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Published OnlineFirst July 26, 2010; DOI: 10.1158/1078-0432.CCR-09-2795
Published OnlineFirst on August 17, 2010 as 10.1158/1078-0432.CCR-09-2795 Published OnlineFirst on August 23, 2010 as 10.1158/1078-0432.CCR-09-2795
Clinical
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clinical and Clinical Evidence that Deoxy-2-[18F]fluoro-D-ose Positron Emission Tomography with Computedography Is a Reliable Tool for the Detection of Early

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ecular Responses to Erlotinib in Head and Neck Cancer

tien Vergez1, Jean-Pierre Delord1,2, Fabienne Thomas1,2, Philippe Rochaix1,2, Olivier Caselles2,
as Filleron2, Séverine Brillouet2, Pierre Canal1,2, Frédéric Courbon2, and Ben C. Allal1,2
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pose: There is a clinical need to identify predictive markers of the responses to epidermal growthreceptor tyrosine kinase inhibitors (EGFR-TKI). Deoxy-2-[18F]fluoro-D-glucose positron emissiongraphy with computed tomography (18FDG-PET/CT) could be a tool of choice for monitoringrly effects of this class of agent on tumor activity.erimental Design: Using models of human head and neck carcinoma (CAL33 and CAL166 cell, we first tested in vitro and in vivo whether the in vivo changes in 18FDG-PET/CT uptake wereated with the molecular and cellular effects of the EGFR-TKI erlotinib. Then, the pathologicorphologic changes and the 18FDG-PET/CT uptake before and after erlotinib exposure in patientsnalyzed.ults: Erlotinib strongly inhibited extracellular signal-regulated kinase-1/2 (ERK-1/2) phosphoryla-oth in the preclinical models and in patients. Western blotting, immunofluorescence, andnohistochemistry showed that erlotinib did not modify Glut-1 expression at the protein level eitherline models or in tumor tissue from mouse xenografts or in patients. Phospho-ERK-1/2 inhibitionssociated with a reduction in 18FDG uptake in animal and human tumors. The biological volumeore accurate than the standardized uptake value for the evaluation of the molecular responses.
was m
Conclusion: These results show that the 18FDG-PET/CT response is a reliable surrogate marker of theeffects of erlotinib in head and neck carcinoma. Clin Cancer Res; 16(17); OF1–12. ©2010 AACR.

fromto selcure adosetreatmDeo

thelial growth factor receptor (EGFR) inhibitors havel activity in various tumor types, including head andquamous cell carcinoma (HNSCC), used alone or inination with cytotoxic agents. Unfortunately, only aup of patients benefit from the use of these targeted(1).

ly, there is a clear medical need for theion of those patients most likely to benefit

mograhas baminiManycouldthat a18FDGof thewithchangfor exvent tical ebleedin melightedictivcorrel

ons: 1Laboratoire de Pharmacologie Clinique etMedicaments Anticancereux, EA 3035, Université

2Institut Claudius Regaud, Toulouse Cedex, France

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

. Allal contributed equally to the supervision of this work.

sented as a poster presentation at the 99th Annualrican Association for Cancer Research, April 13, 2008,ia; poster #422.

uthors: Frédéric Courbon or Ben C. Allal, Institut20-24 rue du pont Saint Pierre, Toulouse 31052,-561424242; Fax: 33-561424631; E-mail: courbon.gaud.fr or [email protected].

0432.CCR-09-2795

ssociation for Cancer Research.

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Research. on April 17, 20clincancerres.aacrjournals.org ed from

this targeted treatment. The development of toolsect these patients could facilitate their therapeuticnd the determination of their biologically effectiveand, subsequently, lead to individualization ofent (2).xy-2-[18F]fluoro-D-glucose positron emission to-phy with computed tomography (18FDG-PET/CT)ecome an important noninvasive technique for ex-ng cancer staging and detecting recurrent neoplasms.clinical trials have shown that 18FDG-PET imagingprovide an early indication of therapeutic responsesre well correlated with clinical outcomes (3–5).-PET could be particularly useful for the evaluationproportion of active tumor cells during treatment

