The association between EGFR variant III, HPV, p16, c-MET, EGFR gene copy number and response to EGFR inhibitors in patients with recurrent or metastatic squamous cell carcinoma of the head and neck.

Abstract

We examine the potential prognostic and predictive roles of EGFR variant III mutation, EGFR gene copy number (GCN), human papillomavirus (HPV) infection, c-MET and p16INK4A protein expression in recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN).
We analyzed the archival tumor specimens of 53 patients who were treated in 4 phase II trials for R/M SCCHN. Two trials involved the EGFR inhibitor erlotinib, and 2 trials involved non-EGFR targeted agents. EGFRvIII mutation was determined by quantitative RT-PCR, HPV DNA by Linear Array Genotyping, p16 and c-MET protein expression by immunohistochemistry, and EGFR GCN by FISH.
EGFRvIII mutation, detected in 22 patients (42%), was associated with better disease control, but no difference was seen between erlotinib-treated versus non-erlotinib treated patients. EGFRvIII was not associated with TTP or OS. The presence of HPV DNA (38%), p16 immunostaining (32%), c-MET high expression (58%) and EGFR amplification (27%), were not associated with response, TTP or OS.
EGFRvIII mutation, present in about 40% of SCCHN, appears to be an unexpected prognostic biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Larger prospective studies are required to validate its significance.

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RESEARCHOpen Access
The association between EGFR variant III, HPV,
p16, c-MET, EGFR gene copy number and
response to EGFR inhibitors in patients with
recurrent or metastatic squamous cell carcinoma
of the head and neck
Nicole G Chau1, Bayardo Perez-Ordonez2, Katherine Zhang3, Nhu-An Pham3, James Ho3, Tong Zhang3,
Olga Ludkovski3, Lisa Wang4, Eric X Chen1, Ming-Sound Tsao3, Suzanne Kamel-Reid3, Lillian L Siu1*
Abstract
Background: We examine the potential prognostic and predictive roles of EGFR variant III mutation, EGFR gene
copy number (GCN), human papillomavirus (HPV) infection, c-MET and p16INK4Aprotein expression in recurrent or
metastatic squamous cell carcinoma of the head and neck (R/M SCCHN).
Methods: We analyzed the archival tumor specimens of 53 patients who were treated in 4 phase II trials for R/M
SCCHN. Two trials involved the EGFR inhibitor erlotinib, and 2 trials involved non-EGFR targeted agents. EGFRvIII
mutation was determined by quantitative RT-PCR, HPV DNA by Linear Array Genotyping, p16 and c-MET protein
expression by immunohistochemistry, and EGFR GCN by FISH.
Results: EGFRvIII mutation, detected in 22 patients (42%), was associated with better disease control, but no
difference was seen between erlotinib-treated versus non-erlotinib treated patients. EGFRvIII was not associated
with TTP or OS. The presence of HPV DNA (38%), p16 immunostaining (32%), c-MET high expression (58%) and
EGFR amplification (27%), were not associated with response, TTP or OS.
Conclusion: EGFRvIII mutation, present in about 40% of SCCHN, appears to be an unexpected prognostic
biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Larger
prospective studies are required to validate its significance.
Background
The epidermal growth factor receptor (EGFR) is over-
expressed in up to 90% of squamous cell carcinoma of
the head and neck (SCCHN) and has been postulated to
be a key molecular target in this malignancy [1]. EGFR
signal transduction leads to cell proliferation, invasion,
angiogenesis and metastasis [2]. EGFR overexpression
and aberrant EGFR gene copy number (EGFR GCN)
have been associated with poorer prognosis and disease-
specific survival in SCCHN [1,3,4]. Therapies targeted
against EGFR have demonstrated modest activity either
alone or in combination with chemotherapy in both
locally advanced [5] and recurrent and/or metastatic
SCCHN [6-10]. No validated biomarkers exist to predict
the response to EGFR inhibitors in SCCHN.
The most common EGFR truncation mutation, EGFR
variant III (EGFRvIII), harbors an in-frame deletion of
exons 2 to 7 (801 bp), resulting in a truncated extracel-
lular EGF-binding domain that is constitutively activated
and ineffectively ubiquinated [11,12]. EGFRvIII is found
in many human cancers and is present in ~40% of glio-
blastomas and 5% of lung squamous cell carcinomas,
where it confers tumorigenicity and dose-dependent
resistance to gefitinib in pre-clinical models [13,14]. The
* Correspondence: lillian.siu@uhn.on.ca
1Division of Medical Oncology and Hematology, Princess Margaret Hospital,
University Health Network, Toronto, Ontario, Canada
Full list of author information is available at the end of the article
Chau et al. Head & Neck Oncology 2011, 3:11
http://www.headandneckoncology.org/content/3/1/11
© 2011 Chau et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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prevalence of EGFRvIII in SCCHN was first reported as
43% in one study of 33 SCCHN tumors [15]. EGFRvIII-
transfected SCCHN cells had decreased apoptosis in
response to cisplatin and decreased growth inhibition
following treatment with the EGFR monoclonal antibody
cetuximab compared with controls [15]. EGFRvIII is an
interesting therapeutic target because unlike wild-type
EGFR, EGFRvIII is not found in normal tissue. EGFRvIII
is proposed to account for limitations in response to
current EGFR inhibitors, however in patients with
SCCHN tumors harboring EGFRvIII response to EGFR
tyrosine kinase inhibition (TKI) is unknown.
HPV infection is a risk factor for the development of
SCCHN. HPV DNA is found in 20-30% of SCCHN and
up to 40-66% of SCCHN of the oropharynx [16,17].
HPV positive oropharyngeal tumors are clinically and
molecularly distinct from HPV negative tumors [18,19]
and associated with a more favorable prognosis [20].
HPV positive status prospectively predicts survival and
response to induction chemotherapy and chemoradia-
tion in stage III or IV oropharynx cancers [21,22] and
better response to radiotherapy alone [23]. The combi-
nation of low HPV titers and high EGFR expression was
associated with worse overall survival in oropharynx
cancer [22]. Inactivation of pRb by HPV E7 protein
results in overexpression of p16 protein, thus p16
immunostaining has served as a surrogate marker for
HPV-associated SCCHN. Patients with tumors lacking
both p16 expression and HPV (p16-/HPV-) had the
worst disease-specific survival compared to tumors with
p16+/HPV+, p16-/HPV+ or p16+/HPV- types [24].
