pEGFR-Tyr 845 expression as prognostic factors in oral squamous cell carcinoma

Abstract

The EGFR (epidermal growth factor receptor) a member of the family of transmembrane protein kinase receptors known as the erbB family shows a significant correlation with the presence of metastases and poorly differentiated oral cancer. Aim of the present work is to define the key-role of EGFR in oral cancer prognosis. We have analyzed the EGFR expression on 149 cases of oral squamous cell cancers (OSCC) and we have found that it was poorly expressed in normal oral epithelium, but its expression was significantly increased in OSCCs. Moreover, we have recorded that both pEGFR-Tyr 845 and pEGFR-Tyr 1068 were mainly distributed in high histological grading and in advanced stages. Western blotting has confirmed the total absence of EGFR phosphorylation in normal oral epithelium and the higher level of protein phosphorylation in representative cases of OSCCs. The EGF-R amplification was found by fluorescence in situ hybridization (FISH) in 14% of OSCC; interestingly, EGF-R amplification was mainly observed in OSCC with higher histological grading (G2 and G3) and advanced stage (pT4) sub-groups. Kaplan-Meyer survival analysis suggested that patients with positive pEGFR-Tyr 845 tumors had a worse prognosis and were bad responders to chemotherapy. These results confirm the central role of EGF-R activation status as a prognostic biomarker in OSCC.

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Cancer Biology & Therapy 13:11, 1-11; September 2012; © 2012 Landes Bioscience
CLINICAL STUDY CLINICAL STUDY
*Correspondence to: Michele Caraglia; Email: michele.caraglia@unina2.it or michele.caraglia@alice.it
Submitted: 03/23/12; Revised: 06/01/12; Accepted: 06/04/12
http://dx.doi.org/10.4161/cbt.20991
Introduction
Oral squamous cell carcinoma (OSCC) remains a significant
cause of morbidity and mortality, with approximately 540,000
in the Western countries.1 Despite recent advances in cancer
treatment, survival rate for oral cancer has not changed signifi-
cantly over the last four to five decades.2,3 The understanding
of the genetic alterations and protein expression profiling in
these tumors might reveal new prognostic factors that accurately
The EGFR (epidermal growth fac tor receptor) a member of the family of transmembr ane protein kinase receptors known as
the erbB family shows a signicant correlation with the presence of metastases and poorly dierentiated oral cancer. Aim
of the present work is to dene the key-role of EGFR in oral cancer prognosis. We have analyzed the EGFR expression on
149 cases of oral squamous cell cancers (OSCC) and we have found that it was poorly expressed in normal oral epithelium,
but its expression was signicantly increased in OSCCs. Moreover, we have recorded that both pEGFR-Tyr 845 and pEGFR-
Tyr 1068 were mainly distributed in high histological grading and in advanced stages. Western blotting has conrmed
the total absence of EGFR phosphorylation in normal oral epithelium and the higher level of protein phosphorylation in
representative cases of OSCCs. The EGF-R amplication was found by uorescence in situ hybridization (FISH) in 14% of
OSCC; interestingly, EGF-R amplication was mainly observed in OSCC with higher histological grading (G2 and G3) and
advanced stage (pT4) sub-groups. Kaplan-Meyer survival analysis suggested that patients with positive pEGFR-Tyr 845
tumors had a worse prognosis and were bad responders to chemotherapy. These results conrm the central role of EGF-R
activation status as a prognostic biomarker in OSCC.
pEGFR-Tyr 845 expression as prognostic factors
in oral squamous cell carcinoma
A tissue-microarray study
with clinic-pathological correlations
Gabriella Aquino,1 Giuseppe Pannone,2 Angela Santoro,3 Giuseppina Liguori,1 Renato Franco,1 Rosario Serpico,4
Gianluca Florio,4 Alfredo De Rosa,4 Marilena Mattoni,2 Valentina Cozza,5 Gerardo Botti,1 Simona Losito,1 Francesco Longo,1
Stefania Staibano,6 Giovanni Cuda,7 Lorenzo Lo Muzio,2 Carolina Sbordone,8 Pantaleo Bufo,2 Anna Grimaldi,9
Michele Caraglia9,* and Marina Di Domenico10,11
1Section of Pa thological Anatomy; Fou rth Section of Ma xillofacial Surger y; Istituto Nazional e per lo Studio e la Cura dei Tumori; Fond azione “G.Pascale ”; Naples, I taly;
2Depart ment of Surgical Science s; Section of Anatomic Pat hology; Second Sec tion of Oral Patholo gy; University of Fog gia; Foggia, Italy; 3Dep artment of Surgical S ciences;
Sectio n of Anatomic Pathology; U niversity of Bari; Bar i, Italy; 4Department of Oral Pathology; Orthodontics and Oral Surgery; Second University of Naples; Naples, Italy;
5Depart ment of Business Managem ent; University of Na ples Parthenope; N aples, Italy; 6Section of Pathological Anatomy; Department of Biomorphological and Functional
Sciences; Uni versity of Naples “Fe derico II”; Naples, Ital y; 7Department of E xperimental and Clin ical Medicine; Univer sity of Magna Graecia ; Medical School; Cata nzaro, Italy;
8Department of Odontostomatology and Maxillofacial Surgery; University of Naples “Federico II”; Naples, Italy; 9Department o f Biochemistry and B iophysics;
Second University of Naples; Naples, Italy; 10Department of General Pathology; Second University of Naples; Naples, Italy; 11Sbarro Insti tute for Cancer Research
and Molecular Medicine and Center for Biotechnology; Temple University; Philadelphia, PA USA
Keywords: EGFR, OSCC, phosphorylated EGFR, targeted therapy
Abbreviations: EGFR, epidermal growth factor receptor; OSCC, oral squamous cell cancers; FISH, f luorescence in situ
hybridization; SH2, Src homology 2; TMA, tissue microarrays; IHC, immunohistochemistry; OS, overall survival; NLS, nuclear
