Preclinical and clinical evidence that 18FDG-PET/CT is a ...clincancerres.aacrjournals.org/content/clincanres/early/2010/07/24/... · This work was presented as a poster presentation

Author manuscripts have been peer reviewed and accepted for publication but have not yet been

edited. Copyright © 2010 American Association for Cancer Research

Preclinical and clinical evidence that 18FDG-PET/CT is a

reliable tool for the detection of early molecular responses

to erlotinib in head and neck cancer

Sébastien Vergez1, Jean-Pierre Delord1,2, Fabienne Thomas1,2, Philippe Rochaix1,2,

Olivier Caselles2, Thomas Filleron2, Séverine Brillouet2 , Pierre Canal1,2, Frédéric

Courbon 2,# * and Ben C. Allal 1, 2 ,#

*

1-Laboratoire de Pharmacologie Clinique et Expérimentale des Médicaments

Anticancéreux, EA 3035, Université Paul Sabatier,

2-Institut Claudius Regaud, 20-24 rue du pont Saint-Pierre, 31052 Toulouse Cedex,

France.

# Corresponding authors

* FC and BCA contributed equally to the supervision of this work.

This work was presented as a poster presentation during the 99th AACR Congress,

San Diego, California, 04/13/2008. poster #422

Published OnlineFirst on July 26, 2010 as 10.1158/1078-0432.CCR-09-2795 Published OnlineFirst on July 26, 2010 as 10.1158/1078-0432.CCR-09-2795

on July 7, 2018. © 2010 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 26, 2010; DOI: 10.1158/1078-0432.CCR-09-2795

http://clincancerres.aacrjournals.org/



STATEMENT OF TRANSLATIONAL RELEVANCE

We show that 18FDG-PET/CT can be used as a surrogate marker for the early

evaluation of EGFR-Tyrosine Kinase Inhibitor efficacy. For this purpose we

developed a preclinical model to validate 18FDG-PET/CT imaging for the early

evaluation of the molecular effects of erlotinib on a Head and Neck Squamous Cell

Carcinoma cell line. We then cross-validated this tool during a clinical trial designed

to assess the pharmacodynamic effects of erlotinib in patients with head and neck

squamous cell carcinoma who received erlotinib as neoadjuvant treatment for a short

time period before surgery. This is the first study providing, from preclinical models to

patients, a reliable proof of efficiency of this method.






Abstract

Purpose: There is a clinical need to identify predictive markers of the responses to

EGFR tyrosine-kinase inhibitors (EGFR-TKI). Positron Emission Tomography of

deoxy-2-[18F]Fluoro-D-glucose (18FDG-PET/CT) could be a tool of choice for

monitoring the early effects of this class of agent on tumor activity.

Experimental design: Using models of human head and neck carcinoma (CAL33

and CAL166 cell lines), we tested first in vitro and in vivo, whether the in vivo

changes in 18FDG-PET/CT uptake were associated with the molecular and cellular

effects of the EGFR-TKI erlotinib. Then, the pathological and morphological changes

and the 18FDG-PET/CT uptake before and after erlotinib exposure in patients were

analyzed.

Results: Erlotinib strongly inhibited ERK-1/2 phosphorylation in both preclinical

models and in patients. Western blotting, immunofluorescence and

immunohistochemistry showed that erlotinib did not modify Glut-1 expression at the

protein level either in cell line models or in tumor tissue from mouse xenografts or in

patients. Phospho-ERK-1/2 inhibition was associated with a reduction in 18FDG

uptake in animal and human tumors. The Biological Volume was more accurate than

the Standardized Uptake Value for the evaluation of the molecular responses.

Conclusion: These results show that the 18FDG-PET/CT response is a reliable

surrogate marker of the effects of erlotinib in head and neck carcinoma.






Introduction

Epithelial growth factor receptor (EGFR) inhibitors have clinical activity in various

tumor types, including head and neck squamous cell carcinoma (HNSCC), used

alone, or in combination with cytotoxic agents. Unfortunately, only a subgroup of

patients benefits from these targeted agents (1).

Consequently, there is a clear medical need for the early identification of those

patients most likely to benefit from this targeted treatment. The development of tools

to select these patients could facilitate their therapeutic cure and the determination of

their biological effective dose, and subsequently lead to individualization of treatment

(2).

Positron emission tomography imaging with [18F] FluoroDeoxyGlucose with

computed tomography (18FDG-PET/CT) has become an important non-invasive

technique for examining cancer staging and detecting recurrent neoplasms. Many

clinical trials have shown that 18FDG-PET imaging could provide an early indication

of therapeutic responses that are well correlated with clinical outcomes (3-5). 18FDG-

PET could be particularly useful for the evaluation of the proportion of active tumor

cells during treatment with EGFR tyrosine-kinase inhibitors (EGFR-TKI). Some

changes in tumor volume are observed late or not at all, for example when intra-

tumoral necrosis and fibrosis prevent tumor shrinkage and could actually cause a

paradoxical expansion of some tumors due to intra-tumoral bleeding or oedema. This

is not the case for the changes in metabolic activity of the tumors which could be

highlighted early. Indeed, some studies have evaluated the predictive value of

18FDG-PET as a consequence of the correlation of early metabolic responses with






the clinical outcome, as has been described for the response to imatinib in

gastrointestinal stromal tumors (5).

Therefore, although some results indicating that molecular imaging with 18FDG-PET

could be a valuable tool for drug development and use (3), it remains to be shown

that the changes in 18FDG uptake correlates with the molecular responses induced

by erlotinib.

Our goal was to establish the rationale of the use of 18FDG-PET/CT as a surrogate

marker for the early evaluation of EGFR-TKI. For this purpose we developed a

preclinical model to validate 18FDG-PET/CT imaging for the early evaluation of the

molecular effects of erlotinib on HNSCC cell lines. EGFR regulates numerous

signalling pathways involved in various cellular mechanisms contributing to the

control of cellular homeostasis and proliferation. In this cellular proliferation pathway

ERK-1/2 is a key downstream effector. This inhibition of the ERK-1/2 could reflect

what we have named the “molecular” and/or “biological” response to the drug.

.

Elsewhere, we also define the “metabolic effects” as the metabolic changes observed

in the cells and resulting in the modification of glucose uptake (18FDG) (6).

Metabolism and proliferation are associated cellular processes, because cell

proliferation is "energy consuming" and metabolism dependent (6).

Finally, we cross-validated this tool during a clinical trial designed to assess the

pharmacodynamic effects of erlotinib in patients with HNSCC who received erlotinib

for a short time period before surgery (7).