Despite the importance of HPV in the pathogenesis and
prognosis of SCCHN in response to chemotherapy and
radiation, the role of HPV DNA and response to EGFR
inhibitors in SCCHN is unclear.
c-MET, a proto-oncogene tyrosine kinase receptor, is
overexpressed in SCCHN, and its ligand, hepatocyte
growth factor (HGF), stimulates cell proliferation, moti-
lity and invasion [25]. c-MET overexpression has been
associated with disease progression in oral squamous
cell carcinoma (OSCC) [26]. Elevated serum HGF is
associated with resistance to chemoradiation and
reduced survival [27]. c-MET amplification and muta-
tions of MET confer an invasive phenotype associated
with metastases in SCCHN [28]. Ligand-independent
constitutive activation of c-MET via its heterodimeriza-
tion with EGFR has been identified as a contributing
mechanism of acquired resistance to cetuximab in
SCCHN [29]. The role of c-MET in response to EGFR
TKI in the clinical setting in SCCHN is unknown.
In this study, we examine the prevalence of EGFRvIII,
HPV, p16, c-MET and EGFR GCN in patients with R/M
SCCHN and explore the potential prognostic and pre-
dictive roles of these biomarkers in patients treated with
or without EGFR TKI. We hypothesized that EGFRvIII
and c-MET would be associated with poorer prognosis
or response to EGFR TKI, while HPV and p16 expres-
sion would predict improved clinical outcomes and
response to treatment.
Methods
Patients
We obtained approval from the University Health Net-
work Research Ethics Board to evaluate the archival for-
malin-fixed paraffin embedded (FFPE) tumor specimens
of patients with R/M SCCHN who were treated in four
phase II trials for R/M SCCHN at Princess Margaret
Hospital conducted from 2000-2005. Two of the four
trials involved the EGFR TKI erlotinib (phase II trial of
erlotinib [8], phase II trial of erlotinib and cisplatin [7])
and the remaining two trials used other non-EGFR tar-
geted agents (phase II trial of the kinesin spindle protein
inhibitor ispinesib [30], phase II trial of the multi-kinase
antiangiogenic inhibitor sorafenib [31]). The medical
records and case report forms were reviewed to obtain
patient demographics, primary tumor site, treatment
details and clinical outcome (response rate, time to pro-
gression and overall survival).
Specimen Characteristics
Archival FFPE tumor specimens were available in 35 of
48 patients (73%) treated with erlotinib and 18 of 37
(49%) patients treated with non-EGFR targeted agents.
H&E stained sections were examined by a histopatholo-
gist (B.P-O.) to confirm the presence of >80% tumor in
the specimens evaluated.
Assay Methods/Molecular Assays
EGFRvIII Mutation Detection
RNA Isolation RNA was isolated in tumor area on the
FFPE slides guided by H&E-stained serial sections. The
tissues were deparaffinized by xylene and ethanol. Total
RNA from paraffin-embedded tissues was extracted
using RecoveryAll™ Total Nucleic Acid isolation Kit
(Ambion Diagnostics, Streetsville, Ontario, Canada).
Real-time RT-PCR Reverse transcription was done
using TaqMan Reverse transcription reagent kit (Roche,
Branchburg, New Jersey) according to the manufac-
turer’s protocol. Reverse transcription reaction was done
in a total volume of 25 mL including RNA template,
1.25 mL random hexamer, reverse transcription buffer,
5.0 mL dNTP, 5.5 mL MgCl, RNase inhibitor, and
M-MLV reverse transcriptase.
Real-time PCR was performed in duplicate in 25 mL
reaction volumes using Platinum SYBR Green qPCR
SuperMix-UDG (Invitrogen, Carlsbad, California) and a
7900HT instrument (Applied Biosystems, Foster City,
California). The amplification conditions were: 50°C for
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2 min., 95°C for 2 min., 40 cycles of 95°C for 15 sec. and
60°C for 1 min.
Data Analysis of real-time PCR A mixture of at least
eight normal FFPE tissue samples was used as a wild-
type, normal control. The EGFRvlll cell line (u373fla-
gEGFRvlll) was used as a positive control.
The relative expression of EGFR exon 4 to EGFR exon
9 was determined using the delta delta Ct (ΔΔCt)
method. All samples were run in duplicate, and the
mean Ct number was used for data analysis. The differ-
ence in Ct values (Δthreshold cycle, ΔCt = Exon 9 Ct -
Exon 4 Ct) was calculated for each RNA sample. The
ΔCt from the normal tissue mixture was then subtracted
from the ΔCt of the test sample to generate a ΔΔCt.
A negative result occurs when the fold change (exon 9:
exon 4 calculated as 2- ΔΔCt) is less than a value of 5.
This value was arbitrarily chosen to ensure that no false
positives were called. A positive result occurs when the
fold change is the same as or greater than that of the
positive control (7). When the fold change of tested
samples falls between that of the normal control and
the positive control (i.e. between 5 and 7) the results are
considered inconclusive.
HPV DNA Detection
The Roche Linear Array HPV Genotyping kit (Roche
Molecular Diagnostics, Pleasanton, California) was used
to detect 37 low- and high-risk HPV types from FFPE
tissues. In brief, FFPE sections were deparaffinized and
DNA was extracted using a column based method
(QIAamp, Qiagen, Valencia, California). HPV detection
was performed using PCR amplification followed by
hybridization of the amplified products to oligonucleo-
tide probes and subsequent colorimetric determination.
All experiments included an HPV positive control and
an HPV negative control.
HPV DNA by in situ hybridization (ISH) using the
INFORM HPV III Family 16 probe (Ventana Medical
Systems Inc., Tucson, Arizona) which detects genotypes
16, 18, 31, 33, 39, 35, 45, 51, 52, 56, 58 and 66, was per-
formed according to the manufacturer’s guidelines using
the Ventana Benchmark automated slide staining sys-
tem. All experiments included an HPV positive control
and an HPV negative control. Slides were scored as
positive if a punctate or diffuse pattern of signal were
observed in the tumor nuclei.