EGFR tyrosine kinase inhibitors (EGFR-TKI). Somees in tumor volume are observed late or not at all;ample, when intratumoral necrosis and fibrosis pre-umor shrinkage and could actually cause a paradox-xpansion of some tumors due to intratumoraling or edema. This is not the case for the changestabolic activity of the tumors, which could be high-d early. Indeed, some studies have evaluated the pre-
e value of 18FDG-PET as a consequence of theation of early metabolic responses with the clinical
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We show that deoxy-2-[18F]fluoro-D-glucose positronemission tomography with computed tomography(18FDG-PET/CT) can be used as a surrogate marker forthe early evaluation of epidermal growth factorreceptor-tyrosine kinase inhibitor efficacy. For this pur-pose, we developed a preclinical models to validate18FDG-PET/CT imaging for the early evaluation of themolecular effects of erlotinib in head and neck squa-mous cell carcinoma cell lines. We then cross-validatedthis tool during a clinical trial designed to assess thepharmacodynamic effects of erlotinib in patients withhead and neck squamous cell carcinoma who receivederlotinib as neoadjuvant treatment for a short timeperiod before surgery. This is the first study providing,from preclinical models to patients, a reliable proof ofeffic

Vergez et al.

Clin COF2


me, as has been described for the response to imati-gastrointestinal stromal tumors (5).refore, although some results indicating that molec-aging with 18FDG-PET could be a valuable tool for

development and use (3), it remains to be showne changes in 18FDG uptake correlate with the mo-r responses (MR) induced by erlotinib.r goal was to establish the rationale of the use of-PET/CT as a surrogate marker for the early evalua-f EGFR-TKI. For this purpose, we developed preclin-odels to validate 18FDG-PET/CT imaging for theevaluation of the molecular effects of erlotinib inC cell lines. EGFR regulates numerous signalingays involved in various cellular mechanisms con-ing to the control of cellular homeostasis and prolif-n. In this cellular proliferation pathway, extracellular-regulated kinase-1/2 (ERK-1/2) is a key downstreamr. This inhibition of the ERK-1/2 could reflect whatve named the “molecular” and/or “biological”se to the drug.where, we also define the “metabolic effects” as theolic changes observed in the cells that result in theication of glucose uptake (18FDG; ref. 6). Metabo-nd proliferation are associated cellular processesse cell proliferation is “energy consuming” andolism dependent (6).lly, we cross-validated this tool during a clinical trialed to assess the pharmacodynamic effects of erloti-patients with HNSCC who received erlotinib for atime period before surgery (7).

rials and Methods

als and agentsale Swiss athymic nude mice, ages 4 to 5 weeks

iency of this method.

les River Laboratories), were maintained in accord-with the standards of the Federation of European

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ancer Res; 16(17) September 1, 2010

Research. on April 17, 20clincancerres.aacrjournals.org Downloaded from

atory Animal Science Associations and were includedtocols following a 2-week quarantine. Erlotinib (OSI-Tarceva) was kindly provided by F. Hoffmann-La, Inc. 18F-FDG (Glucotep) was from Cyclopharma.

odiesantibodies used for Western blotting were as fol-anti–phospho-EGFR/HER-1 (Tyr1173; Euromedex);otal EGFR/HER-1 (Ab-12) and anti–tubulin β (Neo-ers, Interchim); anti–Glut-1 and anti–phospho-/2 pAb (Cell Signaling); anti–ERK-1/2 (c-16; SantaBiotech, Tebu-Bio SA); anti-p27kip1 (DAKO); andidase-conjugated secondary mouse or rabbit anti-(Bio-Rad).antibodies used for immunochemistry were as fol-anti–phospho-EGFR/HER-1 (SC36-9700, Zymed),otal EGFR/HER-1 (EGFr PharmDX, DAKO), anti–1 (RB-9052, NeoMarkers), anti–phospho-ERK-1/283s, Santa Cruz), and anti-p27kip1 (SX53G8, DAKO).

ulture33 and CAL166 cells (human head and neck carci-, Centre Antoine Lacassagne Nice, France; ref. 8)cultured in DMEM containing 10% fetal bovine se-upplemented with 2 mmol/L L-glutamine (culturem; Cambrex Biosciences) at 37°C in a humidifiedphere and 5% CO2.