localization signal; HPFs, high power fields; LSAB-HRP, streptavidin-biotin horseradish peroxidase technique; DFI, disease free
interval; DAPI II, 4,6-diamino-2-phenyindole-2-hydrochloride
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
predict the biological behavior of the disease and allow for a more
accurate prognostic characterization of individual tumors.4 In
addition, these markers might lead to individually targeted ther-
apeutic approaches specifically designed to inhibit several bio-
chemical events in the pathogenesis of this cancer.5-7 The EGFR
(epidermal growth factor receptor) is a member of the family of
transmembrane protein kinase receptors known as the erbB or
HER receptor family: EGFR (HER1 or erbB1), erbB2 (HER2),
erbB3 (HER3) and erb4 (HER4). The EGFR is codified by a
gene localized in locus 7p11.2 of chromosome 7.8 Upon binding

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2 Cancer Biology & Therapy Volume 13 Issue 11
to synthesize DNA in response to EGF, suggesting that this c-Src
mediated phosphorylation site is important for receptor function.
Accumulating evidences indicate that interactions between the
EGFR and the non receptor tyrosine kinase c-Src may contribute
to an aggressive phenotype in multiple human tumors. Aim of
the work was to evaluate EGFR expression and its phosphoryla-
tion in Tyr 845 and Tyr 1068 by immunohistochemistry together
with EGFR amplification by Fluorescence in situ Hybridization
(FISH) in prognostic tissue microarrays (TMA) of paraffin-
embedded tissue specimens from 149 patients who underwent
surgical treatment for squamous cell carcinoma of oral cavity in
the period between 1996–2 0 0 7.
Results
Clinical characteristics of the patients. The OSCC-149 tissue
microarray contained representative tumor samples from 149
patients affected by OSCC. Clinical and pathological data of the
retrospectively studied population have been reported in Table 1.
Interestingly, adjuvant radiotherapy has been used in 110 (74%)
patients and adjuvant chemotherapy in 45 patients (30%); finally,
only 23 (15%) patients in this cohort did not received adjuvant
therapy. The criteria employed to administer adjuvant therapy had
been exclusively clinical, as mentioned in Materials and Methods.
TMA technology has allowed high throughput immunohisto-
chemical analysis of archival material and in particular FISH
technology has been used in order to evaluate the percentage of
EGFR gene amplification in the studied OSCC cases, comparing
gene amplification with immunohistochemical EGFR expression
(EGFR, pEGFR-Tyr 845 and pEGFR-Tyr 1068).
EGFR expression and phosphorylation is associated with
worse grading and prognosis of OSCC. EGFR is expressed in
normal epithelium at low levels and in particular in the basal
and supra-basal proliferating layers (Fig. 1); as expected, EGFR is
highly overexpressed in OSCCs, both in membrane and in cyto-
plasm, and its staining is associated with hig h graded and advanced
staged t umors. Representative IHC findings of EGFR, pEGFR-Tyr
1068 and pEGFR-Tyr 845 can be observed respectively in Figures
1 and 2. We also observed discrete nuclear immunoreactivity of
EGFR as the functional roles of nuclear EGFR were extensively
studied in recent reports. Regarding immunohistochemistry for
pEGFR-Tyr 845 and pEGFR-Tyr 1068 only percentage of cell
staining has been evaluated, irrespective of the sub-cellular local-
ization. The frequencies of OSCC showing phosphorylation of
EGFR on Tyrosine 845 and on Tyrosine 1068 can be observed in
Tabl e 2: both phosphorylations are mainly distributed in high his-
tological degrees and in advanced stages. In fact, 83 (56%) cases
were positive for pEGFR Tyr 845 and 37 (25%) for pEGFR Tyr
1068. Moreover, among pEGFR Tyr 845 positive OSCCs, 80%
were Grade 2–3 tumors (67 cases) and 42% stage 4 (35 cases)
while, among pEGFR Tyr 1068 positive cases, 71% (26 cases)
were G2-3 tumors and 39% (14 cases) were stage 4 cancers. These
data suggested that higher EGFR phosphorylation is correlated
with more advanced OSCC grading and staging. We have also
evaluated EGFR expression and phosphorylation by western blot-
ting of some representative cases of OSCCs and relative normal
of specific polypeptide ligands, including EGF, transforming
growth factorα, beta-cellulin, heparin-binding EGF, epiregulin
and amphiregulin, EGFR undergoes homo- or hetero-dimer-
ization and activation of its intrinsic tyrosine kinase activity.9,10
The primary risk factors of OSCC are tobacco, smoking and
elevated levels of alcohol consumption. Other potential risk fac-
tors include diet, human papillomavirus (particularly HPV16),
and various oral factors, including oral hygiene.11 In fact, high
levels of colonization of OSCC by facultative oral streptococci
were observed in the saliva of OSCC subjects.12,13 More recently,
viable bacteria have been isolated from both superficial and deep
parts of OSCC,14 revealing that the tumor microenvironment
is well suited for bacterial survival. The role of bacteria in the
development of oral cancer has not been delineated, but the per-
sistent presence of bacteria at tumor sites in the oral cavity raises
intriguing questions about the role of bacteria in the progres-
sion of OSCC. The phosphorylation of EGFR activates multiple
biological processes including apoptosis, differentiation, cellular
proliferation, motility, invasion, adhesion, DNA repair and sur-
vival.15 Several strategies have been developed to inactivate the
EGFR pathway including monoclonal antibodies against the
extracellular domain of EGFR.16-18 Expression of EGFR varies
widely in several tumors, including head and neck (80–100%).