P16 and c-MET Detection
Immunohistochemistry (IHC) for p16 and c-MET using
the Ventana Benchmark XT auto-immunostainer
(Tucson, Arizona) was performed on FFPE sections cut
at 4mm thick. Standardized staining protocols were pro-
vided by Ventana for the CINtec p16 Histology kit
(MTM Laboratories Inc, Westborough Massachusetts)
and c-MET antibody (SP44, rabbit monoclonal, Ventana
Medical Systems Inc., Tucson Arizona). Controls were
included in each assay, comprising of positive tissue
controls and negative controls. All p16 and c-MET IHC
slides were reviewed independently by two observers
(B.P.O. and N.G.C.) without knowledge of EGFRvIII,
HPV status or clinical outcome. p16 staining in SCCHN
is generally observed to be dichotomous and scored as
absent (weak or no staining) or present (strong and dif-
fuse staining) [32]. c-MET IHC slides were assigned a
semi-quantitative score based on the product of an
intensity score (0 = no staining or equal to background,
1 = weak, or 2 = strong) and percent of area stained
(0 = 0%, 1 = 1-30%, 2 = 31-60%, 3 = >60%). Sections
with an inter-observer variation were reassessed by a
double-headed light microscope to achieve consensus.
EGFR Gene Copy Number
Archival tumor specimens were analyzed for EGFR
GCN using fluorescent in situ hybridization (FISH) as
previously described [33,34]. One hundred non-overlap-
ping interphase nuclei were scored for EGFR and CEP7
copy number and classified into six categories (Univer-
sity of Colorado Scoring system) by a reviewer blinded
to clinical outcome (O.L.) [35].
Statistical Methods
Descriptive statistics were used to summarize the study
cohort and to estimate the parameters of interest.
Ninety-five percent confidence intervals were obtained
for estimates of the presence of EGFRvIII, HPV, p16,
c-MET and EGFR GCN. Exploratory analyses were per-
formed to characterize the relationships between EGFR-
vIII, HPV, p16, c-MET and EGFR GCN with baseline
patient characteristics and outcomes. Only patients with
conclusive EGFRvIII results were included in the corre-
lation analyses. The Kaplan-Meier method was used to
estimate the overall survival and time to progression. All
biomarkers were examined in univariate analysis of
overall survival and time to progression using Cox pro-
portional hazards model. Only those which were signifi-
cant at 0.10 (two-sided) level in the univariate analysis
were entered in the multivariate analysis and markers
that remained significant at 0.05 (two-sided) level in the
multivariate analysis were considered significant prog-
nostic factors. Statistical analyses were performed using
the SAS 9.1 software package (SAS Institute, Cary,
North Carolina).
Results
Patients
The clinical characteristics of the 53 patients in our
study are described in Table 1. For the entire cohort,
the overall response rate (CR+PR) to study treatment
was 4/53 (7.5%), median time to progression (TTP) was
1.8 months (95% CI 1.6-2.7) and median overall survival
(OS) was 5.9 months (95% CI 4.5-8.7). Patients in the
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erlotinib group had a higher median OS of 7.9 months
(95% CI 4.7-9.8) compared to patients in the non-
erlotinib group with median OS of 4.2 months (95% CI
2.9-7.0) (p = 0.011). The erlotinib group had a higher
TTP than the non-erlotinib group 2.7 months (95% CI
1.6-3.5) vs 1.5 months (95% CI 1.3-1.8) (p = 0.0009).
Expression of EGFRvIII mutation by real-time PCR
As the previously reported immunohistochemistry-suitable
antibody [15] against EGFRvIII is no longer available,
EGFRvIII expression is analysed using the RT-PCR
method. The presence of EGFRvIII mutation was detected
in 22 patients (42%) (Table 2), negative in 19 patients and
inconclusive in 12 patients (Table 3). The median EGFR-
vIII fold change was 6.8 (0.56 to 576.36) for all patients,
15.0 (4.1 to 576.36) for patients in the EGFRvIII positive
group, 1.8 (0.6 to 4.3) for patients in the EGFRvIII negative
group, and 6.5 (6.2 to 6.8) for patients in the inconclusive
group.
Patients with tumors harboring the EGFRvIII mutation
had similar clinical characteristics to patients without
the EGFRvIII mutation (Table 4).
EGFRvIII is associated with disease control In univari-
ate analysis, the presence of EGFRvIII was associated
with better disease control (Table 5). Median EGFRvIII
fold changes were higher for patients with disease con-
trol than patients with progressive disease (11.11 vs.
3.16, p = 0.04). No significant difference was observed
between erlotinib-treated (p = 0.21) versus non-erlotinib
(p = 0.10) treated patients due to the small sample size
(Table 5). The presence of EGFRvIII mutation was not
associated with TTP (HR 0.94 (95% CI 0.33-2.71), p =
0.91) or OS (HR 0.91 (95% CI 0.32-2.60), p = 0.85)
(Figure 1).
HPV DNA
HPV DNA testing by PCR was positive in 20 patients
(38%), negative in 31 patients and inconclusive in 2
patients (Tables 2 and 3). The most prevalent HPV sub-
type found in our analysis was the high-risk HPV-16
(18/20 cases). The majority of HPV-16 positive tumors
were from the oropharynx (12/18). HPV positive tumor
status was not significantly associated with disease con-
trol (Table 6), TTP (HR 1.19 (95% CI 0.46-3.11), p =
0.722) or OS (HR 0.88 (95% CI 0.34-2.29), p = 0.788).
P16
P16 immunoreactivity was detected in 17 patients (32%),
absent in 33 patients and inconclusive in 3 patients
(Tables 2 and 3). The inter-observer variability rate was
6% and discrepant cases were resolved by consensus
review. P16 expression was not associated with disease
control (Table 6), TTP (HR 0.50 (95% CI 0.19-1.32), p =
0.16) or OS (HR 0.61 (95%CI 0.24-1.55), p = 0.30).
The discordance between p16 IHC and HPV DNA by
PCR was 25% (Table 7). To investigate this further, we
performed HPV DNA by ISH. The discordance between
p16 IHC and HPV DNA by ISH was lower at 16%
(Table 7) and all 7 discordant cases were p16-positive/
HPV-ISH-negative. Of these 7 discordant cases, 2 cases
were HPV-16 positive by PCR, 4 cases were HPV nega-
tive by PCR and 1 case was inconclusive by HPV PCR.
C-MET
Forty-nine patients had sufficient tumor samples for
evaluation of c-MET. Eighteen patients (63%) had low
c-MET scores of 0, 1 or 2 and 31 patients had high
c-MET (>2) (Tables 2 and 3). Less than 10% inter-
observer variability was observed and discrepant cases
were resolved by consensus review. High c-MET was
not associated with disease control (Table 6), TTP (HR
1.47 (95%CI 0.56-3.85), p = 0.43) or OS (HR 1.72 (95%
CI 0.65-4.56), p = 0.27).
