell line. Second,we found only a small (1.5-fold) difference in the expo-nential doubling time between the 2 cell lines. Third, wetested the sensitivity of HCT116 and SW620 cells to MTXand 5FU and found a marked difference between the 2cell lines consistent with prior studies (32). Fourth, weperformed immunoblots for TK and TS. The immunoblotsfor TK and TS protein showed slightly lower levels of TK inSW620 cells, but this difference was not considered sig-nificant and may not be an accurate measure of intracel-

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lular enzymatic activity. In total, these in vitro results areconsistent with the imaging results described previously,and are also consistent with SW620 cells relying mainly onthe de novo pathway of TdR synthesis. Although HCT116cells do incorporate TdR at a higher rate than SW620 cells,consistent with the tumor imaging studies, it is not clear ifthe lower EC50 values for MTX and 5FU in the SW620 cellline are attributable only to lower salvage of TdR in this cellline. Further studies will be required to more fully addressthis question. However, inhibition of Aurora kinases A andB has been shown to result in the downregulation ofthymidine kinase 1 (TK1) in HCT116 cells via the Rbpathway (33), through phosphorylation of histone H3and by p53 protein stabilization and induction of p21,and this is consistent with our findings

The wide disparity in [18F]FLT PET imaging betweenuntreated HCT116 and SW620 xenografts (37-fold, back-ground-corrected) compared with a 2.3-fold difference intumor doubling time, respectively, provides a strikingexample of potential limitations associated with [18F]FLT,and [11C]TdR, tumor-proliferation imaging studies. Wesuggest that these results highlight the importance ofdetermining the contribution of the de novo pathway ofthymidine synthesis when [18F]FLT or [11C]TdR PET is usedto image and monitor tumor proliferation. The PET studiesimage/measure the exogenous component of thymidineincorporation into DNA via the salvage pathway. When thefraction of thymidine synthesized via the de novo pathwayand incorporated into DNA increases, the magnitude of[18F]FLT and [11C]TdR uptake in the tumor is correspond-ingly reduced.

Although this issue is frequently ignored in clinicalstudies using [18F]FLT or [11C]TdR PET to image andmeasure tumor proliferation, the initial studies establish-ing the tumor-proliferation imaging paradigms clearlyidentified de novo thymidine synthesis as a potential limita-tion and a confounding factor for quantitation of prolif-eration (31, 34). A review of multiple publications (35–50)and more than 300 cancer patients imaged with [18F]FLTdemonstrated that [18F]FLT was accumulated above back-ground in the vast majority of the reported imaging studies.These studies included a preponderance of lung/thoracic

tumors (130 patients), followed by glioma (n ¼ 52),lymphoma, pancreatic, breast, and colon tumors (n ¼17–34), as well as a smaller numbers of other cancers.Nevertheless, "false negative" [18F]FLT imaging results arerecorded in about 10% of patient studies, and we suspectthat this percentage is actually higher than what has beenreported in the literature. For example, 2 of 23 patients withnon—small-cell lung cancer, 3 of 9 patients with lungmetastases (2 colorectal, 1 melanoma), and 1 of 1 pul-monary carcinoid who had [18F]FLT imaging were classi-fied as "false negative" scans (51). Other studies have notedthat some tumors imaged well with [18F]FDG but not with[18F]FLT or that some tumors with low FLT uptake hadmoderate Ki67 staining, despite an overall correlationbetween these 2 independent measures.

Finally, what remains unexplored inmost [18F]FLT drug–response monitoring studies is whether a particular drugtherapy results in a shift in TdR utilization from the salvagepathway to the de novo synthesis of thymidine or vice versa.This was recently demonstrated in a small number ofpatients (n ¼ 6) with breast cancer (52). This cautionarynote needs wider appreciation and requires further study.

Disclosure of Potential Conflicts of Interests

G.K. Schwartz: income from AstraZeneca. The other authors disclosedno potential conflicts of interest.

Acknowledgments

This work was supported in part by funds from AstraZeneca and NIHgrants CA86438, R24 CA83084 andDOE grant FG03-86ER60407.We thankDrs. Steven Larson and Pat Zanzonico (Memorial Sloan Kettering CancerCenter, New York) for their help and support.

Grant Support

AstraZeneca and NIH grants CA86438, R24 CA83084 and DOE grant FG03-86ER60407.

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 May 26, 2010; revised November 4, 2010; accepted November15, 2010; published OnlineFirst January 18, 2011.

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2011;17:1099-1110. Published OnlineFirst January 18, 2011.Clin Cancer Res   Maxim A. Moroz, Tatiana Kochetkov, Shangde Cai, et al.   F]Fluorothymidine Imaging

18-[′-deoxy-3′F]Fluoro-2-deoxyglucose and 318AZD1152: A Preclinical Analysis of [

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