rn blot analysisday 1, cells (1.5 × 106) were plated in culture medium-mm culture dishes. On day 2, for the EGFR and ERKes cells, were either serum starved or not for 24 hoursen treated with either vehicle or erlotinib at concen-s of 3, 3.5, and 5 μmol/L for 24 to 72 hours. Whenary, cells were treated with 20 ng/mL EGF for the lastnutes of the experiment. The cells were harvested andin lysis buffer [50 mmol/L Tris (pH 8), 150 mmol/L, 0.1% NP40, 5 mmol/L MgCl2, 50 mmol/L NaF,ol/L phenylmethylsulfonyl fluoride, 10 mmol/L2 mmol/L orthovanadate, 5 mg/mL sodium dexoxy-e, 6.4 mg/mL phosphatase substrate; Sigma 104]. ForERK-1/2, Glut-1, p27kip1, or β-tubulin analysis, 70 μgcleared lysates were separated on a 7.5% or 12.5%AGE gel, blotted onto polyvinylidene difluoride mem-s (Amersham), and incubated with specific antibodies.the determination of the effects of erlotinib on Glut-1sion, 24 hours after plating, cells were treated withvehicle or erlotinib as previously described. Cells

harvested by trypsinization and counted. Three mil-ells were pelleted (820 × g, 5 min), lysed, and ana-by Western blot as described earlier (12.5% SDS-, Glut-1 antibody).ection was done using peroxidase-conjugated sec-y antibodies (Bio-Rad) and an enhanced chemilumi-nce detection kit (Amersham Pharmacia Biotech).lots were scanned and analyzed with a Molecular
ics densitometer and ImageQuant software. Results
presentative of three independent experiments.

Clinical Cancer Research



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escenceunofluorescence histology was done as described

here (9). Cells were seeded on glass coverslips inll plates and were treated with either vehicle ornib at 5 μmol/L for 48 hours. Membrane Glut-1orters were detected by incubation with antibodyt Glut-1 plus a rhodamine-labeled secondary anti-and images were recorded with a Princeton camera.ts are representative of three independent experi-in duplicate.

mination of 18FDG uptake by CAL33 cellsday 1, CAL33 cells (1.5 × 106) were plated in 60-mme dishes in culture medium. Twenty-four hours later,ere treated with either glucose (control cells) orMBq/mL 18FDG (treated cells) for 30 minutes. Them was collected; the cells were washed three timesmL of PBS and each wash was collected in separat-es. Cells were trypsinized (500 μL of trypsin) andted in 2.5 mL of culture medium and were pelletedg, 5 min). Trypsin and supernatants were collected.

elleted cells were either reserved (whole cells) or sus-d in 500 μL of PBS, and lysed by three cycles of ther-ock (liquid nitrogen/37°C) followed by 15 minutestrifugation (15,000 × g) to obtain a lysate fraction.pernatants, representing the cytosolic cell fraction,e pellets, representing the membrane plus nuclearaction, were collected. The results are expressed asrcentage of radioactivity (corrected for the physicalof 18F) measured in each fraction versus the 18FDGy at the time of treatment (100%) and are the mean ±f two independent experiments in duplicate.

of erlotinib on cell line xenografts inmice33 or CAL166 xenografts were established by s.c.on of 1 × 107 cells into both mouse flanks. Whenmor size reached ∼200 mm3 (day 4 postimplanta-the mice were pooled and randomly assigned toroups (control and treated with erlotinib) of six toanimals.18FDG-PET/CT imaging, mice were injected throughil vein with 9.85 ± 1.5 MBq of 18FDG and then anes-ed by an i.p. injection of ketamin/xylazin solution5 mg/kg). Anesthesia was maintained for 60 minutesf necessary, before the 18FDG-PET/CT imaging miceechallenged for anesthesia for the further 30 minutesaging [PET-CT Discovery ST, General Electric Healthare (GEHC)], after which the mice were allowed tor. Mice were maintained under a controlled tempera-around 22°C) during all the experiments. Mice werereated orally with either saline (control group) or erlo-t 100mg/kg/d in saline (erlotinib group). Twenty-fourlater, the mice were imaged as described, and in someols, the mice were treated and scanned after 72 hourstment.
perform the PET acquisition, mice were placed in al box enabling four mice to be imaged at once (two
Thethe ch

acrjournals.org


ls and two treated). PET data handlings and recon-ions are shown in Supplementary Table S1. Tracere was measured using the regions of interest (ROI)d on cross-sectional images. ROIs with the same sizedrawn around the tumor and a background regionansaxial slice images to determine the tumor-to-round ratio calculated by dividing the average pixelity within a tumor ROI by the average pixel intensitythe background ROI. Results are representative of at

wo independent experiments with three mice per ki-point.he end of the experiment, the mice were sacrificed;mors were removed and fixed in formalin for path-and immunohistochemical analysis.

of neoadjuvant treatment with erlotinib ints with HNSCC

r team has published a pilot study of neoadjuvantent with erlotinib of nonmetastatic HNSCC (7).ts were eligible if they were candidates for first-lineve surgical treatment or had been scheduled fory by necessity. After diagnosis, patients underwente pan-endoscopy and 18FDG-PET/CT. Treatment50 mg/d erlotinib orally started the following day.ts were treated for 20 days on average (Table 1),ponding to the time between pan-endoscopy andal resection. 18FDG-PET/CT examinations were re-48 hours before surgery at the latest. Pathologic ex-tions with immunostaining were done on biopsiesand after treatment.