In human tumors, high expression of EGFR correlates with a
more aggressive clinical course, and has been reported to be a
useful diagnostic and prognostic marker. In recent years, EGFR
has been considered a promising target for monoclonal antibody
therapy, and in particular recently performed clinical trials have
established the clinical importance of administer monoclonal
antibodies against EGFR together with chemotherapy or radio-
therapy in the treatment of advanced head and neck cancers.19-2 2
However, these treatments are potentially toxic and, as for today,
there are not established criteria to distinguish responsive patients
from non-responders.23 The activation status of EGFR is deter-
mined by its autophosphorylation rate that is responsible for the
triggering of the downstream signaling cascade. Five autophos-
phorylation sites have been identified in vivo in EGFR includ-
ing Tyr1068.24 Tyr 1068 is a classic RTK auto-phosphorylation
site and potentiates docking of growth factor receptor binding
protein Grb1 to EGFR, with subsequent activation of MAPK/
extracellular signal-regulated kinase and phosphoinositide-3-ki-
nase/AKT signaling cascades, respectively.25,26 These events lead
to the autophosphorylation of multiple tyrosine residues in the
COOH-terminal tail of the molecule that serve as binding sites
for cytosolic signaling proteins containing Src homology 2 (SH2)
domains and phosphotyrosine binding domains. Previous results
demonstrated an association between c-Src and EGFR that results
in the appearance of two tyrosine phosphorylations, one of which
is Tyr 845.27 Accumulating evidences indicate that c-Src-medi-
ated phosphorylation of EGFR Tyr 845 is involved in regulation
of receptor function, as well as in tumor progression.2 8,29 Src is
overexpressed in a high percentage of human neoplasms, includ-
ing head and neck cancer, and its deregulation is identified as
one of the major oncogenic signatures found in cancer.30, 31 Early
studies on cells show that cells transiently expressing EGFR bar-
ing a Tyr to Phe mutation at Tyr 845 are impaired in their ability

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patients was 46 months (CI: 0.12–5.07, p = 0.78) vs. 48 months
of negative patients (Fig. 5D). A significant result of the present
study is the demonstration that the survival of patients with early
tumor stages (stage 1 and 2) and of those subjected to adjuvant
chemotherapy can be predicted by the phosphorylation status
of EGFR-Tyr 845. In fact, the median OS of the patients sub-
jected to adjuvant chemotherapy and positive for EGFR-Tyr 845
was 19 months (CI: 1.66–13.15, p = 0.003) vs. 48 months of
negative patients (Fig. 6A). The median OS of the early staged
tumors (T1-T2) positive for EGFR-Tyr 845 was 59 months
(CI: 1.01–10.16; p = 0.048) vs. 69 months of negative patients
(Fig. 6B). No correlation was found between the phosphorylation
mucosal tissues. We have found an increased expression of EGFR
in all the examined OSCCs that was however expressed at sig-
nificant levels also in normal mucosa. These findings were paral-
leled by the total absence of EGFR phosphorylation in normal
oral epithelium while high levels of protein phosphorylation of
both evaluated tyr sites were observed in all representative cases
of OSCCs (Fig. 3). Interestingly, these OSCC cases had trisomic
and amplified EGFR (see below).
Amplification of EGFR correlates with EGFR overexpres-
sion and advanced grading and staging of OSCCs. To more
comprehensively study the relevance of the EGFR amplification
in OSCC (Fig. 4), the protein levels of EGFR were investigated
by immunohistochemistry on the same tissue slides used for
FISH analysis. Overall, no evidence of gene amplification was
found on chromosome 7 in any samples of normal oral epithelia.
Tumor tissues with higher EGFR immunointensity had higher
FISH signal ratios. In the same way, cases with amplified EGFR
showed very strong circumferential staining of the membrane
that appeared remarkable thickened; in addition, cytoplasms
were diffusely stained and in some fields the nuclei appeared
positive. Polysomic cases showed medium-strong immunohis-
tochemical staining with frequent membrane staining inter-
ruptions. Therefore, EGFR immunoreactivity might serve as
a surrogate marker to predict EGFR amplification in OSCC.
Frequencies of gene amplification according to pathological
correlations has been reported in Tabl e 3. EGFR gene ampli-
fication was found in 21 (14%) OSCCs in our series; OSCCs
with amplified EGFR were mainly distributed in higher his-
tological degree (G2-G3, 20 cases, 97%) and advanced stage
(ST4, 15 cases, 67%) OSCCs. We have also assessed the cor-
relations among EGFR immunohistochemical expression, gene
amplification as evaluated by FISH, and pathological findings.
