Squamous cell carcinoma (SCC) of the oral cavity is one of the most frequent cancers
in the world (Vokes et al, 1993). This disease occurs much more frequently in males
(Johnson, 1991). Epidemiologic studies show a strong association between its incidence
and environmental carcinogens, especially the use of tobacco, alcohol, and betel quid
(Franceschi et al, 1990; Ko et al, 1995; Hsieh et al, 2001). In Taiwan, the incidence
of oral carcinoma is one of the highest in the world. The incidence of oral cavity
cancer was 20 per million in our male population, comprising approximately 4–5% of
all malignancies (Chen, 1987). Approximately 85% of all oral cavity cancer patients
habitually use betel quid (Chong, 1966). The majority of oral carcinoma in Taiwan
occurs in buccal mucosa (ICD145), which was relatively less common in Western populations
(Daftary et al, 1991). Such geographical differences in incidence and cancer sites
may result from exposure to different carcinogens, and possibly also from genetic
predisposition.
The overall 5-year survival rate for patients with oral carcinoma is among the lowest
of the major cancers and has not changed during the past two decades (Parker et al,
1996). The standard treatment for patients with this cancer is surgery, radiation,
or multiple modalities for patients at high risk (Clayman et al, 1996). Although standard
care is frequently initially successful in early-stage cancer (stage I or II), disease
relapses still occur in about 20–30%, particularly local tumour or lymph node recurrence
(Vikram, 1994; Clayman et al, 1996). For patients with advanced oral carcinoma (stage
III or IV), standard therapy is far less successful. The recurrent rate in advanced
stage is approximately 50–60% and distant metastasis is 20–35% (Clayman et al, 1996).
Even if there is a good treatment response, patients with advanced disease often suffer
substantial functional and cosmetic morbidity, which decreases the quality of life.
The identification of prognostic factors that may affect disease outcome may lead
to improvements in adjuvant systemic therapy and better control of the disease.
The tyrosine kinase receptor, epidermal growth receptor (EGFR) family proteins EGFR
and Her-2 have been reported to be overexpressed in many cancers. They are often associated
with a poor prognosis, suggesting that they are potential molecular targets for anticancer
therapy. This family of proteins consists of four closely related transmembrane receptors,
including EGFR (erbB1), HER-2 (erbB2), HER-3 (erbB3), and HER-4 (erbB-4) (Olayioye
et al, 2000; Simon, 2000). Several ligands, such as EGF and amphiregulin, bind to
EGFR, whereas there is no known high-affinity ligand binding to Her-2. However, both
EGFR and Her-2 interact with other members of the family by heterodimerisation, resulting
in activation of their intrinsic kinase activity (Olayioye et al, 2000). Overexpression
of EGFR and Her-2 have been reported to be associated with higher grades or reduced
survival in a variety of cancers, including breast, colorectal, and head and neck
cancers (for a review, see Klijn et al, 1992; Salomon et al, 1995). Several molecular
therapeutic agents against EGFR or Her-2, such as Cetuximab and Herceptin, have been
studied in clinical trials (Colomer et al, 2001; Robert et al, 2002).
Although several studies have found overexpression of EGFR and Her-2 in head and neck
cancers, the clinical relevance of the finding varies. For example, Storkel et al
(1993) found that overexpression of EGFR was associated with shortened survival, but
Werkmeister et al (2000) reported that Her-2 was strongly associated with survival.
Christensen et al (1995) and Khan et al, (2002) could not find significant correlation
of either EGFR or Her-2 with clinicopathological features or prognosis; however, Bei
et al (2001) and Xia et al (1999) found colocalisation of both molecules in oral cancer
tissues and the combined use of these molecules is a stronger predictor for the cancer
prognosis. We therefore designed this study to investigate EGFR and Her-2 in paired
grossly normal and cancerous tissues from squamous cell carcinoma of oral cavity patients.
Our aims were to determine their levels of expression and see if these levels correlated
with clinicopathological variables, and if they were useful prognostically.
MATERIALS AND METHODS
Patients, tissues and cells
Fifty-nine consecutive patients seen in 1999 at the Otorhinolaryngology or Head and
Neck Surgery clinics at Chang Gung Memorial Hospital (Taoyuan, Taiwan) were enrolled
for the study. Written informed consent was obtained from all patients participating
in this study. A questionnaire was filled out by each patient before the first clinical
visit investigating whether or not the patient was a habitual betel net chewer (daily
chewer), cigarette smoker (daily smoker), and/or regular alcohol drinker (daily drinker).
The standard treatment was radical surgery for early-stage patients and adjuvant radiotherapy
for patients with intermediate risk, such as a close margin or lymph node metastases.