Materials and Methods

Animals and agents. Female Swiss athymic nude mice, 4 to 5 weeks old (Charles

River Laboratories, L’Arbresele, France) were maintained in accordance with the

standards of the Federation of European Laboratory Animal Science Associations.

and included in protocols following 2-weeks quarantine. Erlotinib (OSI-774,

Tarceva) was kindly provided by F. Hoffmann-La Roche, Inc (Basel, Switzerland).

18F-FDG (Glucotep®) was from Cyclopharma (Toulouse France).

Antibodies. The antibodies used for Western blotting were: Anti-Phospho-

EGFR/HER-1 (Tyr1173, Euromedex, Mundolsheim, France); anti-total EGFR/HER-1

(Ab-12) and anti-tubulin beta (NeoMarkers Ab, Interchim, Montluçon, France); anti-

Glut1, anti-phospho-ERK-1/2 pAb, (cell signaling Ab, Ozyme-Saint-Quentin-en-

Yvelines, France), anti-ERK-1/2 (c-16) (Santa Cruz Biotech, Tebu-Bio SA, Le Perray

en Yvelines, France); anti p27Kip1 (Dako, Trappes, France), peroxidase-conjugated

secondary mouse or rabbit antibodies (Bio-Rad, Marnes la Coquette, France).

The antibodies used for immunochemistry: Anti-Phospho-EGFR/HER-1 (SC36-9700,

Zymed); anti-total EGFR/HER-1 (EGFr PharmDX™, Dako); anti-Glut-1 (RB-9052,

Neomarkers, Fremont CA), anti phospho-ERK-1/2 (SC7383s, Santa Cruz), and anti

p27kip1 (SX53G8, Dako).

Cell culture. CAL33 and CAL166 cells (human head and neck carcinoma, Centre

Antoine Lacassagne Nice, France (8)) were cultured in Dulbecco Modified Eagles

medium (DMEM) containing 10% Fetal bovine serum (FBS), supplemented with 2






mM L-glutamine (culture medium; Cambrex biosciences, Emerainville, France) at

37°C in a humidified atmosphere and 5% CO2.

Western blot analysis. On day 1, 1.5x106 cells were plated in culture medium in 100

mm culture dishes. On day 2 for the EGFR and ERK analyses cells were or not

serum-starved for 24h and then treated with either vehicle or erlotinib at

concentrations of 3, 3.5 or 5 µM for 24 to 72 hours. When necessary, cells were

treated with 20 ng/mL EGF for the last 15 minutes of the experiment. The cells were

harvested and lysed in lysis buffer (Tris 50 mM pH 8, NaCl 150 mM, 0.1% NP40, 5

mM MgCl2, 50 mM NaF, 2 mM PMSF, 10 mM DTT, 2 mM orthovanadate, 5 mg/mL

sodium dexoxycholate, 6.4 mg/mL phosphatase substrate; Sigma 104®). For EGFR,

ERK-1/2, Glut-1, p27kip1, or beta-tubulin analysis, 70 µg of the cleared lysates were

separated on a 7.5% or 12.5% SDS-PAGE gel, blotted onto PVDF membranes

(Amersham, Orsay, France) and incubated with specific antibodies.

For the determination of erlotinib effects on Glut-1 expression, 24 hours after plating,

cells were treated with either vehicle or erlotinib as previously described. Cells were

harvested by trypsinization and counted. Three million cells were pelleted (820g, 5

min), lysed and analyzed by western blot as described here before (12.5% SDS-

PAGE, Glut-1 antibody).

Detection was performed using peroxidase-conjugated secondary antibodies (Bio-

Rad) and an enhanced chemiluminescence detection kit (Amersham Pharmacia

Biotech). The blots were scanned and analyzed with a Molecular Dynamics

densitometer and ImageQuant software. Results are representative of three

independent experiments.






Fluorescence. Immunofluorescence histology was performed as described

elsewhere (9). Cells were seeded on glass cover-slips in six-well plates, and treated

with either vehicle or erlotinib at 5 µM erlotinib for 48 hours. Membrane Glut-1

transporters were detected by incubation with antibody against Glut-1 plus a

rhodamine-labeled secondary antibody and images recorded with a Princeton

camera. Results are representative of three independent experiments in duplicate.

Determination of 18FDG uptake by CAL33 cells. On day 1, 1.5x106 CAL33 cells

were plated in 60 mm culture dishes in culture medium. 24 hours later cells were

treated with either glucose (control cells) or 24.42 MBq/ml of 18FDG (treated cells) for

30 minutes. The medium was collected, the cells were washed three times with 3 ml

of PBS and each wash collected in separated tubes. Cells were trypsinized (500 µl of

trypsin) and collected in 2.5 ml of culture medium and pelleted (850g, 5 min). Trypsin

and supernatants were collected. The pelleted cells were either reserved (whole

cells) or suspended in 500µl PBS and lysed by three cycles of thermal shock (liquid

nitrogen/37°C) followed by 15 minutes centrifugation (15000g) to obtain a lysate

fraction. Supernatants, representing the cytosolic cell fraction and the pellets,

representing the membrane plus nuclear cell fraction were collected. The results are

expressed as the percentage of radioactivity (corrected for the physical decay of 18F)

measured in each fraction versus the 18FDG activity at time of treatment (100%) and

are the mean ± SEM of 2 independent experiments in duplicate.






Effect of erlotinib on cell lines xenografts in nude mice. CAL33 or CAL166

xenografts were established by the subcutaneous injection of 1x107 cells into both

mouse flanks. When the tumor size reached around 200 mm3 (day 4 post

implantation), the mice were pooled and randomly assigned to 2 groups (control and

treated with erlotinib) of six to eight animals.

For the 18FDG-PET/CT imaging mice were injected via the tail vein with 9.85±1.5MBq

of 18FDG and then anesthetized by an intra-peritoneal injection of ketamin/xylazin

solution (100 mg/kg / 5 mg/kg). Anesthesia was maintained for 60 minutes and if

necessary, before the 18FDG-PET/CT imaging mice were re-challenged for

anesthesia for the further 30 minutes of imaging (PET-CT Discovery ST General

Electric Health and Care (GEHC) Milwaukee USA) after which the mice were allowed

to recover. Mice were maintained under a controlled temperature (around 22°C)

during all the experiments. Mice were then treated per-os with either saline (control

group) or Erlotinib at 100 mg/kg/day in saline (erlotinib group). 24 hours later the

mice were imaged as described, and in some protocols the mice were treated and

scanned after 72 hours treatment.

To perform the PET acquisition, mice were placed in a special box enabling 4 mice to

be imaged at once (2 control and 2 treated). PET data handlings and reconstructions

are shown in the supplementary data. Tracer uptake was measured using the regions

of interest (ROI) selected on cross-sectional images. ROI with the same size were

drawn around the tumor and a background region on transaxial slice images to

determine the Tumor-to-Background Ratio (TBR) calculated by dividing the average

pixel intensity within a tumor ROI by the average pixel intensity within the background






ROI. Results are representative of at least 2 independent experiments with 3 mice

per kinetic point.