Interestingly, women have higher percentage of EGFR expres-
sion in euploid not amplified tumors, but lower percentage in
polysomic and/or amplified cancers.. In not EGFR amplified
OSCCs EGFR overexpression is more evident in advanced and
undifferentiated tumors, while in polysomic and amplified can-
cers this correlation is lost.
EGFR activating phosphorylations and survival analysis.
Clinical outcome of the patients showing EGFR activating
phosphorylations compared to the respective phosphorylation
negative cases has been studied by Kaplan-Meier curves; these
topics are reported in Figures 5 and 6. The survival curves
showing percentage survival according to EGFR-p-Tyr 845 in
males and females (Fig. 5A and B), and EGFR-p-Tyr 1068 in
males and females (Fig. 5C and D) were not statistically sig-
nificant. In fact, the median OS of positive EGFR-p-Tyr 845
males was 59 months (CI: 0.37–1.81; p = 0.836) vs. 69 months
of negative patients (Fig. 5A). However, inside females affected
by OSCC with EGFR-p-Tyr 845 negative staining a trend for a
better survival was observed (median OS: 69 months) if com-
pared to the patients positive for EGFR-p-Tyr 845 (median OS:
24 months, CI: 0.80 –13.62; p = 0.098) (Fig. 5B). The median
OS of positive EGFR-p-Tyr 1068 male patients was 33 months
(CI: 0.37–1.79, p = 0.812) vs. 59 months of negative patients
(Fig. 5C). The median OS of positive EGFR-p-Tyr 1068 female
Tab le 1. Clinic-pathological characteristic of study population
Clinical-pathological characteristics of 149 patients selected for TMA
Age
Range 31-108 Mean 66
Sex
M♂ 104 (70%) F♀ 45 (30%)
Site
Lip 4 (2.5%)
Tongue 81 (54%)
Buccal mucosa 3 (1.5%)
Floor of mouth (FOM) 19 (13 %)
Gum 10 (7%)
Palate 0 (0%)
Retromolar tritone 12 (8% )
Multisites 20 (14%)
Total 149
TNM Staging
T N0 N+ M0 M+
T1 18 (12%) 10 (7%) 28 (19% ) 0
T2 42 (28%) 27 (18%) 69 (47 %) 1 (1%)
T3 9 (5.5%) 15 (10 %) 24 (16%) 0
T4 9 (5.5%) 19 (13%) 27 (18%) 0
Total 78 (52%) 71 (48%) 148 (99%) 1 (1%)
Stage
St 1 St 2 St 3 St 4
18 (12%) 42 (28%) 33 (22%) 56 (38%)
Grade
G1 G2-3
25 (17 %) 124 (83%)
Radiotherapy
+ -
110 (74%) 39 (26%)
Chemotherapy
+ -
45 (30%) 104 (70%)
No adjuvant therapy
23 (15% )

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4 Cancer Biology & Therapy Volume 13 Issue 11
Discussion
OSCC represents one of the major health issues, with over
540,000 new cases reported in western Countries annually.31-35
Though improvements in screening and early diagnosis have dra-
matically reduced the incidence of this neoplasm in recent years,
the 5-year disease-free survival is still poor, despite the great sci-
entific and financial efforts. For many years, the main prognostic
factors of OSCC have been the conventional grading, staging
and site of tumor. The molecular mechanisms involved in the
oral carcinogenesis are not yet fully understood and the complete
genetic profile of the cancer cells is still to be characterized.
Recent reports show a significant correlation among the amplifi-
cation status of EGFR, and both the presence of metastases and
poorly differentiated tumors. Therefore, EGFR overexpression in
OSCC has been suggested as a valuable prognostic marker for
shortened survival and metastatic spread.36 ,37 On the other hand,
current literature on prognostic role of EGFR has been limited by
small series of cases characterized by low time of follow-up and
lacking of complete and detailed information about diagnosis
and therapy. The present retrospective study investigates about
the correlation among EGFR expression, EGFR phosphoryla-
tion, amplification or polysomic status and the clinical and path-
ological characteristics in a large series of oral cancers of different
grade and stage, by combining different types of molecular diag-
nostic methods, as immunohistochemistry, FISH and western
Blotting on TMA. TMA are a high-throughput technology that
allows the simultaneous in situ analysis of a large number of
tumors at the protein, DNA and RNA levels using immunohis-
tochemistry or in situ hybridization techniques.38-40 In the recent
years molecular technologies such as Fluorescent in situ
Hybridization (FISH) has become an essential tool in the diag-
nosis and management of a variety of solid tumors and hemato-
logic malignancies in the clinical setting, as well as an aid in the
identification of particular genetic disorders. FISH yielding
information about chromosomal changes can also be considered
an innovative method of choice to select patients for individual-
ized targeted cancer therapies. In our study, combining different
molecular techniques and morphological/histological informa-
tion, the key role of EGFR and its phosphorylated forms has been
brought out. Amplification of 7p11.2 is frequently found in many
cancer types, including lung and breast cancer, and glioblasto-
mas. Our study showed that the 7p11.2 amplification and the
centromere polysomy were the most common DNA copy number
status of EGFR and the lymph nodal status of disease. The study
of survival for pEGFR-Tyr 1068 has given rise to negative statisti-
cal results as regard to the clinical outcome (data not shown). All
the remaining correlations with other clinical and pathological
factors were all not significant (data not shown).