Table 1 Clinical characteristics of the entire study cohort
(n = 53)
Clinical CharacteristicNumber
Median Age (Range)56 (15-78)
GenderFemale:Male12:41
ECOG Performance Status0:1:215:34:4
Locoregional RecurrenceYes:No45:8
Distant MetastasesYes:No19:34
Primary Tumor SiteOropharynx
Larynx
Oral cavity
Hypopharynx
Neck mass unknown
Paranasal sinus
20
14
10
2
4
3
Histologic GradeWell differentiated
Moderately differentiated
Poorly differentiated
Infiltrating basaloid
5
33
14
1
Prior TherapyChemotherapy
Radiation Therapy
Surgery
21
51
41
RaceAsian
Black
Caucasian
Other
8
42
9
1
SmokerCurrent
Former
Never
Unknown
33
3
15
2
ErlotinibYes: No35:18
Best ResponsePartial response
Stable disease
Progressive disease
Inevaluable
4
20
24
5
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EGFR Gene Copy Number
Forty-five patients had sufficient tumor samples for eva-
luation of EGFR GCN by FISH. High EGFR GCN
(amplification and high polysomy) was detected in 13
patients and low EGFR GCN (disomy, low polysomy)
was detected in 33 patients (Tables 2 and 3). High
EGFR GCN was not predictive for TTP (HR 0.99, p =
0.822) or OS (HR 1.10, p = 0.644). High EGFR GCN
was not associated with the presence of EGFRvIII (p =
0.14 Fisher’s exact test).
Discussion
To the best of our knowledge, this is the first study to
evaluate the role of EGFRvIII in a cohort of patients
with R/M SCCHN treated with or without EGFR TKI.
This study confirms that EGFRvIII mutation is common
Table 2 EGFRvIII mutation positive detected by RT-PCR (n = 22)
Case TreatmentPrimary SiteSpecimen SiteEGFRvIII by
RT-PCR
EGFRvIII Fold
Changes
HPV DNA by
Linear Array
P16
IHC
MET score
by IHC
EGFR FISH
1 Erlotinibhypopharynx Untreated
primary
+11.33--High Low
polysomy
2 Erlotiniboral cavityUntreated
primary
+ 7.01-- HighDisomy
3 Erlotinib larynxUntreated
primary
+ 61.77-- LowLow
polysomy
4Erlotinib larynxLocal
recurrence
+26.64--LowHigh
polysomy
5Erlotinibneck mass
unknown primary
Untreated
lymph node
+60.0433 ++HighLow trisomy
6Erlotiniboral cavityUntreated
lymph node
+218.49--HighAmplification
7ErlotiniboropharynxUnknown
primary
+15.6616 ++HighLow trisomy
8Erlotiniboral cavityLocal
recurrence
+8.94--HighLow
polysomy
9Erlotinib +
Cisplatin
neck mass
unknown primary
Untreated
lymph node
+127.8416+NENELow trisomy
10Erlotinib +
Cisplatin
larynxNode
recurrence
+576.3616++HighDisomy
11Erlotinib +
Cisplatin
larynxUntreated
primary
+8.26--LowFailed
12Erlotinib +
Cisplatin
larynxUntreated
primary
+17.38--HighLow
polysomy
13Erlotinib +
Cisplatin
oropharynxUntreated
primary
+14.2816++HighDisomy
14Erlotinib +
Cisplatin
oral cavityUntreated
primary
+69.68-NENELow
polysomy
15Erlotinib +
Cisplatin
oral cavityUntreated
primary
+11.1153+, 58+, 6+, 52+-HighLow
polysomy
16SorafeniboropharynxUntreated
primary
+7.7116 ++LowLow
polysomy
17SorafeniboropharynxUntreated
primary
+7.9316 ++LowLow trisomy
18Ispinesibneck mass
unknown primary
Untreated
lymph node
+4.1216+, 53+, 51 ++HighHigh
polysomy
19IspinesiboropharynxUntreated
primary
+15.6116+, 53+, 33+, 51
+, 58 +
-HighHigh
polysomy
20IspinesiblarynxUntreated
primary
+
218.26--HighDisomy
21IspinesiboropharynxLocal
recurrence
+29.2516+, 53+, 51++LowDisomy
22IspinesibhypopharynxLocal
recurrence
+9.3116+, 53+, 58+, 52
+
-HighLow
polysomy
Abbreviations: +, positive; -, negative; NE, not evaluable.
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Table 3 EGFRvIII mutation negative detected by RT-PCR (n = 19) and inconclusive cases (n = 12)
CaseTreatmentPrimary SiteSpecimen
Site
EGFRvIII by
RT-PCR
EGFRvIII Fold
Changes
HPV DNA by
Linear Array
P16
IHC
MET score
by IHC
EGFR FISH
23Erlotinib larynxLocal
recurrence
- 1.76-- HighLow polysomy
24Erlotinib larynxUntreated
primary
- 0.95-- NENE
25 Erlotiniboropharynx Untreated
primary
Incon.NE 16 +- Low Failed
26Erlotinib larynxUntreated Incon. NE--NE NE
27Erlotinib oropharynx Local
recurrence
-2.5616 +-HighFailed
28Erlotinibneck mass
unknown primary
Local
recurrence
- 1.81--HighLow polysomy
29Erlotinibparanasal sinusUntreated
primary
-0.56--HighHigh polysomy
30ErlotiniboropharynxUntreated
primary
-3.1616 ++HighLow trisomy
31ErlotiniblarynxUntreated
primary
-3.17--HighLow polysomy
32ErlotiniboropharynxLocal
recurrence
NENE-+LowLow polysomy
33ErlotiniblarynxUntreated
primary
-3.16--HighHigh trisomy
34Erlotinib +
Cisplatin
oropharynxUntreated
primary
Incon.NEIncon.+LowNE
35Erlotinib +
Cisplatin
larynxUntreated
primary
-4.29--LowLow polysomy
36Erlotinib +
Cisplatin
oral cavityUntreated
primary
-0.9--LowHigh polysomy
37Erlotinib +
Cisplatin
paransal sinusLocal
recurrence
Incon.NE16+, 53+Incon.LowLow polysomy
38Erlotinib +
Cisplatin
larynxLocal
recurrence
Incon.NE16+, 53+, 33+, 51
+
+HighDisomy
39Erlotinib +
Cisplatin
paranasal sinusLocal
recurrence
-2.596+-HighNE
40Erlotinib +
Cisplatin
oral cavityUntreated
primary
-2.01--HighDisomy
41Erlotinib +
Cisplatin
oropharynxUntreated
primary
Incon.NE-+HighLow trisomy
42Erlotinib +
Cisplatin
oral cavityUntreated
primary
Incon.NE--HighHigh polysomy
43Sorafeniboral cavityUntreated
primary
Incon.6.8--LowLow polysomy
44SorafeniboropharynxUntreated
primary
-0.99-+HighAmplification/
High trisomy
45Sorafeniboral cavityLocal
recurrence
Incon.