At the end of the experiment, the mice were sacrificed and the tumors removed; fixed

in formalin for pathological and immunohistochemical analysis.

Effect of neoadjuvant treatment with erlotinib in patients with HNSCC

Our team has published a pilot study of neoadjuvant treatment with erlotinib of non-

metastatic HNSCC (7). Patients were eligible if they were candidates for first-line

curative surgical treatment or had been scheduled for surgery by necessity. After

diagnosis, patients underwent routine pan-endoscopy and 18FDG-PET/CT. Treatment

with oral erlotinib 150 mg/day started the following day. Patients were treated for 20

days on average (Table1), corresponding to the time between pan-endoscopy and

surgical resection. 18FDG-PET/CT examinations were repeated 48 hours before

surgery at the latest. Pathological examinations with immunostaining were done on

biopsies before and after treatment.

18F-FDG PET/CT acquisitions and interpretations

Serum glucose was measured before intra-venous injection of 370MBq of 18F-FDG

(Glucotep® Cyclopharma Toulouse France). A whole-body (from skull to pelvis)

18FDG-PET/CT acquisition was carried out (GEHC). Acquisitions were performed in

two-dimensional mode (2D), (5 minutes/bed position). 2D sinograms were

reconstructed in 256*256 matrix size, with a field of view of 50 cm and corrected for

attenuation, random and scatter. CT imaging was performed for Attenuation

Correction and anatomical correlation with a 200 mA tube current, 140 kV tube






voltage, a helical pitch of 0.75 :1 and a reconstructed slice thickness of 3.75 mm for

an interval of 3.27 mm between slices.

The 2D18FDG-PET/CT data corrected for attenuation were transferred to an

Advantage Workstation 4.2® (GEHC Buc sur Yvette France). The maximum

Standard Uptake value (SUVmax) (10) and the Biological Volume of the tumor (BV)

were determined using commercial software (PET VCAR® GEHC). SUVmax were

corrected for the Body Weight (SUVBW). The biological volume was determined using

a fixed threshold between the maximum pixel counts within the tumor and the

background. We previously carried out a control study, and determined that for the

PET system and the acquisition protocol used, a threshold of 35% was the most

accurate.

The metabolic response was expressed according to the change in either the SUV

and BV determined as follows:

Δ SUV = (SUVa-SUVb)/SUVb x 100 and δBV= (BVa-BVb) / BVb x100 (b stands for

before treatment and a for after treatment).

Immunohistochemistry

Analyses were done on 4 µm-thick formalin-fixed paraffin sections of patient tumors

and cell lines xenografts according to procedure described elsewhere (7). For Glut-1

immunostaining the antibody dilution was ready to use and antigen retrieval methods

used 10 mmol/L citrate buffer, pH 6 Microwave heat 750W 5 min x 3. Immunostaining

semi-quantitative assessment was performed regarding the staining intensity using a

four points scale (i.e.: 0=negative; +=weak; ++=moderate; +++=strong) and the

percentage of labeled cells. Immunostaining analyses were evaluated using the






ImmunoReactive Score (IRS), according to Remmele et al (11). The IRS (range 0–

12) is the product of the scores for staining intensity (0–3 scale) and percentage of

cells stained (0–4 scale).

Statistical analysis. All results are expressed as mean ± standard error of the mean

(SEM). Results were analyzed using Student’s t tests and a P value <0.05 was

accepted as statistically significant. SUVmax and BV after and before treatment were

compared using a paired non-parametric two-tailed test. Comparisons between IRS

score before and after treatment were performed using Wilcoxon signed rank test for

paired data.

Δ SUV and δBV were compared between two groups of patients defined by their

molecular response [Molecular Response (MR) versus non-Molecular Response

(nMR)] using an unpaired non-parametric two-tailed test.

Receiver operating characteristic (ROC) analyses were done to evaluate the overall

performance of SUVmax and BV values and the relative remaining ΔSUV and δBV as

a prognostic test for the molecular responses.






Results

Effects of erlotinib on cell lines proliferation. Firstly, we checked the absence of

somatic mutations in the tyrosine kinase domain of the EGFR in the CAL33 cell lines

(data not shown). We then studied the dose and time course effects of erlotinib on

CAL33 proliferation and showed that erlotinib inhibited cell proliferation in a dose-and

time-dependent manner with an IC50 of 4 ± 0.6 µM (data not shown). This growth-

inhibitory effect of erlotinib was paralleled by an inhibition of EGFR and ERK-1/2

phosphorylation associated with the up-regulation of the cyclin dependent kinase

inhibitor p27kip1 (Figure-1). These results showed that erlotinib inhibited its molecular

target and regulated the proliferation pathway via the inhibition of phospho-ERK-1/2,

which we concentrated on as the marker of the molecular effects of erlotinib,

translating its “biological response” we also named “molecular response” for the

following experiments and in the rest of the manuscript.

We used a supplementary cell line, CAL166, described in the literature for

overexpressing EGFR (12, 13). Nevertheless we have quantified its EGFR

expression versus the CAL33 and showed an expression two times higher (Suppl

Figure-7A). We then showed that erlotinib inhibited CAL166 proliferation in a dose-

and time-dependent manner with an IC50 of 9.6±0.2 µM (data not shown), showing a

lower sensitivity to erlotinib compared to CAL33.

Effect of erlotinib on CAL33 Glut-1 transporter expression.

18FDG is transported into cells by the Glut glucose transporter proteins, and so we

investigated their modification of expression under erlotinib treatment in a dose- (3 to






5 µM) time-dependant (24 to 72h) manner. The levels of the Glut transporter proteins

were determined by Western blotting. CAL33 and CAL166 cells expressed a

detectable and similar Glut-1 (Suppl Figure-7A) but not Glut-3 and Glut-4 transporters

(data not shown). CAL33 treatment with 3 to 5 µM of erlotinib for 24 to 72 hours did

not decrease Glut-1 levels from 3x106 whole cell lysates (Figures-2A, B). Moreover,

the immunohistochemical study of CAL33 (Figures-4C, D) and CAL166 (Suppl

Figure-7B) tumors from xenografted mice showed a comparable level of Glut-1

before and after treatment. These results showed that erlotinib did not modify glucose

transport and consequently 18FDG uptake in these cell lines. Moreover, the data also

suggests that the glucose transport capability is not altered in remnant malignant

cells, and that the cytostatic effect of erlotinib is not mediated by any inhibition of the

glycolytic activity in malignant cells.

18FDG uptake by CAL33 cells. We then studied the 18FDG uptake by CAL33 cells.

