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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
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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.
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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.
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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
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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).
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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
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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.
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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.
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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
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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
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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
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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.
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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
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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
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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
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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
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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%.
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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
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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
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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
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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.
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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.
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edited. Copyright © 2010 American Association for Cancer Research
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.
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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
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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
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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
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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).
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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
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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.
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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
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(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.
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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
bbD
1 5
2
2.5
3
3.5
4
4.5
se o
f con
trol
ver
sus
erlo
tinib
arbi
trar
y un
it)
1 5
2
2.5
3
3.5
4
4.5
se o
f con
trol
ver
sus
erlo
tinib
arbi
trar
y un
it)
D10% FBS
0
0.5
1
1.5
24 h 48 h 72 h
Time in hours
p27
fold
incr
eas (a
0
0.5
1
1.5
24 h 48 h 72 h
Time in hours
p27
fold
incr
eas (a
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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
activ
ity 12,6
10
12
8383,3C
DG
activ
ity 12,6
10
12
8383,3
DG
activ
ity 12,6
10
12
8383,3
Perc
enta
geof
FD
4,1
0 0
5,04
7,56
0
2
4
6
8
Perc
enta
geof
FD
4,1
0 0
5,04
7,56
0
2
4
6
8
Perc
enta
geof
FD
4,1
0 0
5,04
7,56
0
2
4
6
8
0
Medium
Was
h-ou
t1
Was
h-ou
t2
Was
h-ou
t3
Who
le ce
llsCyto
solic
Membr
ane
C ell fractionation
0
Medium
Was
h-ou
t1
Was
h-ou
t2
Was
h-ou
t3
Who
le ce
llsCyto
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Membr
ane
C ell fractionation
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Was
h-ou
t1
Was
h-ou
t2
Was
h-ou
t3
Who
le ce
llsCyto
solic
Membr
ane
C ell fractionation
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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
DG
upt
ake
in tu
mor
(
TB
R)
-C
AL1
66
* *
-60
-40
-20
0
% o
f var
iatio
n of
FD
Gvs
. bac
kgro
und
(T
D4 D5 D7 D4 D5 D7
-20
-10
0
10
% o
f var
iatio
n of
Fvs
. ba
ckgr
ound
D4 D5 D4 D5
* p<0.01
** p<0.0001
*** p<0.05
Student’s t tests
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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%
Staining intensity +++Percentage of stained cells 100%
PatientsBefore Erlotinib After Erlotinib
IRS 12 IRS 12
Glut1
E FStaining intensity+ +++Percentage of stained cells 80%
Staining intensity +++Percentage of stained cells 80%
P-ERK
Percentage of stained cells 80%IRS 8
Percentage of stained cells 80%IRS 8
G HStaining intensity 0Percentage of stained cells 0IRS 0
Staining intensity ++Percentage of stained cells 80%IRS 6
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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
25
50*p= 0.0109
**
**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
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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
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Figure- 7
CAL166
EGFR
CAL33A
Glut-1
Tubulin
CAL33 CAL166EGFR 0,433 0,701Glut-1 10,85 12,64
Expression (Arbitrary Unit)
B
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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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