NEIncon.-LowLow polysomy
46SorafeniboropharynxUntreated
primary
Incon.6.2216 ++LowLow trisomy
47Sorafeniblarynx Local
recurrence
-2.28-+HighHigh polysomy
48SorafeniboropharynxUntreated
primary
-0.64--HighLow polysomy
49SorafeniboropharynxLocal
recurrence
-1.4--HighHigh polysomy
50SorafeniboropharynxLocal
recurrence
-1.3--HighHigh polysomy
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in R/M SCCHN, and may play a role in prognosis. We
identified EGFRvIII mutation in 42% of 53 R/M SCCHN
tumors. This is in keeping with the first description of
EGFRvIII expression by IHC and RT-PCR in 42% of 33
SCCHN tumors sampled [15]. In vitro studies suggest
that EGFRvIII mutated SCCHN cell lines are resistant
to the anti-EGFR monoclonal antibody cetuximab [15].
In this study, EGFRvIII was not associated with an infer-
ior response to erlotinib therapy. Importantly, we
observed a significant association between the presence
of EGFRvIII (mean fold change and copy number by
RT-PCR) with greater disease control, regardless of
treatment with erlotinib, suggesting that perhaps EGFR-
vIII may have a prognostic role.
The prognostic or predictive significance of the EGFR-
vIII mutation in response to systemic therapy in patients
with SCCHN has not been previously described. The
potential prognostic role of EGFRvIII appears to be
independent of any clinicopathologic characteristics.
This is consistent with another study where EGFRvIII
detected by IHC in 234 of 681 locally advanced SCCHN
tumors (34%) was associated with increased tumor size
but not stage or other clinical factors [36]. In our study,
EGFRvIII was not associated with overall survival or
TTP. To our knowledge, EGFRvIII has not been linked
to survival in SCCHN. EGFRvIII has been described
more extensively in glioblastoma where it results in
enhanced proliferation and reduced apoptosis effects
that are mediated through increased levels of activated
Ras [37] and activation of the PI3K pathway [38]. How-
ever, the role of EGFRvIII as a prognostic or predictive
marker of response to EGFR inhibitors in glioblastoma
remains controversial. EGFRvIII and PTEN co-expres-
sion was associated with response to EGFR TKI in 26
patients out of a cohort of 49 patients with recurrent
glioma and a validation set of 33 patients [39]. EGFRvIII
has been reported as a prognostic marker for poorer
survival in some studies [40,41], but not in others
[42,43]. Conflicting results have been attributed to small
sample sizes with incomplete clinical data and varying
methods to detect EGFRvIII.
The presence of activating mutations conferring a bet-
ter prognosis has been reported with EGFR mutations
in non-small cell lung cancer (NSCLC) [44] and with
PIK3CA mutations in breast cancer [45]. Somatic acti-
vating mutations (exon 19 deletion and 21 point muta-
tion) in the EGFR tyrosine kinase domain confer
sensitivity to EGFR inhibitors in NSCLC. Patients with
Table 3 EGFRvIII mutation negative detected by RT-PCR (n = 19) and inconclusive cases (n = 12) (Continued)
51Sorafenib oropharynxLocal
recurrence
-1.76-+ LowMonosomy/
Disomy
52Ispinesib oropharynxUntreated
lymph node
Incon.NE--HighLow polysomy
53IspinesiboropharynxNode
recurrence
-4.0516 +-LowAmplification
Abbreviations: +, positive; -, negative; Incon., inconclusive; NE, not evaluable.
Table 4 Presence of the EGFRvIII mutation is not significantly associated with any clinical characteristics
Clinical CharacteristicEGFRvIII absent (n = 19)EGFRvIII present (n = 22)p-value
Male - no., (%)16 (84%)18 (82%)0.839 (Fisher’s)
Age - mean, (+/-SD)53.5 (+/- 11.6)55.1 (+/- 14.1)0.685 (t-test)
Oropharynx - no., (%)8 (42%)6 (27%)0.318 (Chi-square)
Larynx - no., (%)6 (32%)6 (27%)0.763 (Chi-square)
Oral Cavity - no., (%)2 (11%)5 (23%)0.271 (Fisher’s)
Distant metastasis - no., (%)4 (21%)12 (55%)0.053 (Fisher’s)
Locoregional recurrence - no., (%)18 (95%)16 (73%)0.099 (Fisher’s)
Well-moderately differentiated - no., (%)15 (79%)14 (64%)0.325 (Fisher’s)
Poorly differentiated - no., (%)4 (21%)8 (36%)
Prior chemotherapy - no., (%)9 (47%)8 (36%)0.476 (Chi-square)
Prior radiotherapy - no., (%)17 (89%)22 (100%)0.209 (Fisher’s)
Prior surgery - no., (%)17 (89%)17 (77%)0.419 (Fisher’s)
Caucasian - no., (%)15 (79%)18 (82%)0.562 (Fisher’s)
Erlotinib treatment - no., (%)12 (63%)15 (68%)0.735 (Chi-square)
No erlotinib treatment - no., (%)7 (38%)7 (32%)
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these mutations also had improved survival and
response to chemotherapy alone [46] or placebo [47].
This suggests that EGFR mutations in NSCLC are a
good prognostic factor independent of EGFR TKI, hence
it may be more difficult to demonstrate the value of
EGFR mutations as predictors of benefit to EGFR TKI
[44]. The prognostic value of EGFRvIII in SCCHN
needs to be verified, and its role as a predictive marker
of response to EGFR inhibitor should remain a relevant
therapeutic question.
In this study, the prevalence of HPV, p16 and c-MET
expression (38%, 32% and 63% respectively) was in
keeping with the literature. We did not observe HPV,
p16 and c-MET expression to be predictive of disease
control, TTP or OS. This may be due to limitations of a
small sample size. Consistent with prior reports [21],
HPV-16 was the most common HPV subtype in our
study. c-MET is a poor prognostic marker in OSCC
[48], however the small proportion (11%) of OSCC in
our study precludes any meaningful association.