The 18FDG uptake was determined via the radioactivity measurement in the cell

lysates after subcellular fractionation, in membrane and cytosol fractions, of CAL33

cells exposed to 18FDG for 30 minutes. The radioactivity values reported in Figure-

2C, were established at time of treatment and were corrected for the physical decay

of 18F. We showed that 18FDG was detectable in the cytosolic fraction of CAL-33 cells

exposed to 18FDG suggesting that in CAL-33 cells, glucose transporters can

translocate 18FDG from the extracellular domain to the cytosol.

Evaluation of 18FDG-PET/CT imaging to evaluate the early response of nude

mice xenografts to erlotinib. First we checked our human PET-CT spatial






resolution and sensitivity in order to validate its capabilities in imaging, detection and

18FDG uptake activity by 1x107 cell lines 4 days after their xenografting. The

pharmacokinetic study of the 18FDG mice tumor uptake lead us to determine the

optimal window for in vivo imaging which was between 60 and 80 minutes following

the 18FDG injection (data not shown).

We then studied whether a rapid decrease in 18FDG uptake in erlotinib-treated mice

could be detected by 18FDG-PET imaging. Both visual and semi-quantitative

analyses were carried out and are shown in Figure-3 .

We performed a series of protocols in which we imaged each time four mice bearing

CAL33 or CAL166 tumors in both flanks (2 control mice and 2 erlotinib-treated)

before and after erlotinib treatment lasting 24 to 72 hours (72h only for CAL33). Our

data showed significant and dramatic reductions in 18FDG uptake of 48 % (p<0.001)

and 36% (p<0.001) for respectively CAL33 and CAL166 after 24 hours of erlotinib

treatment. This inhibition persists at a significant level (p<0.05) until 72h (64%

inhibition). Here, we have shown that 18FDG-PET imaging enabled the early (24h) in

vivo evaluation of erlotinib treatment effects resulting in 18FDG uptake inhibition

underlying the inhibition of the cellular metabolism, which is linked to the tumor

inhibition. As observed for EGFR activity inhibition, the 18FDG uptake inhibition in

CAL166 is lower than for CAL33 (36% versus 48%) certainly in relation with the lower

sensitivity to erlotinib of this cell line.

Pathological analysis of both pre-clinical and clinical studies.

The immunohistochemistry analyses revealed a significant reduction of the

phosphorylated form of the ERK-1/2 when we compared patient before versus after






erlotinib treatment (p=0.047 ; Figure-4). Phospho-EGFR showed a reproducible and

important reduction in the mice model also observed in patients surprisingly non

statistically significant (Figure-4 and Suppl-Figure-7 for CAL166). Whereas no

significant change was observed in the levels of total-EGFR (p=0.61). More

interestingly no change was observed on Glut-1 expression level (p=0.2) even we

compared “molecular responder” patients to “molecular non-responder” patients

(respectively p=0.42 and 0.33 Figure-4). These results are in total agreement with

our previous in vitro data and comfort the rationale for using 18FDG-PET as a

surrogate marker of erlotinib biological effect.

Patient study. This study concerned 18 patients (1 female, 17 male) with a mean

age of 58.5 ±10.4 years. Table-1 summarizes the patients’ characteristics and clinical

outcomes after the treatment. Capillary blood glucose level at the time of 18FDG

injection was on average 1.02±0.21 g/mL [0.6-1.43 g/mL].

The parameters related to the 18FDG uptake within the entire tumors and their

alterations after the exposure to erlotinib are summarized in table-2 (supplementary

data). On average, the treatment led to a statistically significant reduction by 17.9%,

and 28.8% of the SUVbwmax and BV respectively (Figure-5A).

Among these 18 patients, immunohistochemical analyses of the phospho-ERK1/2

demonstrated that 11 patients had a molecular response and 7 were considered as

non-molecular responders. Alterations are more pronounced for BV than the

SUVbwmax (Figure-5C). Thus, considering the alteration in phospho-ERK1/2 protein

as the gold standard, the accurate way to assess the metabolic response relied on






δBV rather than the ΔSUV variations, as δBV appeared significantly higher for

patients having a molecular response p=0.0109 (Figure-5B).

Using ROC analysis of the performance of δBV values for the prediction of a

molecular response an area under the curve of 0.92 (95% CI, 0.769-1.07; p=0.003)

was observed (Figure-5D). A cutoff value of δBV=-16% gives the diagnosis of

molecular response with sensitivity of 100% and specificity of 86%.






Discussion

In non-small cell lung cancers and colorectal cancer, it is well-established that EGFR

or Kras mutations are predictive factors for the effects of EGFR inhibitors (EGFR-TKI)

(14). In Head and Neck Squamous Cell Carcinoma (HNSCC) there are no currently

established markers or surrogate markers of the responses to EGFR targeted

therapies e.g. erlotinib (2, 15). EGFR mutations appear to be relatively rare in

HNSCC (16) and indeed neither in the clinical study we published (7) nor in the

CAL33 cell line we used in this study we find any relevant mutations of the EGFR

catalytic domain. Kras mutations are relatively low or absent in HNSCC (17, 18). In

this study, we looked at the in vitro effects of erlotinib on its molecular target (EGFR)

and regulated proliferation pathways on the HNSCC cell line CAL33. As described in

the literature, we showed that erlotinib-inhibited EGFR phosphorylation. This

inhibition is associated in vivo and in vitro with the inhibition of the proliferation signal

transduction pathway resulting in the p27kip1 up-regulation and the phospho-ERK-1/2

inhibition, leading to cell growth inhibition (19). These results allowed us to fix ERK-

1/2 phosphorylation as a marker of the biological and molecular effect of erlotinib in

our model, defining the cell “molecular response” under erlotinib treatment for the

whole of the study.

A major problem for oncologists is being able to detect early any specific biological

effects of the targeted therapies in patients (2).

The conventional radiographic modalities are not adapted for the early evaluation of

the therapeutic effectiveness of cytostatic drugs. Because these agents prevent

tumor growth without necessarily inducing significant tumor regression, assessment






based strictly on sequential measurement of tumor size may not accurately reflect the

viable tumor cell fraction in a residual mass. Warburg’s findings underpin the

principles of tumor imaging with [18F]-FluoroDeoxyGlucose positron emission

tomography (18FDG-PET) (6). The increased metabolism of tumors for glucose and

its analogs, such as 18FDG, is the basis for PET imaging in oncology. Glucose and its

analogs are transported into the cell by membrane transporters of the Glut family.

Thus, the evaluation of glycolytic activity as an indicator of the effects of EGFR

targeted therapies seems relevant as a link between EGFR inhibition and the control

of cell proliferation. For instance, the phosphatidyl-Inositol-3-Kinase

(PI3K)/Akt/mTOR (mammalian target of rapamycine) cascade is one of the signaling

pathways activated by tyrosine kinase receptors (EGFR in particular) which regulates

anti-proliferative and apoptotic functions, and is also involved in the regulation of cell

metabolism (3).