Figure 1. EGFR expression in normal peri-tumor oral epithelium as
evaluated by immunohistochemistry and in representative cases of
OSCC’s with amplied EGFR. (A) EGFR shows a high expression limited
to the basal proliferative layer, whereas spinous epithelial layer demon-
strated faint-intermediate expression at membrane malpighian bridges
(LSAB-HRP, original magnication x63, inset original magnication
x100). (B)Photos A and B show two dierent cases with strong EGFR ex-
pression. Amplied cases showed very strong circumferential staining
of the membrane that appeared remarkable thickened; in addition, cy-
toplasms were stained (A1 and B1) and in some elds (A1) the nuclei ap-
peared positive (LSAB-HRP, nuclear counterstaining with haematoxylin;
Ventana pre-diluted Ab not specic for activating phosphorylations).

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growth of the primary papillary carcinoma and related to
decreased recurrence-free survival in surgically treated tumours.44
Cytoplasmic staining has also been associated with high renal
tumour stage and high renal tumor grade. It has been suggested
that the cytoplasmic overexpression of EGFR plays a significant
role in the progression of pancreatic ductal adenocarcinoma,
change also in OSCC and provided cogent evidence
that EGFR was the key gene contributing to OSCC
development. We identified amplification and poly-
somic status in OSCC tissues, by validated FISH
analysis, a sensitive and specific method of assessing
gene copy number, on a large series of samples (n =
149). By comparing genetic contents and expression/
phosphorylation levels, we concluded that the key
“cancer-driving” gene, EGFR plays an important role
in oral cancer development. The present study con-
firms that high EGFR expression is present in OSCCs
and that an uncontrolled tumor growth may be
mediated by abnormal EGFR expression. EGFR
expression extent and intensity scores suggest that
EGFR expressing carcinomas display pathological
features of more aggression which may be attribut-
able to the activation of different signaling pathways
that control diverse biological processes. As reported
by other investigators, EGFR expression involved all
epithelial layers in OSCC specimens while in normal
oral epithelia it was localized to the basal cell layer.
Similar results were reported by other investigators.
Since the squamous epithelium maintains a continu-
ous physiological regeneration in normal conditions,
it is reasonable that the basal cells interpret signals of
EGF by binding to EGFR, while its expression
beyond basal localization in cancerous tissue suggests
that a correlation between EGFR and tumor progres-
sion may exist. The expression was mainly localized
to the peripheries of highly undifferentiated tumor
nests. This finding confirms the presence of this
receptor on more undifferentiated cells and explains
that the staining reaction varies with cellular differ-
entiation. Moreover, it may explain that peripheral
tumor cells receive a signal from EGF resulting in the
additional proliferation of cancer tissues. Other aim
of our work has been to assess whether heterogeneity
of EGFR immunoreactivity in OSCC is related to
non-standardized criteria for staining evaluation.
Different methods of immunohistochemical evalua-
tion led to different results, strengthening the need
for standardization, especially against a background
of rapidly evolving EGFR targeted cancer treatment
strategies. In numerous immonohistochemical stud-
ies, EGFR staining has been observed in the cyto-
plasm and in nucleus as well as in the membrane, but
the subcellular localization of EGFR has so far
received little attention.41,42 In a series of benign and
malignant skin tumors, loss of membrane staining
and increased cytoplasmic accumulation of EGFR
were observed in malignant cutaneous epithelial tumors when
compared to normal tissue. Furthermore, this alteration in EGFR
distribution was predominant in less well-differentiated tumors,
indicating increased cytoplasmic localization with increasing
ma lig n a nc y.43 Some authors showed that strong cytoplasmic
EGFR staining was significantly associated with extra-thyroidal
Figure 2. Phosphorylated EGFR-p-tyr1068 and p-tyr845 in oral cancer as evaluated
by TMA-based immunohistochemistry. (A) A TMA core of positive p-EGFR-tyr 1068
is showed in (A), at larger magnication in (B), demonstrating strong cytoplasmic
expression of the activated receptor (LSAB-HRP, nuclear counterstaining with hae-
matoxylin). (B) Phosphorylation of EGFR on Tyrosine 845 in oral cancer as evaluated
by TMA-based IHC. A, A1, A2 show high cytoplasmic expression of p-845 EGFR in a
representative case of OSCC with vascular invasion. B, B1, B2 show p-845 EGFR in a
case of OSCC with poor dierentiation (A, A1, A2, B, B1, B2: IHC-LSAB-HRP, nuclear
counterstaining with haematoxylin; phosphorylated tyr845 EGFR Ab).