Limitations of this study include its small sample
size, potential bias towards patients with available
tumor specimens (larger tumor size), potentially vari-
able fixation and quality of the archival tissues and
Table 5 EGFRvIII mutation is associated with disease control
EGFRvIIIBest Response
Progressive DiseaseDisease Control (Partial Response or Stable Disease)
EGFRvIII absent by ΔΔCt 12 (67%)6 (33%) P = 0.0099
EGFRvIII present by ΔΔCt5 (25%)15 (75%) (Chi-square)
EGFRvIII mean fold change10.6263.76P = 0.04
EGFRvIII median fold change3.1611.11 (Wilcoxon)
Erlotinib treated patients
EGFRvIII absent by ΔΔCt6 (55%) 5 (45%) P = 0.21
EGFRvIII present by ΔΔCt3 (33%)10 (77%)(Fisher’s)
Non-erlotinib treated patients
EGFRvIII absent by ΔΔCt6 (86%)1 (14%)P = 0.21
EGFRvIII present by ΔΔCt2 (29%)5 (71%)(Fisher’s)
05101520253035
0.0
0.2
0.4
0.6
0.8
1.0
months
Survival proportion
EGFRvIII+
EGFRvIII-
Figure 1 Overall survival by EGFRvIII mutation status (HR = 0.91, 95% CI: 0.32-2.60, p = 0.85).
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potential variation in marker status of primary tumor
compared with recurrent or metastatic tumors (to our
knowledge, this is theoretical and has not been
described). Due to the absence of an untreated control
group in this study (’control’ patients received sorafe-
nib or ispinesib), our results cannot conclusively con-
firm the prognostic versus predictive value of a
biomarker. Although our methods did not use an anti-
body for EGFRvIII detection, we acknowledge that the
use of RT-PCR in FFPE samples has demonstrated
superior accuracy relative to IHC tests [49] and may
allow greater applicability to settings where frozen tis-
sue is unavailable.
Conclusion
Predictors of response to EGFR inhibitors in SCCHN
remain elusive. Biomarkers are desperately needed to
guide patient selection in SCCHN. EGFRvIII remains an
interesting tumor-specific target worthy of further
exploration as a prognostic or predictive marker of
response to EGFR inhibitor therapy in SCCHN. Larger
prospective randomized studies are required to distin-
guish the prognostic and predictive significance of
EGFRvIII, HPV, p16, c-MET and EGFR GCN in
SCCHN treated with EGFR inhibitors.
Acknowledgements
This work was supported by an unrestricted grant from Pfizer Canada, Inc.
Author details
1Division of Medical Oncology and Hematology, Princess Margaret Hospital,
University Health Network, Toronto, Ontario, Canada.2Department of
Pathology, Princess Margaret Hospital, University Health Network, Toronto,
Ontario, Canada.3Advanced Molecular Profiling Laboratory, Princess
Margaret Hospital, University Health Network, Toronto, Ontario, Canada.
4Department of Biostatistics; Princess Margaret Hospital, University Health
Network, Toronto, Ontario, Canada.
Authors’ contributions
NC participated in the study design, data acquisition, immunohistochemical
interpretation and drafted the manuscript. BP-O participated in the
histological examination, immunohistochemical interpretation and
manuscript preparation. KZ, NAP, JH, TZ, M-ST, SK-R carried out
immunostaining, ISH, PCR analysis, and prepared and reviewed the
manuscript. OL performed the FISH analysis and reviewed the manuscript.
LW performed the statistical analysis. EC participated in patient management
and reviewed the manuscript. LS conceived of the study, participated in its
design and coordination, and prepared and revised the manuscript. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 31 December 2010 Accepted: 27 February 2011
Published: 27 February 2011
References
1.Grandis JR, Tweardy DJ: Elevated levels of transforming growth factor
alpha and epidermal growth factor receptor messenger RNA are early
markers of carcinogenesis in head and neck cancer. Cancer Res 1993,
53:3579-3584.
2.Woodburn JR: The epidermal growth factor receptor and its inhibition in
cancer therapy. Pharmacol Ther 1999, 82:241-250.
3.Temam S, Kawaguchi H, El-Naggar AK, Jelinek J, Tang H, Liu DD, Lang W,
Issa JP, Lee JJ, Mao L: Epidermal growth factor receptor copy number
alterations correlate with poor clinical outcome in patients with head
and neck squamous cancer. J Clin Oncol 2007, 25:2164-2170.
4.Chung CH, Ely K, McGavran L, Varella-Garcia M, Parker J, Parker N, Jarrett C,
Carter J, Murphy BA, Netterville J, et al: Increased epidermal growth factor
receptor gene copy number is associated with poor prognosis in head
and neck squamous cell carcinomas. J Clin Oncol 2006, 24:4170-4176.
5.Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU,
Sur R, Raben D, Jassem J, et al: Radiotherapy plus cetuximab for
squamous-cell carcinoma of the head and neck. NEnglJ Med 2006,
354:567-578.
6.Cohen EE, Rosen F, Stadler WM, Recant W, Stenson K, Huo D, Vokes EE:
Phase II trial of ZD1839 in recurrent or metastatic squamous cell
carcinoma of the head and neck. J Clin Oncol 2003, 21:1980-1987.
7.Siu LL, Soulieres D, Chen EX, Pond GR, Chin SF, Francis P, Harvey L, Klein M,
Zhang W, Dancey J, et al: Phase I/II trial of erlotinib and cisplatin in
patients with recurrent or metastatic squamous cell carcinoma of the
head and neck: a Princess Margaret Hospital phase II consortium and
National Cancer Institute of Canada Clinical Trials Group Study. J Clin
Oncol 2007, 25:2178-2183.
8.Soulieres D, Senzer NN, Vokes EE, Hidalgo M, Agarwala SS, Siu LL:
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.
9.Burtness B, Goldwasser MA, Flood W, Mattar B, Forastiere AA: Phase III
randomized trial of cisplatin plus placebo compared with cisplatin plus
cetuximab in metastatic/recurrent head and neck cancer: an Eastern
Cooperative Oncology Group study. J Clin Oncol 2005, 23:8646-8654.
10.Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki A, Rottey S, Erfan J,
Zabolotnyy D, Kienzer HR, Cupissol D, et al: Platinum-based chemotherapy
plus cetuximab in head and neck cancer. N Engl J Med 2008,
359:1116-1127.