Experimental models of xenografts of gastrointestinal stromal tumors have

demonstrated that FDG-PET might enable alterations in glucose metabolism to be

observed before the cytostatic effects of imatinib mesylate, an EGFR-TKI (20).

Cullinane et al have shown, on another xenograft model of gastrointestinal stromal

tumors, that imatinib treatment induced a decrease in FDG uptake together with the

early (4 hours post treatment) decrease in Glut-1 transcription and expression (21).

This metabolic effect, which has not been observed on the resistant cell line,

preceded the cell cycle block and apoptosis of the treated cells. Moreover Su et al.

have observed that, in non-small cell lung cancer lines treated with the TKI gefitinib

there is an immediate “metabolic” response (after four hours of treatment) linked to a

decrease in FDG uptake but associated with the Glut-3 transporter expression






inhibition together with inhibition of EGFR phosphorylation and Akt phosphorylation

(22).

In this study we evaluated Glut-1 expression. Whatever the experimental technique

used, western-blot, immunofluorescence assay or immunohistochemical analysis of

the cell lines, mice xenografts and patients' tumors, we did not observe any

significant decrease in Glut-1 expression. Moreover the representation to the

membrane of the Glut-1 transporter was also studied by immunofluorescence using a

disconsolation procedure (data not shown). Until now in HNSCC there are no reports

showing Glut-1 decreasing under the influence of EGFR-TKI. Elsewhere there are

studies reporting that there are no significant correlations between 18FDG

accumulation and Glut-1 expression in HNSCC (23, 24). These findings suggest that

18FDG-PET does not underestimate the residual disease after HNSCC treatment with

erlotinib since Glut expression appears to be conserved in remnant malignant cells.

Here both animal model and patient tumor data demonstrated that the alterations in

18FDG uptake are associated to tumor responses at a molecular level, with the down

regulation of P-ERK1-2, and also clinical benefits to patients (7) without

underestimation of the residual disease because of the non-modification of the Glut-1

expression in malignant cells from patients and tumor xenografts. Taken together our

results demonstrate that FDG uptake can be considered as an early and reliable

marker to assess the efficacy of an EFGR-TKI. However, evaluation according to the

variation of the (ΔSUV) or the variation of the biological volume (δBV) compared to

molecular response led to conflicting conclusions. Using P-ERK1-2 inhibition as

control, ΔSUVmax seemed less accurate than δBV for the diagnosis of the molecular

response to erlotinib with 18FDG-PET/CT. Despite its limitations, the SUV is the most






frequently encountered parameter used for treatment monitoring with PET (10).

Boucek et al demonstrated on phantom studies that the evaluation of the metabolic

volume was more accurate than the SUVmax (25). Moreover the study of Daisne et

al demonstrating that BV defined by FDG-PET provided a reliable evaluation of the

real volume of head and neck cancer is in agreement with our conclusion concerning

the accuracy of the use of BV instead of SUV (26).

It is clear that there are many methodological approaches to perform PET images

segmentations. We acknowledge that the method used in this study may be less

accurate than the one used by Daisne et al, or Boucek et al, as the influence of the

different noise-to-signal ratios is not taken into account for threshold determination

(21,22). Nevertheless the method used in this study is available on commercialized

clinical software and according to Krak et al. it appeared to be a good compromise

between simplicity, user independence, reproducibility and accuracy (27).

There are still some discrepancies between the molecular and metabolic responses.

Smith-Jones et al. showed that 18FDG-PET may be limited in detecting the cytostatic

response to targeted therapies because of its lack of specificity (28). This could lead

to the use of a more specific marker of cell proliferation such as 3-deoxy-3-18F-

fluorothymidine (FLT) (29). Indeed, Atkinson et al reported that FLT allowed anti-

EGFR inhibitor therapy in squamous cell carcinoma to be monitored (30). However

FLT is not commercially available. Moreover, the present study provided strong

evidence that FDG is a suitable marker of the effect of tyrosine kinase inhibitors.






Conclusion

18FDG-PET/CT enables the early evaluation of the efficacy of erlotinib treatment to

be achieved both in preclinical models of HNSCC and in patients. This is the first

preclinical and clinical study assessing that 18FDG-PET/CT using the daily

conventional clinical procedures is a reliable way to assess the early biological

effects of erlotinib. In this head and neck cancer model the inhibition of 18FDG uptake

was in agreement with the molecular response (inhibition of ERK phosphorylation).

Moreover the differential molecular responses and the metabolic effects observed

implicate EGFR pathway disruption (ERK1-2 inhibition) as the mechanism driving

18FDG-PET/CT changes. The expression of glucose transporters was not altered in

malignant cells whether from patients or tumor xenografts. These data establish the

use of the metabolic tumor volume on 18FDG-PET/CT for early evaluation of the

biological effect of EGFR-TKI in HNSCC.






References

1. Soulieres D, Senzer NN, Vokes EE, et al. Multicenter phase II study of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with recurrent or metastatic squamous cell cancer of the head and neck. J Clin Oncol 2004;22:77-85.

2. Goodin S. Erlotinib: optimizing therapy with predictors of response? Clin Cancer Res 2006;12:2961-3.

3. Kelloff GJ, Hoffman JM, Johnson B, et al. Progress and promise of FDG-PET imaging for cancer patient management and oncologic drug development. Clin Cancer Res 2005;11:2785-808.

4. Kostakoglu L, Goldsmith SJ. PET in the assessment of therapy response in patients with carcinoma of the head and neck and of the esophagus. J Nucl Med 2004;45:56-68.

5. Van den Abbeele AD, Badawi RD. Use of positron emission tomography in oncology and its potential role to assess response to imatinib mesylate therapy in gastrointestinal stromal tumors (GISTs). Eur J Cancer 2002;38 Suppl 5:S60-S65.

6. Warburg O. On the origin of cancer cells. Science 1956;123:309-14.

7. Thomas F, Rochaix P, Benlyazid A, et al. Pilot study of neoadjuvant treatment with erlotinib in nonmetastatic head and neck squamous cell carcinoma. Clin Cancer Res 2007;13:7086-92.

8. Gioanni J, Fischel JL, Lambert JC, et al. Two new human tumor cell lines derived from squamous cell carcinomas of the tongue: establishment, characterization and response to cytotoxic treatment. Eur J Cancer Clin Oncol 1988;24:1445-55.

9. Allal C, Favre G, Couderc B, et al. RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription. J Biol Chem 2000;275:31001-8.