G PET/CT acquisitions and interpretationsum glucose was measured before i.v. injection ofBq of 18F-FDG (Glucotep Cyclopharma). A whole-(from skull to pelvis) 18FDG-PET/CT acquisition wasd out (GEHC). Acquisitions were done in a two-sional mode (5 minutes per bed position). Two-

nsional sinograms were reconstructed in a 256 ×atrix, with a field of view of 50 cm and corrected foration, random, and scatter. CT imaging was doneenuation correction and anatomic correlation with aA tube current, a 140-kV tube voltage, a helical pitch5:1, and a reconstructed slice thickness of 3.75 mmn interval of 3.27 mm between slices.two-dimensional 18FDG-PET/CT data corrected foration were transferred to an Advantage WorkstationEHC). The maximum standardized uptake valueax; ref. 10) and the biological volume of the tumorwere determined using commercial software (PETGEHC). SUVmax was corrected for body weight

BW). BV was determined using a fixed thresholden the maximum pixel counts within the tumore background. We previously carried out a controland determined that for the PET system and thesition protocol used, a threshold of 35% was theaccurate.
metabolic response was expressed according toange in either the SUV or the BV determined as
Clin Cancer Res; 16(17) September 1, 2010 OF3



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Vergez et al.

Clin COF4


s:ΔSUV = (SUVa − SUVb)/SUVb × 100 and δBV =BVb)/BVb × 100 (“b” stands for before treatment

a” for after treatment).

nohistochemistrylyses were done on 4-μm-thick formalin-fixed paraf-tions of patient tumors and cell lines xenografts ac-g to a procedure described elsewhere (7). For Glut-1nostaining, the antibody dilution was ready to use.n retrieval was done using 10 mmol/L citrate buffer) and 750-W microwave heat for 5 minutes × 3.nostaining intensity was assessed semiquantitativelya four-point scale (i.e., 0, negative; +, weak; ++,rate; +++, strong) and the percentage of labeledImmunostaining analyses were evaluated using thenoreactive score (IRS) according to Remmele et al.The IRS (range, 0-12) is the product of the scoresining intensity (0-3 scale) and percentage of cellsd (0-4 scale).

tical analysisresults are expressed as mean ± SEM. Results were
ed using Student's t test, and P < 0.05 was acceptedistically significant. SUVmax and BV after and before
effectEGFR



ent were compared using a paired nonparametriciled test. Comparisons between IRS scores beforefter treatment were done using Wilcoxon signed-rankr paired data.V and δBV were compared between two groups ofts defined by their MR (MR versus non-MR) usingpaired nonparametric two-tailed test.eiver operating characteristic analyses were done tote the overall performance of SUVmax and BV valuese relative remaining ΔSUV and δBV as a prognosticr the MRs.

lts

s of erlotinib on cell line proliferationt, we checked the absence of somatic mutations inrosine kinase domain of the EGFR in the CAL33 celldata not shown). We then studied the dose andcourse effects of erlotinib on CAL33 proliferationhowed that erlotinib inhibited cell proliferationose- and time-dependent manner with an IC50 of6 μmol/L (data not shown). This growth-inhibitory

1. Patien
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cavity 1,950 2 36 DF Or cavity 3,000 2 48 DF Or cavity 3,750 1 48 DF Or cavity 2,700 2 18 DRD
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pharynx 2,850 2 7 DRD Or cavity 2,700 0 48 DF Oro harynx 3,300 2 35 DRD Or cavity 3,450 2 48 DF L rynx 4,050 0 38 DF
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harynx 3,600 1 37 DuRD Or cavity 3,000 1 36 DF Or cavity 1,650 3 12 LR Oro harynx 3,450 1 34 DF
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ean
3,091.7 Mean 29.2 SD 928.9 SD 14.3 Max 4,950.0 Max 48.0Min 750.0 Min 6.0
E: The tumor localization, cumulated dose of erlotinib, toxicity, and follow-up are detailed for 18 patients with nonmetastaticCC who received neoadjuvant treatment with erlotinib between panendoscopy and surgical treatment.