Table 6 HPV by PCR, p16 by IHC, c-MET by IHC are not
associated with disease control
Best Response
Progressive
Disease
Disease Control
(Partial Response or
Stable Disease)
HPV PCR
negative
14 (50%)14 (50%)P = 0.86
HPV PCR
positive
9 (47%)10 (53%)(Chi-square)
P16 IHC
negative
14 (47%)16 (53%)P = 0.67
P16 IHC
positive
8 (53%)7 (47%)(Chi-square)
c-MET IHC
< or = 2
9 (56%)7 (44%)P = 0.39
c-MET IHC >212 (43%)16 (57%)(Chi-square)
Table 7 Concordance of p16 IHC status with HPV by PCR
and HPV by ISH
P16 IHC
positive
P16 IHC
negative
HPV PCR positive
105P = 0.0016
HPV PCR
negative
525(Chi-
Square)
HPV ISH positive
60P = 0.0002
HPV ISH negative 731(Fisher’s)
Chau et al. Head & Neck Oncology 2011, 3:11
http://www.headandneckoncology.org/content/3/1/11
Page 9 of 11

Page 10

11.Grandal MV, Zandi R, Pedersen MW, Willumsen BM, van DB, Poulsen HS:
EGFRvIII escapes down-regulation due to impaired internalization and
sorting to lysosomes. Carcinogenesis 2007, 28:1408-1417.
Huang HS, Nagane M, Klingbeil CK, Lin H, Nishikawa R, Ji XD, Huang CM,
Gill GN, Wiley HS, Cavenee WK: The enhanced tumorigenic activity of a
mutant epidermal growth factor receptor common in human cancers is
mediated by threshold levels of constitutive tyrosine phosphorylation
and unattenuated signaling. J Biol Chem 1997, 272:2927-2935.
Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, Huang HJ: A
mutant epidermal growth factor receptor common in human glioma
confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994,
91:7727-7731.
Ji H, Zhao X, Yuza Y, Shimamura T, Li D, Protopopov A, Jung BL,
McNamara K, Xia H, Glatt KA, et al: Epidermal growth factor receptor
variant III mutations in lung tumorigenesis and sensitivity to tyrosine
kinase inhibitors. Proc Natl Acad Sci USA 2006, 103:7817-7822.
Sok JC, Coppelli FM, Thomas SM, Lango MN, Xi S, Hunt JL, Freilino ML,
Graner MW, Wikstrand CJ, Bigner DD, et al: Mutant epidermal growth
factor receptor (EGFRvIII) contributes to head and neck cancer
growth and resistance to EGFR targeting. Clin Cancer Res 2006,
12:5064-5073.
Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L,
Zahurak ML, Daniel RW, Viglione M, Symer DE, et al: Evidence for a causal
association between human papillomavirus and a subset of head and
neck cancers. J Natl Cancer Inst 2000, 92:709-720.
Shi W, Kato H, Perez-Ordonez B, Pintilie M, Huang S, Hui A, O’Sullivan B,
Waldron J, Cummings B, Kim J, et al: Comparative prognostic value of
HPV16 E6 mRNA compared with in situ hybridization for human
oropharyngeal squamous carcinoma. J Clin Oncol 2009, 27:6213-6221.
Gillison ML, D’Souza G, Westra W, Sugar E, Xiao W, Begum S, Viscidi R:
Distinct risk factor profiles for human papillomavirus type 16-positive
and human papillomavirus type 16-negative head and neck cancers. J
Natl Cancer Inst 2008, 100:407-420.
Westra WH, Taube JM, Poeta ML, Begum S, Sidransky D, Koch WM: Inverse
relationship between human papillomavirus-16 infection and disruptive
p53 gene mutations in squamous cell carcinoma of the head and neck.
Clin Cancer Res 2008, 14:366-369.
Weinberger PM, Yu Z, Haffty BG, Kowalski D, Harigopal M, Brandsma J,
Sasaki C, Joe J, Camp RL, Rimm DL, Psyrri A: Molecular classification
identifies a subset of human papillomavirus–associated oropharyngeal
cancers with favorable prognosis. J Clin Oncol 2006, 24:736-747.
Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H, Forastiere A,
Gillison ML: Improved survival of patients with human papillomavirus-
positive head and neck squamous cell carcinoma in a prospective
clinical trial. J Natl Cancer Inst 2008, 100:261-269.
Kumar B, Cordell KG, Lee JS, Worden FP, Prince ME, Tran HH, Wolf GT,
Urba SG, Chepeha DB, Teknos TN, et al: EGFR, p16, HPV Titer, Bcl-xL and
p53, sex, and smoking as indicators of response to therapy and survival
in oropharyngeal cancer. J Clin Oncol 2008, 26:3128-3137.
Lassen P, Eriksen JG, Hamilton-Dutoit S, Tramm T, Alsner J, Overgaard J:
Effect of HPV-associated p16INK4A expression on response to
radiotherapy and survival in squamous cell carcinoma of the head and
neck. J Clin Oncol 2009, 27:1992-1998.
Smith EM, Wang D, Kim Y, Rubenstein LM, Lee JH, Haugen TH, Turek LP:
P16INK4a expression, human papillomavirus, and survival in head and
neck cancer. Oral Oncol 2008, 44:133-142.
Knowles LM, Stabile LP, Egloff AM, Rothstein ME, Thomas SM, Gubish CT,
Lerner EC, Seethala RR, Suzuki S, Quesnelle KM, et al: HGF and c-Met
participate in paracrine tumorigenic pathways in head and neck
squamous cell cancer. Clinical Cancer Research 2009, 15:3740-3750.
Chen YS, Wang JT, Chang YF, Liu BY, Wang YP, Sun A, Chiang CP:
Expression of hepatocyte growth factor and c-met protein is
significantly associated with the progression of oral squamous cell
carcinoma in Taiwan. Journal of Oral Pathology & Medicine 2004,
33:209-217.
Allen C, Duffy S, Teknos T, Islam M, Chen Z, Albert PS, Wolf G, Van WC:
Nuclear factor-kappaB-related serum factors as longitudinal biomarkers
of response and survival in advanced oropharyngeal carcinoma. Clin
Cancer Res 2007, 13:3182-3190.
Di Renzo MF, Olivero M, Martone T, Maffe A, Maggiora P, Stefani AD,
Valente G, Giordano S, Cortesina G, Comoglio PM: Somatic mutations of
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
the MET oncogene are selected during metastatic spread of human
HNSC carcinomas. Oncogene 2000, 19:1547-1555.
Wheeler DL, Huang S, Kruser TJ, Nechrebecki MM, Armstrong EA,
Benavente S, Gondi V, Hsu KT, Harari PM: Mechanisms of acquired
resistance to cetuximab: role of HER (ErbB) family members. Oncogene
2008, 27:3944-3956.
Tang PA, Siu LL, Chen EX, Hotte SJ, Chia S, Schwarz JK, Pond GR,
Johnson C, Colevas AD, Synold TW, et al: Phase II study of ispinesib in
recurrent or metastatic squamous cell carcinoma of the head and neck.