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6 Cancer Biology & Therapy Volume 13 Issue 11
known target of c-Src45, 46 and c-Src has been correlated with the
progression and poor prognosis of OSCC. In fact, it has been
recently reported that CD133 (a stem cell marker) via Src activa-
tion mediates tumor initiating property and epithelial-mesenchy-
mal transition of head and neck cancer.47 Moreover, high
expression of Src protein (labeling indices > 50%) was correlated
with larger tumor size (p = 0.017), positive lymph node metasta-
sis (p = 0.030), more advanced clinical stages (p = 0.007), and
recurrence (p < 0.001) of OSCC.48 Our data suggest that the
detection of phosphorylation in Tyr 845 of EGFR could be a sur-
rogate marker of activation of Src in OSCC and could have a role
in predicting poor prognosis in the early stages discriminating
patients who require a more aggressive therapeutic strategy
despite the initial extension of the disease. This is still more
important if we consider that choose of the best treatment strat-
egy is presently based exclusively on the grading and staging of
OSCC. Finally, we have also found that pTyr 845 EGFR can be
a predictive marker of resistance to chemotherapy in OSCC.
This finding discloses a new scenario in which the use of Src
inhibitors such as dasatinib should be explored in the treatment
of pTyr845 EGFR positive OSCCs. In fact, it was recently dem-
onstrated in preclinical models that the simultaneous inhibition
of Scr and Met signaling can be an attractive and effective new
strategy in the treatment of OSCC.49 HPV infection plays an
important role in pathogenesis of OSCC as recently reviewed by
our group.50 Moreover, there is according evidence that HPV
positive cases are more responsive to chemo- and radiation treat-
ments whereas HPV negative are associated with worse prognosis
and need further research to detect future targeted treatments.
However, the cohort of cases analyzed in this study is mainly
constituted by HPV negative cases. In the present study we have
analyzed only cancers of the oral cavity carefully excluding the
oropharyngel cancers that, in turn, show a very high HPV inci-
dence. In fact, consecutive slides of TMA used for EGFR and
p-EGFR were studied by p-16 immunohistochemistry, and in
situ hybridization in order to detect LR-HPV and
HR-HPV and they resulted HR-HPV negative.51
Further studies based on consensus PCR to detect
HR-HPV confirmed that the cases were HPV nega-
tive. This finding is in agreement with some Italian
retrospective studies showing a very low frequency of
HPV in squamous cell carcinoma limited to the oral
cav it y.52 Since the cohort of OSCC cases studied by
TMA was mainly HPV negative the percentage of
cases showing EGFR amplification was lower than
that reported in other studies.
In conclusion, the search for molecular prognos-
tic markers for cancer is still a major clinical and
therapeutic issue. The determination of the expres-
sion of Ghrelin, type I interferons and IGF-I may
be important prognostic markers and promising
approaches for novel treatment strategies, even if
the literature characterizes better the role of these
markers in neuroendocrine tumors rather than
squamous.53-56 However, detailed studies will be
required for better understanding of the complex
especially in the invasion and acquisition of aggressive clinical
behavior. Taken together, the available information concerning
EGFR expression and subcellular localization are consistent with
an aberrant function of the EGFR located in the cytoplasm. Our
results are consistent with the existent model of translocation of
membranous EGFR to the cytoplasm and afterwards to the
nucleus acting as a transcriptional factor and fostering cell prolif-
eration. The exact mechanism by which cell-surface EGFR trans-
locates into the cytoplasm and the cell nucleus remains largely
unknown. EGFR co-localizes and interacts with importins α1/
β1, carriers that are critical for macromolecules nuclear import.
EGFR variant mutated at the nuclear localization signal (NLS) is
defective in associating with importins and in entering the nuclei
indicating that EGFR’s NLS is critical for EGFR/importins
interaction and EGFR nuclear import. Moreover, our data sug-
gest that a critical factor determining the prognosis of cancers is
the activation status of EGFR. In fact, we have found that pTyr
845 is correlated with a worse prognosis in early stage OSCCs
and in tumors subjected to adjuvant chemotherapy. Tyr845 is a
Table 2. Clinic-pathological characteristics of pEGFR Tyr 845/1068
positive and negative OSCCs
Variables (%) pEGFRTyr 845 pEGFR-Tyr 1068
% Positive cases (n.) % Positive cases (n.)
56 (83) 25 (37)
M75 (62) 81 (30)
F25 (21) 19 (7)
G1 20 (16) 2 9 (11)
G2-3 80 (67) 71 (26)
Stage 1 12 (10) 16 (6)
Stage 2 27 (22) 16 (6)
Stage 3 19 (16) 29 (11)
Stage 4 42 (35) 39 (14 )
Figure 3. Western blotting of representative normal epithelia, EGFR-trisomic and
EGFR-amplied OSCC’s using phosphorylated Tyr 845 EGFR antibody. Both trisomic
and amplied OSCC’s show EGFR phosphorylation on both Tyr sites, whereas normal
epithelia are negative. For further details see Materials and Methods section.

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whenever possible, one core of normal mucosa of the same tissue
block were arrayed for each case. All the donor cores were for-
matted into one recipient block. H&E staining of a 4-μm TMA
mechanism of carcinogenesis relating to OSCCs and
to improve chemoradiotherapy through the discov-
ery of new therapeutic markers. Our data lead sup-
port to the need of detecting the expression of EGFR
and its phosphorylated isoforms in clinical practice
and diagnostic management of OSCCs.57 The study
of EGFR Tyr phosphorylation could be also a useful
surrogate marker of activation of downstream signal
transduction pathways in the cell and could be help-
ful in guiding the therapeutic decisions in OSCC
patients. In fact, target-based therapies are widely
accepted as the future of cancer treatment58,59 and
detection of EGFR status in oral cancer patients may
identify patients who will benefit from the use of new
anti-cancer agents.60,61
Materials and Methods
Study cases. Paraffin blocks from OSCC resections
were retrieved from the archives of National Cancer
Institute of Naples. A single block for each case
was selected for use in the construction of the tis-
sue microarray (TMA). Tissues from 149 cases were
included, representing different clinical and patho-
logical categories. Clinical information including
demographic, therapeutic and clinical outcome vari-
ables were retrieved from patient medical records and
were considered for correlation analysis (Ta ble 1).