n inhibition ofociated with the

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ulation of the cyclin-dependent kinase inhibitor1 (Fig. 1). These results showed that erlotinib inhib-ts molecular target and regulated the proliferationay through inhibition of phospho-ERK-1/2, whichncentrated on as the marker of the molecular effectstinib, translating its “biological response” which weamed “molecular response” for the following experi-and in the rest of the article.used a supplementary cell line, CAL166, describedliterature for overexpressing EGFR (12, 13). Never-s, we have quantified its EGFR expression versus thatL33 and found it to be two times higher (Supple-ry Fig. S2). We then showed that erlotinib inhibited6 proliferation in a dose- and time-dependent man-ith an IC50 of 9.6 ± 0.2 μmol/L (data not shown),ting a lower sensitivity to erlotinib compared with.

of erlotinib on CAL33 Glut-1 transporterssionDG is transported into cells by the Glut glucoseorter proteins, and thus we investigated their modi-n of expression under erlotinib treatment in a dose-dent (3-5 μmol/L) and time-dependent (24-72 hours)er. The levels of the Glut transporter proteins were
ined by Western blotting. CAL33 and CAL166 cellssed detectable and similar levels of Glut-1 (Supple-
cells,extrac

cation), ERK-1/2 (B), and p27 (C and D; fold increase of variation versus contat 20 ng/mL during the 15 last min of the experiment). Data are representative o

acrjournals.org


ry Fig. S2) but not Glut-3 and Glut-4 transportersnot shown). CAL33 treatment with 3 to 5 μmol/L ofib for 24 to 72 hours did not decrease Glut-1 levelscell lysate of 3 × 106 cells and on the membrane ofcell (Fig. 2A and B). Moreover, the immunohisto-

cal study of CAL33 (Fig. 4C and D) and CAL166 (Sup-ntary Fig. S2) tumors from xenografted mice showedarable levels of Glut-1 before and after treatment. Theseshowed that erlotinib did not modify glucose trans-nd, consequently, 18FDG uptake in these cell lines.ver, the data also suggest that the glucose transport ca-ty is not altered in the remnant malignant cells, ande cytostatic effect of erlotinib is not mediated by antion of the glycolytic activity in malignant cells.

uptake by CAL33 cellsthen studied the 18FDG uptake by CAL33 cells. Theuptake was determined by measuring the radio-

ty in the cell lysates after subcellular fractionationbrane and cytosol fractions) of CAL33 cells exposedDG for 30 minutes. The radioactivity values reported. 2C were established at the time of treatment andcorrected for the physical decay of 18F. We showed8FDG was detectable in the cytosolic fraction of3 cells exposed to 18FDG, suggesting that in CAL-33
glucose transporters can translocate 18FDG from theellular domain to the cytosol.
Effects of erlotinib on the CAL33 proliferation pathway. Effects of erlotinib treatment (5 μmol/L) on P-EGFR (A; % of variation versus controlkip1
rol quantification) using Western blot (when indicated, EGF was
f three independent experiments. FBS, fetal bovine serum.




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Clin COF6


ation of 18FDG-PET/CT imaging to evaluaterly response of nude mouse xenograftsotinibt, we checked the spatial resolution and sensitivity ofuman PET-CT to validate its capabilities in imagingetecting the 18FDG uptake activity of 1 × 107 cells 4fter their xenografting. The pharmacokinetic study ofFDG mouse tumor uptake led us to determine theal window for in vivo imaging, which was betweend 80 minutes after the 18FDG injection (data not).
then studied whether a rapid decrease in 18FDG up-erlotinib-treated mice could be detected by 18FDG-
72 ho18FDG



maging. Both visual and semiquantitative analysesarried out and are shown in Fig. 3.performed a series of protocols in which we imagedime four mice bearing CAL33 or CAL166 tumors inflanks (two control mice and two erlotinib-treatedbefore and after erlotinib treatment lasting 24 tours (72 hours only for CAL33). Our data showed sig-nt and dramatic reductions in 18FDG uptake of 48%.0001) and 36% (P < 0.001) for CAL33 and CAL166,tively, after 24 hours of erlotinib treatment. Thistion persisted at a significant level (P < 0.05) until