Concomitant chemoradiotherapy was given in patients with lymph node metastases and
extracapsular spread (ECS). All cancers were histologically graded as well differentiated,
moderately differentiated, or poorly differentiated, according to the World Health
Organization (WHO) classification (Shanmugaratnam, 1991). For each sample, the presence
of bone or nerve invasion, lymphatics, blood vessels, tumour depth, and the presence
or absence of lymph node ECS were specifically recorded. Tumour pathological staging
was classified according to the AJCC system (Fleming et al, 1998). Biopsies of cancerous
and grossly normal mucosa tissue were obtained from each subject before chemo- or
radiotherapy. A portion of each tissue sample was stored in liquid nitrogen until
use for molecular assay. An oral cancer cell line OC2 (Wong et al, 1990) was used
as a positive control. OC2 cells were grown at 37oC, 5% CO2 in RPMI medium containing
10% fetal bovine serum and antibiotics (100 U ml−1 penicillin, 100 Uml−1 streptomycin,
and 0.25 μg ml−1 amphotericin B).
Extraction of cellular proteins
The investigators were blinded as to the source and type of tissue being assayed.
Tissue samples (∼50 mg) were homogenised in 300 μl of a lysis buffer (10 mM Tris-HCI,
pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS (Pharmacia Ontario, Canada), 10% glycerol,
5 mM mercaptoethanol, and 0.1 mM phenylmethylsulphonyl fluoride (PMSF) in Kontes tubes
with matching pestles rotated at 450 r.p.m. After 30 min at 4°C, the lysate was centrifuged
at 15 000 r.p.m. for 30 min at 4°C. The supernatants of the protein extracts were
used for the EGFR and Her-2 ELISA assay. The protein concentration of each tissue
sample was determined using Coomassie protein assay reagent (Pierce).
Determination of EGFR and Her-2 protein levels
An enzyme-linked immunosorbent assay (ELISA) was used to detect tissue EGFR and Her-2
protein expression. The ELISA kits were purchased from CalBiochem Inc. (CA, USA).
A total of 10 μg cellular protein was used in each assay, performed according to the
manufacturer's protocol. All samples were analysed in duplicate and the average of
the two was recorded. To define the relative expression of EGFR or Her-2, both cancer
tissue and the normal counterpart samples were assayed. The cutoff value for gene
overexpression was defined as the level of mean plus two s.d. in the normal tissue
expression values, and was designated as one-fold of overexpression.
Statistical analysis
The Pearson χ
2 test was used to look for the association between EGFR or Her-2 expression and clinicopathological
parameters, including tumour extent (T, N, and overall stage) and the pathological
findings (degree of differentiation, tumour depth, or ECS). For prognostic factors
analysis, the Kaplan–Meier method was used for single-variant analysis and the Cox
logistic regression model was used for multivariant analysis. All P-values presented
were two-sided, and the significance level was set at <0.05.
RESULTS
Patient characteristics
The patient characteristics are summarised in Table 1
Table 1
Characteristics of patients with oral cancer
Characteristic
Number
Percentage (%)
Total
59
100
Sex
Female
0
0
Male
59
100
Age
⩽40 years
13
22
41–60 years
31
53
>60 years
15
25
Habits
Alcohol drinking
35
59
Smoking
53
90
Betel quid chewing
53
90
Cancer site
Buccal mucosa
24
41
Tongue
18
31
Others
17
29
Cancer histological grade
Well differentiated
20
34
Moderately differentiated
33
56
Poorly differentiated
6
10
. Their median age was 48.0 years, ranging from 31 to 78, and all were male. A total
of 59% of the patients consumed alcohol, 90% smoked tobacco, and 90% chewed betel
quid. Cancers included 24 (41%) in the buccal mucosa, 18 (31%) in the tongue, and
17 (29%) in other sites. All cancers were SCC, with 20 (34%) well differentiated,
33 (56%) moderately differentiated, and six (10%) poorly differentiated. The disease
staging is summarised in Table 2
Table 2
Tumour and node stage distribution
Stage
T1
T2
T3
T4
Total
N0
7
15
4
11
37
N1
0
2
0
3
5
N2
0
7
5
5
17
Total
7
24
9
19
59
. Of the 59 patients, seven (12%) had stage I disease (T1N0), 15 (25%) had stage II
(T2N0), six (10%) had stage III (T3N0, T2N1), and had 31 (53%) stage IV (T4N0, T4N1,
T2N2, T3N2, T4N2).