This tissue microarray has been named OSCC-149.
The study was approved by the Research Ethics
Boards of Fondazione “G. Pascale.” All the patients
included in the OSCC-149 have been treated accord-
ing to standard diagnostic and therapeutic criteria.
Briefly, for oral cancers trans-oral resections plus
sentinel lymphadenectomy have been performed
for T1-2/N0 while resection of primary plus neck
lymphadenectomy (levels I–IV; for any T with N+, resection of T
plus neck lymphadenectomy) have been made for T3-4/N0. For
maxillary and ethmoidal tumors of any T, total maxillectomy or
extended maxillectomy was performed while for the same tumors
of any T with N+ total maxillectomy or extended maxillectomy
plus neck lymphadenectomy (levels I–IV) was made. Adopted
criteria for adjuvant therapy after primitive cancer removal and
neck dissection were the following: (1) positive margins or close
(< 5 mm); (2) T3-T4 primary tumor; (3) vascular invasion; (4)
perineural invasion; (5) N > 1 (more than one positive lymph-
node including micrometastasis and extracapsular spreads).
Tissue microarray based immunohistochemistry. For tissue
microarray construction, areas of interest rich in non-necrotic
tumoral cells were identified on corresponding haematoxylin
and eosin-stained sections and marked on the source paraffin
block. The source block was cored and a 0.6 mm core trans-
ferred to the recipient master block using Galileo TMA CK
3500 Tissue Microarrayer (ISE TMA Software, Integrated
System Engineering). Moreover, two cores from different areas (a
superficial one and one representative of the deep invasion) and,
Figure 4. FISH analysis for the detec tion of EGFR amplication. (A) An OSCC showing
euploid chromosome 7 and not amplied EGFR; (B) a case of OSCC showing triploid
chromosome 7 and three copies of EGFR; (C) a case of OSCC demonstrating aneu-
ploidy at chromosome 7 and multiple copies of EGFR (polysomic not amplied EGFR);
(D)a case of OSCC showing euploid chromosome 7 and multiple copies of EGFR visu-
alized as nuclear clusters (amplied EGFR gene) [FISH: LSI EGFR Dual-Color Probe-Hyb
Set, LSI EGFR Spectrum Orange/Cep-7 Spectrum Green; DAPI II (4,6- diamino-2-pheny-
indole-2-hydrochloride) was used for chromatin counterstaining; a ratio of LSI EGFR
Spectrum Orange/Cep-7 Spectrum Green > 2 has been considered as amplied; for
further details see Materials and Methods].
Tab le 3. Frequencies of FISH amplification according to pathological
correlations
Variables % N. A. (n) % N.A.
polysomic (n)
% Amplified
(n)
Patients (total) 80 .5 (12 0) 5.5 (8) 14 (21)
M70 (84) 67 (5) 87 (18)
F30 (36) 3 (3) 13 (3)
G1 20 (24) 0 (0) 7 (1)
G2-3 80 (96) 10 0 (8) 97 (20)
Stage 1 12 (15 ) 17 (1) 6. 5 (1)
Stage 2 25.5 (30) 33 (3) 20 (4)
Stage 3 25.5 (30) 33 (3) 6 .5 (1)
Stage 4 37 (45) 17 (1) 6 7 (15)
N.A., not amplified.

© 2012 Landes Bioscience.
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8 Cancer Biology & Therapy Volume 13 Issue 11
(OLYMPUS BX41, at x40) and were selected for EGFR positive
tumor cells with an average of 1,000 tumor cells per case and 200
tumor cells per field. The topographical staining pattern was also
evaluated and recorded as membranous (M), cytoplasmic (C), or
mixed and nuclear (N). For each case, the cumulative percentage
of positive cells among all sections examined was determined.
Since till now there is not standardized criteria for EGFR stain-
ing evaluation, we have chosen to grade and score the extent of
EGFR immunostaining as follows: 0 points for negative staining
of the considered cells, (1) < 10%, (2) 10–50%, (3) 51–80% and
(4) ≥ 80% positive staining of the considered cells. The intensity
of staining was scored as 0, no staining; +, weak; ++, moderate;
+++, strong. For pEGFR-Tyr 845 and pEGFR-Tyr 1068 immu-
nohistochemical evaluation, we selected a dichotomized indica-
tor variable. In order to determine the best logical cut-off point
section was used to verify all samples. Immunohistochemical
analysis on 4-μm TMA serial sections was performed by using
Ventana Benchmark XT autostainer and/or manual standard
linked streptavidin-biotin horseradish peroxidase technique
(LSAB-HRP), according to the best protocol for each anti-
body used in our laboratory: pre-diluted primary anti-EGFR
(clone3C6); primary anti-p-EGFR Tyr 1068 (code2234 phos-
phoTyr 1068-EGF receptor, Cell Signaling) diluted 1:400 in
PBS and primary anti-p-EGFR Tyr 845 (code2231 phosphoTyr
845-EGF receptor Cell Signaling) diluted 1:400 with PBS and
incubated overnight. Negative control slides without primary
antibodies were included for each staining. The results of the
immunohistochemical staining were evaluated separately by
two observers (RF, GB). In each tissue section 10 representative
high power fields (HPFs) were analyzed at optical microscope
Figure 5. Kaplan Meier curvesof patients positive for p-Tyr 845 and 1068EGFR stratied for gender. Survival curves showing percentage survival ac-
cording to sex and EGFR-p-Tyr 845 (A and B), and EGFR-p-Tyr 1068 (C and D). Interestingly, inside the group of females aected by OSCC the EGFR-p-Tyr
845 negative subgroup showed a trend for a better survival if compared to the EGFR-p-Tyr 845 positive subgroup (for details, see text).