10. Young H, Baum R, Cremerius U, et al. Measurement of clinical and subclinical tumour response using [18F]-fluorodeoxyglucose and positron emission tomography: review and 1999 EORTC recommendations. European Organization for Research and Treatment of Cancer (EORTC) PET Study Group. Eur J Cancer 1999;35:1773-82.

11. Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 1987;8:138-40.






12. Barriere J, Fischel J, Formento P, et al. Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against tumor cell lines characterized for EGFR expression and K-ras mutation. J Clin Oncol 2009;27:(May 20 suppl)e14583.

13. Lo NC, Maffi M, Fischel JL, et al. Impact of erythropoietin on the effects of irradiation under hypoxia. J Cancer Res Clin Oncol 2009;135:1615-23.

14. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer - molecular and clinical predictors of outcome. N Engl J Med 2005;353:133-44.

15. Niu G, Li Z, Xie J, Le QT, Chen X. PET of EGFR Antibody Distribution in Head and Neck Squamous Cell Carcinoma Models. J Nucl Med 2009;50:1116-23.

16. Cooper JB, Cohen EE. Mechanisms of resistance to EGFR inhibitors in head and neck cancer. Head Neck 2009;31:1086-94.

17. Chang SS, Califano J. Current status of biomarkers in head and neck cancer. J Surg Oncol 2008;97:640-3.

18. Sheikh Ali MA, Gunduz M, Nagatsuka H, et al. Expression and mutation analysis of epidermal growth factor receptor in head and neck squamous cell carcinoma. Cancer Sci 2008;99:1589-94.

19. Moyer JD, Barbacci EG, Iwata KK, et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res 1997;57:4838-48.

20. Prenen H, Deroose C, Vermaelen P, et al. Establishment of a mouse gastrointestinal stromal tumour model and evaluation of response to imatinib by small animal positron emission tomography. Anticancer Res 2006;26:1247-52.

21. Cullinane C, Dorow DS, Kansara M, et al. An in vivo tumor model exploiting metabolic response as a biomarker for targeted drug development. Cancer Res 2005;65:9633-6.

22. Su H, Bodenstein C, Dumont RA, et al. Monitoring tumor glucose utilization by positron emission tomography for the prediction of treatment response to epidermal growth factor receptor kinase inhibitors. Clin Cancer Res 2006;12:5659-67.

23. Li SJ, Guo W, Ren GX, et al. Expression of Glut-1 in primary and recurrent head and neck squamous cell carcinomas, and compared with 2-[18F]fluoro-2-deoxy-D-glucose accumulation in positron emission tomography. Br J Oral Maxillofac Surg 2008;46:180-6.

24. Tian M, Zhang H, Nakasone Y, Mogi K, Endo K. Expression of Glut-1 and Glut-3 in untreated oral squamous cell carcinoma compared with FDG accumulation in a PET study. Eur J Nucl Med Mol Imaging 2004;31:5-12.






25. Boucek JA, Francis RJ, Jones CG, et al. Assessment of tumour response with (18)F-fluorodeoxyglucose positron emission tomography using three-dimensional measures compared to SUVmax--a phantom study. Phys Med Biol 2008;53:4213-30.

26. Daisne JF, Duprez T, Weynand B, et al. Tumor volume in pharyngolaryngeal squamous cell carcinoma: comparison at CT, MR imaging, and FDG PET and validation with surgical specimen. Radiology 2004;233:93-100.

27. Krak NC, Boellaard R, Hoekstra OS, et al. Effects of ROI definition and reconstruction method on quantitative outcome and applicability in a response monitoring trial. Eur J Nucl Med Mol Imaging 2005;32:294-301.

28. Smith-Jones PM, Solit D, Afroze F, Rosen N, Larson SM. Early tumor response to Hsp90 therapy using HER2 PET: comparison with 18F-FDG PET. J Nucl Med 2006;47:793-6.

29. von Forstner C, Egberts JH, Ammerpohl O, et al. Gene expression patterns and tumor uptake of 18F-FDG, 18F-FLT, and 18F-FEC in PET/MRI of an orthotopic mouse xenotransplantation model of pancreatic cancer. J Nucl Med 2008;49:1362-70.

30. Atkinson DM, Clarke MJ, Mladek AC, et al. Using fluorodeoxythymidine to monitor anti-EGFR inhibitor therapy in squamous cell carcinoma xenografts. Head Neck 2008;30:790-9.






Acknowledgments

We would like to acknowledge the Institut Claudius Regaud animal facility and the medical

writing support of John Woodley and Daniela Oswald. We also would like to acknowledge

Julia Nalis and Cyril Jaudet for TEP imaging acquisitions.






Table-1

Pts N# Primary Tumor

Cumulated dose of erlotinib (150mg *n.days)

Cutaneous Toxicity

Follow up

(month)events/clinical status

1 oral cavity 1950 2 36 DF 2 oral cavity 3000 2 48 DF 3 oral cavity 3750 1 48 DF 4 oral cavity 2700 2 18 DRD 5 hypopharynx 2850 2 7 DRD 6 oral cavity 2700 0 48 DF 7 oropharynx 3300 2 35 DRD 8 oral cavity 3450 2 48 DF 9 larynx 4050 0 38 DF

10 oropharynx 3600 1 37 DuRD 11 oral cavity 3000 1 36 DF 12 oral cavity 1650 3 12 LR 13 oropharynx 3450 1 34 DF 14 larynx 750 3 18 DF 15 larynx 3000 1 14 DRD 16 larynx 3600 1 16 DuRD 17 oral cavity 3900 1 27 DF 18 oral cavity 4950 1 6 DRD

Mean 3091.7 Mean 29.2 SD 928.9 SD 14.3

Max 4950.0 Max 48.0 Min 750.0 Min 6.0






Supplementary data: Table-2

Parameters related to the FDG uptake under erlotinib treatment

Patient N#SUVbw max

beforeSUVbw max

after BV before

(mL) BV after (mL)SUVbw

Response (%) BV response (%)molecular response

6 9.9 8.6 12.7 7.7 -13.1 -39.4 no1 14.0 16.0 9.7 9.6 14.3 -1.0 no10 12.0 7.5 9.8 12.7 -37.5 29.6 no12 11.2 9.6 4.3 4.1 -14.3 -5.3 no15 16.0 17.0 21.0 26.0 6.3 23.8 no18 16.7 10.2 14.7 17.0 -38.9 15.6 no14 17.0 11.0 6.8 6.6 -35.3 -2.9 no11 8.0 2.4 1.2 0.8 -70.0 -33.3 yes13 4.5 3.7 15.4 11.2 -17.8 -27.3 yes8 7.4 5.2 6.3 1.1 -29.7 -82.5 yes7 26.7 20.9 7.3 1.6 -21.7 -78.1 yes5 18.5 11.4 20.1 3.0 -38.4 -85.1 yes2 6.2 7.7 3.7 2.2 24.2 -41.2 yes3 10.6 9.4 24.2 10.5 -11.3 -56.6 yes16 7.2 9.3 2.6 1.7 29.2 -34.6 yes4 41.3 30.0 7.8 5.4 -27.4 -30.8 yes9 30.3 24.3 11.2 7.7 -19.8 -31.3 yes17 15.4 12.1 11.8 7.3 -21.4 -38.1 yes

Mean 15.2 12.0 10.6 7.6 -17.9 -28.8SD 9.2 7.0 6.3 6.3 23.8 33.2Max 41.3 30.0 24.2 26.0 29.2 29.6Min 4.5 2.4 1.2 0.8 -70.0 -85.1






Legend to figures and table

Figure-1 : Effects of erlotinib on the CAL33 proliferation pathway .