Invest New Drugs 2008, 26:257-264.
Elser C, Siu LL, Winquist E, Agulnik M, Pond GR, Chin SF, Francis P,
Cheiken R, Elting J, McNabola A, et al: Phase II trial of sorafenib in
patients with recurrent or metastatic squamous cell carcinoma of the
head and neck or nasopharyngeal carcinoma. J Clin Oncol 2007,
25:3766-3773.
Weinberger PM, Yu Z, Haffty BG, Kowalski D, Harigopal M, Sasaki C,
Rimm DL, Psyrri A: Prognostic significance of p16 protein levels in
oropharyngeal squamous cell cancer. Clin Cancer Res 2004, 10:5684-5691.
Agulnik M, da Cunha SG, Hedley D, Nicklee T, Dos Reis PP, Ho J, Pond GR,
Chen H, Chen S, Shyr Y, et al: Predictive and pharmacodynamic
biomarker studies in tumor and skin tissue samples of patients with
recurrent or metastatic squamous cell carcinoma of the head and neck
treated with erlotinib. Journal of Clinical Oncology 2007, 25:2184-2190.
Tsao MS, Sakurada A, Cutz JC, Zhu CQ, Kamel-Reid S, Squire J, Lorimer I,
Zhang T, Liu N, Daneshmand M, et al: Erlotinib in lung cancer - molecular
and clinical predictors of outcome. N Engl J Med 2005, 353:133-144.
Cappuzzo F, Hirsch FR, Rossi E, Bartolini S, Ceresoli GL, Bemis L, Haney J,
Witta S, Danenberg K, Domenichini I, et al: Epidermal growth factor
receptor gene and protein and gefitinib sensitivity in non-small-cell lung
cancer. J Natl Cancer Inst 2005, 97:643-655.
Eriksen JGLP, Overgaard J: The EGFRvIII variant in squamous cell
carcinomas of the head and neck: Expression and correlation with
clinico-pathological parameters in 681 patients from the randomised
DAHANCA 6/7 study. ECCO 15 - 34th ESMO Multidisciplinary Congress Berlin;
2009, 472, September 2009.
Prigent SA, Nagane M, Lin H, Huvar I, Boss GR, Feramisco JR, Cavenee WK,
Huang HS: Enhanced tumorigenic behavior of glioblastoma cells
expressing a truncated epidermal growth factor receptor is mediated
through the Ras-Shc-Grb2 pathway. J Biol Chem 1996, 271:25639-25645.
Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK,
Furnari FB, White FM: Quantitative analysis of EGFRvIII cellular signaling
networks reveals a combinatorial therapeutic strategy for glioblastoma.
Proc Natl Acad Sci USA 2007, 104:12867-12872.
Mellinghoff IK, Wang MY, Vivanco I, Haas-Kogan DA, Zhu S, Dia EQ, Lu KV,
Yoshimoto K, Huang JH, Chute DJ, et al: Molecular determinants of the
response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 2005,
353:2012-2024.
Pelloski CE, Ballman KV, Furth AF, Zhang L, Lin E, Sulman EP, Bhat K,
McDonald JM, Yung WK, Colman H, et al: Epidermal growth factor
receptor variant III status defines clinically distinct subtypes of
glioblastoma. J Clin Oncol 2007, 25:2288-2294.
Shinojima N, Tada K, Shiraishi S, Kamiryo T, Kochi M, Nakamura H, Makino K,
Saya H, Hirano H, Kuratsu J, et al: Prognostic value of epidermal growth
factor receptor in patients with glioblastoma multiforme. Cancer Res
2003, 63:6962-6970.
Heimberger AB, Hlatky R, Suki D, Yang D, Weinberg J, Gilbert M, Sawaya R,
Aldape K: Prognostic effect of epidermal growth factor receptor and
EGFRvIII in glioblastoma multiforme patients. Clin Cancer Res 2005,
11:1462-1466.
Liu L, Backlund LM, Nilsson BR, Grander D, Ichimura K, Goike HM, Collins VP:
Clinical significance of EGFR amplification and the aberrant EGFRvIII
transcript in conventionally treated astrocytic gliomas. J Mol Med 2005,
83:917-926.
Eberhard DA, Giaccone G, Johnson BE: Biomarkers of response to
epidermal growth factor receptor inhibitors in Non-Small-Cell Lung
Cancer Working Group: standardization for use in the clinical trial
setting. J Clin Oncol 2008, 26:983-994.
Kalinsky K, Jacks LM, Heguy A, Patil S, Drobnjak M, Bhanot UK, Hedvat CV,
Traina TA, Solit D, Gerald W, Moynahan ME: PIK3CA mutation associates
with improved outcome in breast cancer. Clin Cancer Res 2009,
15:5049-5059.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
Chau et al. Head & Neck Oncology 2011, 3:11
http://www.headandneckoncology.org/content/3/1/11
Page 10 of 11

Page 11

46.Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens SL, Herbst RS,
Ince WL, Janne PA, Januario T, Johnson DH, et al: Mutations in the
epidermal growth factor receptor and in KRAS are predictive and
prognostic indicators in patients with non-small-cell lung cancer treated
with chemotherapy alone and in combination with erlotinib. J Clin Oncol
2005, 23:5900-5909.
Shepherd FA, Tsao MS: Unraveling the mystery of prognostic and
predictive factors in epidermal growth factor receptor therapy. J Clin
Oncol 2006, 24:1219-1220, author reply 1220-1211.
Lo ML, Leonardi R, Mignogna MD, Pannone G, Rubini C, Pieramici T,
Trevisiol L, Ferrari F, Serpico R, Testa N, et al: Scatter factor receptor (c-
Met) as possible prognostic factor in patients with oral squamous cell
carcinoma. Anticancer Research 2004, 24:1063-1069.
Yoshimoto K, Dang J, Zhu S, Nathanson D, Huang T, Dumont R,
Seligson DB, Yong WH, Xiong Z, Rao N, et al: Development of a real-time
RT-PCR assay for detecting EGFRvIII in glioblastoma samples. Clin Cancer
Res 2008, 14:488-493.
47.
48.
49.
doi:10.1186/1758-3284-3-11
Cite this article as: Chau et al.: The association between EGFR variant III,
HPV, p16, c-MET, EGFR gene copy number and response to EGFR
inhibitors in patients with recurrent or metastatic squamous cell
carcinoma of the head and neck. Head & Neck Oncology 2011 3:11.
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