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www.landesbioscience.com Cancer Biology & Therapy 9
Statistical analysis. The data were analyzed by the Stanton
Glantz stat istical soft ware 3 (MS-DOS) and Graph Pad Prism soft-
ware version 4.00 for Windows (Graph Pad software). Differences
between the groups were determined using the one-way analysis
of variance (ANOVA) and the Student-Newman-Keuls test. Only
p values < 0.05 were considered significant. Disease free interval
(DFI) and overall survival (OS) were calculated from the date of
for the presence and absence of expression, we used
a specific model formally known as the Martingale
residuals. In this way we have established a cut-off
point at 5%: the cells were considered positive when
≥ 5% of them showed a cytoplasm staining, and
negative when no staining was observed or < 5%
of cells stained for the marker. Inter-rate reliability
between the two investigators blindly and indepen-
dently examining the immunostained sections was
assessed by the Cohen’s K-test, yielding K values
higher than 0.70 in almost all instances.
Tissue microarray based FISH. The interphase
FISH was performed on representative sections of
Prognostic OSCC TMA. The sections were cut
onto positively charged slides at 5 μm thickness.
Deparaffinization of sections was carried out with
two 10 m immersion in bio-clear, followed by three
3 m immersion in ethanol 100, 70 and 50%. The
slides were rinsed in distilled water by immersing
the slides in citrate buffer (pH 6) for 15 m at 90°C.
The slides were then rinsed in distilled water for
5 m twice. The slides pre-treatment and protease
incubation were performed according the manu-
factures illustrated in datasheet of Vyses (paraffin
pre-treatment reagent kit II). The used probes were
the commercial LSI EGFR Dual-Color Probe-Hyb
Set (Vysis/Abbott Molecular) LSI EGFR Spectrum
Orange/Cep-7 Spectrum Green in order to simul-
taneously visualize EGFR gene and chromo-
some 7 copy number according to manufacturer’s
instructions. DAPI II (4,6-diamino-2-phenyin-
dole-2-hydrochloride) was used for chromatin coun-
terstaining. The fluorescence signals (orange for LSI
EGFR, green for Cep-7 and blue for nuclear chro-
matin) were evaluated under epifluorescence micro-
scope (Olympus). Image acquisition was done by
CCD microscopy camera (Olympus). Signals were
evaluated by two independent evaluators (RF, GB)
scoring at least 100 interphase nuclei in four dif-
ferent high power fields (HPF). The FISH results
were scored as follow: specimens with the ratio LSI
EGFR/CEP-7 ≥ 2.0 were considered as amplified;
polysomic were considered cases showing three or
more CEP-7 signals per cell in more than 30% of
the evaluated cells.31,32
Tissue microarray based western blotting.
Cancer cells and normal epithelial tissues have been
microdissected from the donor blocks punching two
cores of 1 mm each from the same fields selected for
IHC and FISH with the help of H&E stained slides. Expression
levels of EGFR, pEGFR-Tyr 1068 and pEGFR-Tyr 845 proteins
were determined by immunoblotting,33 using anti pEGFR-Tyr
1068 and pEGFR-Tyr 845 antibodies. Representative cases of
normal epithelia together with trisomic and amplified EGFR
OSCC have been selected for this analysis. All the donor cores
were dewaxed in xylene.
Figure 6. Kaplan-Meier curves of patients positive forp-Tyr-845 EGFR expression sub-
jected to adjuvant chemotherapy (A) or with early stage tumours (B). Among patients
receiving adjuvant chemotherapy p-Tyr 845 negative cases had a better sur vival (p <
0.05). Among T1-T2 tumors p-Tyr 845 negative cases had a better survival (p < 0.05).
See text for details.

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10 Cancer Biology & Therapy Volume 13 Issue 11
Acknowledgments
First of all, we thank all patients or their relatives for their volun-
tary participation in this study. Then, a particular thanks to the
financial support by Fondazione Banca del Monte Foggia-Italy
and Associazione Italiana Ricerca sul Cancro (AIRC). Michele
Caraglia received a grant from MIUR PRIN 2009 and FIRB
Programma quadro 2011.
pathological diagnosis to the date of recurrence or death or the last
event. Estimates of the survival probability were obtained using
Kaplan-Meier method, by Chi-square test and reporting values of
the Hazard Ratio, at 95% CI and the median survival in months.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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