Effects of erlotinib treatment (5µM) on P-EGFR (A, % of variation versus control

quantification), ERK-1/2 pathway (B) and p27kip1 (C,D fold increase of variation

versus control quantification) using Western blot (when indicated EGF was added at

20ng/ml during the 15 last minutes of the experiment). Data are representative of 3

independent experiments.

Figure-2 : Effects of erlotinib on CAL33 Glut-1 expression and 18FDG uptake.

Time course (24 to 48 hours) and dose (3 to 5µM) effects of erlotinib treatment on the

CAL33 Glut-1 transporter using Western blotting (A) and immunofluorescence (B).

Data are representative of 3 independent experiments.

CAL33 cells were treated with either glucose (control cells) or 18FDG (24.42 MBq/ml ,

treated cells) for 30 minutes. Cells where then washed, lysed and fractionated or not

(whole cell). The radioactivity content of collected samples (medium, PBS washes,

trypsin, cell fractionation or not) was measured. Results are percentage of

radioactivity measured in each fraction versus the 18FDG activity of treatment

(100%). Results are expressed as the mean of 2 independent experiments in

duplicate (C).

Figure-3: PET study of the effects of erlotinib on tumor 18FDG capture.

A, Nude mice bearing ( ) subcutaneous CAL33 or CAL166 tumors and visualized

by PET using an intra-venous injection of 9.85 ± 1.5MBq of 18FDG 4 days post

implantation (before) and 24 to 72h post (after) either saline (control group) or






erlotinib (erlotinib group at 100mg/kg) per-os treatment. B-(CAL33) and C-(CAL166) ,

histogram representation of the variation of the TBR (tumor to background ratio of

FDG uptake signal using regions of interest of the same size drawn on cross

sectional images) before and after (24h to 72h) saline or erlotinib treatment per-os.

Data are representative of 4 independent experiments for CAL33 and 2 for CAL166,

each performed in triplicate.

Figure-4: Immunohistochemical study of the effects of erlotinib on tumor tissue.

Thick sections of 4µm of formalin fixed and paraffin embedded tumors from mice

(A,B,C,D) and patients (E,F,G,H) before and after erlotinib treatment, were stained

with antibody against P-EGFR (A,B), Glut-1 (C,D,E,F), P-ERK(G,H). The IRS (range

0–12) is the product of the scores for staining intensity (0=negative; +=weak;

++=moderate; +++=strong scale) and percentage of cells stained (0–4 scale) (11).

Mice data are representative of 4 independent experiments performed in triplicate.

Figure-5: Effects of erlotinib on human tumor tissue.

A. Metabolic response with the mean tumor value ± 1Standard Deviation of SUVbw

(square), BV (circle) before (filled symbol) and after (open symbol) erlotinib treatment

* Two tailed Wilcoxon signed rank test, of pooled data

(SUV= maximum Standardized Uptake Value, BW = Body Weight, BV = Biological

Volume)

B. Facial sagittal section of a 18FDG-PET-CT fusion of a patient with an

oropharyngeal neoplasm before (I) and after 21 days of erlotinib (II).






Pts# 13 Molecular responder and metabolic responder [ΔSUV = -17.8% and δBV = -

27.3%]

C Metabolic response (mean ±SEM) assessed using the SUVmax (ΔSUVbw) and the

Biologic Volume (δBV) between two groups of patients, those with a significant

alteration of the phospho-ERK-1/2 protein (Molecular responder MR) and those

without significant alteration of the phospho-ERK-1/2 protein (non-Molecular

responder nMR).

D. ROC curve of the performance of δBV for the prediction of molecular response

(δBV>16.29%, sensitivity of 100%)

Table-1. Patients characteristics. The tumor localization, the cumulated dose of

erlotinib, the toxicity and the follow-up are detailed for 18 patients with non-metastatic

HNSCC who received neoadjuvant treatment with erlotinib between panendoscopy

and surgical treatment. (DF: disease free, DRD: death related to the disease, DuRD:

death unrelated to the disease, LR: local relapse)

Supplementary data legend to figure 6 and table 2 plus commentary to PET

data handling and reconstruction in the preclinical studies and interpretation to

supplementary figure 6.

Table-2. Parameters related to the FDG uptake under erlotinib treatment.

The tumoral 18FDG uptake reported by SUVBW and BV, before and after treatment

are listed in the columns 1 to 4. The percentage of variation of SUVBW and BV of 18

patients treated with erlotinib are listed in the columns 5 and 6 respectively. The






column 7 indicates if the patient were molecular responders . (SUV= maximum

Standardized Uptake Value, BW = Body Weight, BV = Biological Volume).

Figure-6: Ki67 immunohistochemical study of the effects of erlotinib on tumor

xenograft tissue.

Thick sections of 4µm of formalin fixed and paraffin embedded tumors from CAL33

cell line xenografted in nude mice, before and after 24 hours of erlotinib treatment,

were stained with hemalun-eosin (A,B respectively) or using antibody against Ki67,

before and after erlotinib treatment, with low magnification (C,D respectively) or high

magnification (E,F respectively). Mice data are representative of 4 independent

experiments performed in triplicate.

Figure-6 interpretation: Erlotinib treatment induced an early thinning of the

proliferative basal cell layer. And at the opposite the maturated cell layer (narrows)

appeared bigger after treatment. The overall Ki67 staining decreased in this kind of

tumors however the percentage of labeled cells in the proliferative basal cell layer

remained constant.

PET data handling and reconstruction in the preclinical studies for review only

The PET acquisitions were performed in two-dimensional mode (2D). 2D sinograms

were reconstructed in 256*256 matrix size, with a field of view (FOV) of 20 cm and

corrected for attenuation, random and scatter. Slice thickness was 3.27 mm every

3.27 mm. CT imaging was performed for Attenuation Correction (AC) and anatomical

correlation with a 200 mA tube current, 80 kV tube voltage, 512*512 matrix size, and

a reconstructed slice thickness of 1.25 mm for an interval between slices of 0.67 mm.