FigGluTim(3-5treatranandrepexpwith18Fforlysecellcolwasnotperin eactaretwodup

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20. © 2010 American Ass

Effects of erlotinib on CAL33expression and 18FDG uptake.ourse (24-48 h) and doseol/L) effects of erlotinibnt on the CAL33 Glut-1rter using Western blotting (A)munofluorescence (B). Data arentative of three independentents. CAL33 cells were treatedher glucose (control cells) or(24.42 MBq/mL; treated cells)in. Cells were then washed,nd fractionated or not (wholehe radioactivity content ofd samples (medium, PBS, trypsin, cell fractionation ors measured. Results aretage of radioactivity measuredfraction versus the 18FDGof treatment (100%). Resultsressed as the mean ofependent experiments in

ere, we have shown thate early (24 hours) in vivo

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PET study of the effects of erlotinib on tumor 18FDG capture. A, nude mice bearing (—▸) subcutaneous CAL33 or CAL166 tumors and visualizedusing an i.v. injection of 9.85 ± 1.5 MBq of 18FDG 4 d after implantation (Before) and 24 to 72 h after oral treatment (After) with either saline (controlor erlotinib (erlotinib group at 100 mg/kg). B and C, histogram representations for CAL33 (B) and CAL166 (C) of the variation of the TBRto-background ratio of FDG uptake signal using ROIs of the same size drawn on cross-sectional images) before and after (24-72 h) saline or
treatment orally. Data are representative of four (CAL33) or two (CAL166) independent experiments, each done in triplicate.
Clin Cancer Res; 16(17) September 1, 2010acrjournals.org OF7

Research. on April 17, 2020. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from


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tion of erlotinib treatment effects resulting in 18FDGe inhibition, which underlies the inhibition ofar metabolism, which in turn is linked to tumortion. As observed for EGFR activity inhibition, theuptake inhibition in CAL166 is lower than that

L33 (36% versus 48%), which is related to the lower treatm



logic analysis of both preclinical andal studiesimmunohistochemical analyses revealed a signifi-eduction of the phosphorylated form of ERK-1/2we compared the patients before and after erlotinib
ent (P = 0.047; Fig. 4). Phospho-EGFR showed a
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Immunohistochemicalf the effects of erlotinib onissue. Sections (4 μm thick)alin-fixed and paraffin-ded tumors from mice (A-D)tients (E-H) before and aftertreatment were stained

tibodies against P-EGFRB), Glut-1 (C-F), and P-ERKH). The IRS (range, 0-12)roduct of the scores forintensity (0, negative;; ++, moderate; +++, strongnd the percentage of cells(0-4 scale; ref. 11). Mousee representative of fourndent experiments done in

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as also observed in patients; surprisingly, the re-n was statistically nonsignificant (Supplementary2 for CAL166; Fig. 4). No significant change wased in the levels of total EGFR (P = 0.61). More inter-ly, no change was observed on the Glut-1 expression(P = 0.2) even when we compared “molecularder” patients to “molecular nonresponder” patients.42 and P = 0.33, respectively; Fig. 4). These results areal agreement with our previous in vitro data andrt the rationale for using 18FDG-PET as a surrogater of the biological effects of erlotinib.

t studys study included 18 patients (1 female and 17 male)mean age of 58.5 ± 10.4 years. Table 1 summarizestients' characteristics and clinical outcomes after theent. Capillary blood glucose level at the time ofinjection was, on average, 1.02 ± 0.21 g/mL (0.6-

/mL).parameters related to the 18FDG uptake within thetumors and their alterations after the exposure tonib are summarized in Supplementary Table S1.erage, the treatment led to a statistically significanttion by 17.9% and 28.8% of SUVbwmax and BV,tively (Fig. 5A).ong these 18 patients, immunohistochemical analysesspho-ERK1/2 showed that 11 patients had a molecu-ponse (MR) and 7were considered as non-MR. Altera-are more pronounced for BV than for SUVbwmax

C). Thus, considering the alteration in phospho-2 protein as the gold standard, the accurate way tothe metabolic response relied on the δBV rather

the ΔSUV variations, as δBV seemed significantlyfor patients having a MR (P = 0.0109; Fig. 5B).

ng receiver operating characteristic analysis of themance of δBV values for the prediction of a MR,a under the curve of 0.92 (95% confidence interval,-1.07; P = 0.003) was observed (Fig. 5D). A cutoff
of δBV = −16% gives the diagnosis of MR a sensitiv- (20).
graftimatingetherGlut-1whichprecedcells.smallthereof trebut istogethand AIn t

not owithblot,

100% and a specificity of 86%.

ssion

on–small cell lung cancers and colorectal cancer, itl established that EGFR or Kras mutations are predic-ctors for the effects of EGFR inhibitors (EGFR-TKIs;4). In HNSCC, there are no currently establishedrs or surrogate markers of the responses to EGFR-ed therapies (e.g., erlotinib; refs. 2, 15). EGFRtions seem to be relatively rare in HNSCC (16),deed, we did not find any relevant mutations ofFR catalytic domain, neither in the clinical studyblished (7) nor in the CAL33 cell line we used in this. Kras mutations are relatively low or absent inC (17, 18). In this study, we looked at the in vitro
of erlotinib on its molecular target (EGFR) and reg-proliferation pathways in the HNSCC cell line
cal antumo

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3. As described in the literature, we showed thatib inhibited EGFR phosphorylation. This inhibitionciated in vivo and in vitro with the inhibition of theeration signal transduction pathway, which results inp1 upregulation and phospho-ERK-1/2 inhibition,g to cell growth inhibition (19). These resultsed us to fix ERK-1/2 phosphorylation as a markerbiological and molecular effects of erlotinib inodel, defining the cell MR to erlotinib treatmente whole of the study.ajor problem for oncologists is the detection of anyspecific biological effects of the targeted therapies ints (2).conventional radiographic modalities are not

ed for the early evaluation of the therapeutic effec-ss of cytostatic drugs. Because these agents preventr growth without necessarily inducing significant tu-egression, assessment based strictly on sequentialrement of tumor size may not accurately reflect

able tumor cell fraction in a residual mass. Warburg'sgs underpin the principles of tumor imaging with-PET (6). The increased metabolism of tumors fore and its analogues, such as 18FDG, is the basis foraging in oncology. Glucose and its analogues areorted into the cell by membrane transporters oflut family. Thus, the glycolytic activity as an indicatoreffects of EGFR-targeted therapies seems relevant asbetween EGFR inhibition and the control of cell

eration. For instance, the phosphatidyl-inositol-3-e/Akt/mammalian target of rapamycin cascade isf the signaling pathways activated by tyrosine kinaseors (EGFR in particular) that regulate antiprolifera-nd apoptotic functions and is also involved in thetion of cell metabolism (3).erimental models of xenografts of gastrointestinalal tumors have shown that FDG-PET might enabletions in glucose metabolism to be observed beforetostatic effects of imatinib mesylate, an EGFR-TKICullinane et al. (21) have shown, in another xeno-model of gastrointestinal stromal tumors, thatib treatment induced a decrease in FDG uptake to-with an early (4 hours posttreatment) decrease intranscription and expression. This metabolic effect,has not been observed in the resistant cell line,ed the cell cycle block and apoptosis of the treatedMoreover, Su et al. (22) have observed that, in non–cell lung cancer lines treated with the TKI gefitinib,is an immediate “metabolic” response (after 4 hoursatment) that is linked to a decrease in FDG uptakealso associated with Glut-3 transporter expressioner with the inhibition of EGFR phosphorylationkt phosphorylation.his study, we evaluated Glut-1 expression. We didbserve any significant decrease in Glut-1 expressionany of the experimental techniques used: Westernimmunofluorescence assay, or immunohistochemi-
alysis of cell lines, mouse xenografts, and patients'rs. Moreover, the representation to the membrane



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Glut-1 transporter was also studied by immunoflu-nce using a disconsolation procedure (data not
). Until now, in HNSCC, there are no reports show-decrease in Glut-1 by EGFR-TKI. Elsewhere, there are
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s reporting that there are no significant correlationsen 18FDG accumulation and Glut-1 expression in

Figtissmeandafte*, twpoosagfusneoerloandand(pothebeta s1/2thophomochaδBV16.