2D18F-FDG PET/CT data corrected from attenuation (AC) were transferred to an

Advantage Workstation 4.2® (GEHC Buc sur Yvette France).

Figure-7: Preclinical study with the CAL166 cell line.

A, EGFR and Glut-1 expression on CAL33 and CAL166 cell lines using western-blot.

Data are representative of 2 independent experiments.

B, Immunohistochemical study of the effects of erlotinib on nude mice CAL166

tumors.

Thick sections of 4µm of formalin fixed and paraffin embedded tumors from mice

from control group (A,C,E) and after erlotinib treatment (B,D,F), were stained with

antibody against EGFR (A,B), P-EGFR (C,D) and Glut-1 (E,F). The IRS (range 0–12)

is the product of the scores for staining intensity (0=negative; +=weak; ++=moderate;

+++=strong scale) and percentage of cells stained (0–4 scale) (11). Mice data are

representative of 2 independent experiments performed in duplicate.

Figure 7 interpretation:

A. The CAL166 cell line is described in the literature for overexpressing EGFR (12,

13). Nevertheless we have characterized its EGFR status of expression by western

blot versus the CAL33 cells. Our western blot results have been quantified and

showed that the CAL166 cell express around two times more EGFR compared to

CAL33. Moreover the western blot study of the CAL166 Glut-1 level of expression

has shown that this cell line has a similar Glut-1 level of expression as the CAL33

(representative of 2 independent experiments).

B. The immunohistochemistry analyses revealed no significant reduction of the total

form of the EGFR when we compared mice tumors control versus erlotinib treatment






(p=0.01). Whereas significant (p<0.01) change in the levels of Phospho-EGFR (P-

EGFR) was observed. But compared to the CAL33 cell the inhibition of the P-EGFR

observed was not complete. Indeed a slight staining persists in 10% of the cells,

corroborating the erlotinib difference of sensitivity.

Moreover, the immunohistochemical and the immunofluorescence (data not shown)

studies of respectively CAL166 tumors from xenografted nude mice and in vitro

cultured CAL166 cells showed a similar level of Glut-1 expression before and after

erlotinib treatment. These results are in total agreement with our previous data on

CAL33 cells.




EGF 24h40%

50%

Figure- 1

Cont 24h

Erlotinib 24hErlotinib+EGF

24h

-30%

-20%

-10%

0%

10%

20%

30%

% o

f P-E

GFR

var

iatio

n A0% FBS

B24h 48h 72h

-40%

-30%

B

P-ERK-1/2

ERK-1/2 -tot

0% FBS

C24h 48h 72h

10% FBS

Tubulin

p27kip

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2

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3

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Time in hours

p27

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incr

eas (a

0

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1

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24 h 48 h 72 h

Time in hours

p27

fold

incr

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Figure- 2Figure- 2

Glut-1

Erlotinib (µM) 0

24h

3 3.5 5

48h

3 3.5 5A

Glut-1

Erlotinib (µM) 0

24h

3 3.5 5

24h

3 3.5 5

48h

3 3.5 5

48h

3 3.5 5A

B Control Erlotinib (24h)B Control Erlotinib (24h)

C

DG

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ity 12,6

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h-ou

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h-ou

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A Control

group

Erlotinib

group

Figure- 3

Erlotinib

group

CAL33 CAL166

CT

Before

tumor

Fusion PET/CT

After

Control group Erlotinib groupB C

20

40

60

80

G u

ptak

e in

tum

or

TB

R)

–C

AL3

3

***

***

10

20

30

40

50

60

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* p<0.01

** p<0.0001

*** p<0.05

Student’s t tests




Control group Erlotinib (24h) groupMice

Figure- 4

A B

P-EGFR

Staining intensity ++Percentage of stained cells 95%

Staining intensity 0Percentage of stained cells 0

Glut1

Percentage of stained cells 95%IRS 8

Percentage of stained cells 0IRS 0

G ut

C D

Staining intensity +++Percentage of stained cells 100%


PatientsBefore Erlotinib After Erlotinib

IRS 12 IRS 12

Glut1

E FStaining intensity+ +++Percentage of stained cells 80%


P-ERK



G HStaining intensity 0Percentage of stained cells 0IRS 0

Staining intensity ++Percentage of stained cells 80%IRS 6




A

Figure- 5

15

20

25

15.017.520.022.525.027.5*P= 0.006

**P= 0.015U

V m

ax BV (m

Two tailed Wilcoxonsigned rank test

A

0

5

10

0.02.55.07.510.012.5

SUVbw BV

SU

mL)

BSUVbw max before (cm/mL) SUVbw max after (cm/mL) BV before BV after

C -25

0

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**

**p= 0.029

**

**

BV

r (%

)

Unpaired t test withwelch’s correction

C

Mr BVr NMr BVr Mr SUVr NMr SUVr-100

-75

-50

40

60

80

100Sensitivity%

D

0 10 20 30 40 50 60 70 80 900

20

40

100% - Specificity%

D




Table-1

Pts N# Primary Tumor

Cumulated dose of

erlotinib (150mg *n.days)

Cutaneous Toxicity

Follow up (month) events/clinical status

1 oral cavity 1950 2 36 DF 2 oral cavity 3000 2 48 DF 3 oral cavity 3750 1 48 DF 4 oral cavity 2700 2 18 DRD 5 hypopharynx 2850 2 7 DRD 6 oral cavity 2700 0 48 DF 7 oropharynx 3300 2 35 DRD 8 oral cavity 3450 2 48 DF 9 larynx 4050 0 38 DF 10 oropharynx 3600 1 37 DuRD 11 oral cavity 3000 1 36 DF 12 oral cavity 1650 3 12 LR 13 oropharynx 3450 1 34 DF 14 larynx 750 3 18 DF 15 larynx 3000 1 14 DRD 16 larynx 3600 1 16 DuRD 17 oral cavity 3900 1 27 DF 18 oral cavity 4950 1 6 DRD

Mean 3091.7 Mean 29.2 SD 928.9 SD 14.3

Max 4950.0 Max 48.0 Min 750.0 Min 6.0




Figure- 7

CAL166

EGFR

CAL33A

Glut-1

Tubulin

CAL33 CAL166EGFR 0,433 0,701Glut-1 10,85 12,64

Expression (Arbitrary Unit)

B




Published OnlineFirst July 26, 2010.Clin Cancer Res Sébastien Vergez, Jean Pierre Delord, Fabienne Thomas, et al. in head and neck cancertool for the detection of early molecular responses to erlotinib Preclinical and clinical evidence that 18FDG-PET/CT is a reliable

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