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20. © 2010 American A

Effects of erlotinib on human tumorA, metabolic response with theumor value ± 1 SD of SUVbw (square)(circle) before (filled symbol) andpen symbol) erlotinib treatment.ailed Wilcoxon signed-rank test ofdata. BW, body weight. B, facialsection of a 18FDG-PET-CT

of a patient with an oropharyngealm before (I) and after (II) 21 d of. Patient #13, molecular respondertabolic responder (ΔSUV = −17.8%V = −27.3%). C, metabolic responsemean; bars, SEM) assessed usingVmax (ΔSUVbw) and the BV (δBV)n two groups of patients, those withicant alteration of the phospho-ERK-tein (Mr, molecular responder) andithout significant alteration of theo-ERK-1/2 protein (nMr, non–lar responder). D, receiver operatingteristic curve of the performance ofr the prediction of MR (δBV >

ings suggest that 18FDG-PETesidual disease after HNSCC


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ent with erlotinib because Glut expression seems toserved in remnant malignant cells.e, both the animal model and the patient tumorhowed that the alterations in 18FDG uptake are asso-with tumor responses at the molecular level, withwnregulation of P-ERK-1/2, and with the clinical

its observed in patients (7) without underestimatingsidual disease because of the nonmodification ofexpression in malignant cells from patients and tu-

enografts. Taken together, our results show that FDGe can be considered as an early and reliable markeress the efficacy of an EGFR-TKI. However, evaluationing to the variation in SUV (ΔSUV) or the variation(δBV) compared with MR led to conflicting conclu-Using P-ERK1-2 inhibition as control, ΔSUVmax

d to be less accurate than δBV for the diagnosis ofR to erlotinib with 18FDG-PET/CT. Despite its limita-the SUV is the most frequently encountered param-sed for treatment monitoring with PET (10). Boucek(25) demonstrated in phantom studies that thetion of the metabolic volume was more accuratethe SUVmax. Moreover, the study of Daisne et al.howing that BV defined by FDG-PET provided a re-evaluation of the real volume of head and neck can-rees with our conclusion about the accuracy of theBV instead of SUV.clear that there are many methodologic approachesforming PET image segmentations. We acknowledgeemethod used in this studymay be less accurate thane used by Daisne et al. (26) or Boucek et al. (25), asfluence of the different noise-to-signal ratios is notinto account for threshold determination (21, 22).theless, the method used in this study is availablemmercialized clinical software, and according tot al. (27), it seemed to be a good compromise be-simplicity, user independence, reproducibility, andcy.re are still some discrepancies between the molecu-d metabolic responses. Smith-Jones et al. (28)d that 18FDG-PET may be limited in detecting the
atic response to targeted therapies because of itsf specificity. This could lead to the use of a more
Recepublish

esophagus. J Nucl Med 2004;45:56–68.n den Abbeele AD, Badawi RD. Use of positron emission tomog-hy in oncology and its potential role to assess response to ima-

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ic marker of cell proliferation, such as 3-deoxy-3-orothymidine (29). Indeed, Atkinson et al. (30) re-that 3-deoxy-3-18F-fluorothymidine allowed anti-inhibitor therapy in squamous cell carcinoma tonitored. However, 3-deoxy-3-18F-fluorothymidinecommercially available. The present study providesevidence that FDG is a suitable marker of the effects

Is.

lusion

G-PET/CT enables the early evaluation of the effica-erlotinib treatment both in preclinical models ofC and in patients. This is the first preclinical andl study to assess that 18FDG-PET/CT using the dailyntional clinical procedures is a reliable way to deter-the early biological effects of erlotinib. In the headeck cancer model used in this study, the inhibition ofuptake was in agreement with the MR (inhibition

K phosphorylation). Moreover, the differential MRse metabolic effects observed implicate EGFR path-isruption (ERK1-2 inhibition) as the mechanismg 18FDG-PET/CT changes. The expression of glucoseorters was not altered in malignant cells whetherpatients or tumor xenografts. These data establishse of the metabolic tumor volume in 18FDG-PET/r the early evaluation of the biological effects of-TKI in HNSCC.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

thank the Institut Claudius Regaud animal facility, John Woodleyniela Oswald for medical writing support, and Julia Nalis andudet for the TEP imaging acquisitions.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

ived 10/21/2009; revised 06/23/2010; accepted 07/13/2010;ed OnlineFirst 08/17/2010.

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Published OnlineFirst July 26, 2010.Clin Cancer Res Sébastien Vergez, Jean-Pierre Delord, Fabienne Thomas, et al. CancerEarly Molecular Responses to Erlotinib in Head and NeckComputed Tomography Is a Reliable Tool for the Detection of F]fluoro-D-glucose Positron Emission Tomography with

18Preclinical and Clinical Evidence that Deoxy-2-[

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