The distribution of EGFR and Her-2 in oral cancer tissues
The relative expression of EGFR and Her-2 in all 59 patients were examined and plotted
in Figure 1
Figure 1
Relative expression of Her-2 and EGFR. (A) Relative expression of EGFR (A) and Her-2
(B) in normal and cancer tissue samples. The horizontal bar in the figure indicates
the cut-off value (designated as one-fold)
. Similar results were obtained when we assayed EGFR and Her-2 levels using different
sites of the same tumour. As shown in the Figures, EGFR was overexpressed in two (3%)
normal mucosa tissues and 34 (58%) cancer tissues. Similarly, Her-2 was overexpressed
in one (2%) normal tissue and 24 (41%) cancer tissues. Compared to all normal samples,
the average expression of EGFR in cancer tissue was 3.48-fold, with an s.d. of 2.01,
while the average expression of Her-2 was 1.51-fold, with an s.d. of 0.45. Thus, although
the levels of both EGFR and Her-2 expression differed significantly between normal
and cancer tissues (P<0.001), EGFR had greater overexpression than Her-2 on average
in all the cancer patients. The distribution of EGFR and Her-2 expression in the 59
oral cancer tissues is summarised in Table 3
Table 3
EGFR and Her-2 overexpression in oral cancer tissues
EGFR overexpression
Her-2 overexpression
No
Yes
Total
No
17
18
35
Yes
8
16
24
Total
25
34
59
. Of the patients, 17 (29%) had normally expressed in both molecules, 16 (27%) had
overexpressed in both molecules, and 26 (44%) had overexpressed in either molecule.
EGFR and Her-2 were not significantly coexpressed (P=0.245).
Correlation of EGFR or Her-2 with clinicopathological parameters
The correlations of the expression levels of EGFR or Her-2 with clinicopathological
parameters are shown in Table 4
Table 4
Association of EGFR and Her-2 status with clinicopathological parameters
EGFR overexpression
Her-2 overexpression
Parameter
N
No(%)
Yes(%)
P-value
No(%)
Yes(%)
P-value
T stage
T1–T2
31
18 (58)
13(42)
0.010
17(55)
14 (45)
0.461
T3–T4
28
7 (25)
21(75)
18(64)
10(36)
Nstage
N=0
37
20 (54)
17 (46)
0.019
22 (60)
15 (41)
0.978
N>0
22
5 (23)
17(77)
13(59)
9 (41)
Overall stage
I–II
22
15 (68)
7 (32)
0.002
12 (55)
10 (46)
0.565
III–IV
37
10(27)
27(73)
23(62)
14(38)
Differentiation
Well
20
9 (45)
11 (55)
0.852
13 (65)
7 (35)
0.050
Moderate
33
13(39)
20(60)
16(49)
17(52)
Poor
6
3(50)
3(50)
6(100)
0 (0)
Tumour depth>10 mm
No
26
15 (58)
11 (42)
0.035
13 (50)
13 (50)
0.196
Yes
33
10(30)
23(70)
22(67)
11(33)
ECS of lymph node
No
43
22 (51)
21 (49)
0.025
25 (58)
18 (42)
0.762
Yes
16
3(19)
13(81)
10(66)
6(38)
Total
59
25(42)
34(58)
35(59)
24(41)
. As shown in the table, significant correlations were found between EGFR expression
and tumour extent (T stage) (P=0.010), lymph node status (N stage) (P=0.019), clinical
overall stage (P=0.002), tumour depth (P=0.035), and ECS of lymph node (P=0.025).
However, there was no association between Her-2 expression with cancer stage or any
other clinicopathological parameters. The results indicate that EGFR has an association
with the aggressiveness of oral cancer.
Evaluation of possible prognostic factors associated with oral cancer
As shown in Table 5
Table 5
Univariant analysis of prognostic factors in oral cancer
Parameter
Group
2-year survival (week)
P-value
T stage
T1-2/T3-4
73/22
0.101
N stage
N0-1/N2-3
80/22
<0.001
Overall stage
I–II/III–IV
86/51
0.008
Tumour depth(mm)
⩽10/>10
84/48
0.002
LN ECS
No/yes
79/19
<0.001
EGFR overexpression
(−)/(+)
88/45
0.001
Her-2 overexpression
(−)/(+)
64/62
0.928
, for the 2-year survival, there were significant correlations with N stage (P=0.000),
overall stage (P=0.008), tumour depth (P=0.002), ECS of lymph node (P=0.000), and
the expression levels of EGFR (P =0.001). To define the role of the above factors
further, multivariant analysis was conducted and has been demonstrated in Table 6
Table 6
Multivariant analysis of prognostic factors in oral cancer
Parameter
Risk ratio
95% CIa
P-value
T stage
0.781
0.22–2.36
0.583
N stage
4.215
1.21–14.74
0.024
Overall stage
0.601
0.009–4.16
0.606
Tumour depth
3.854
0.87–17.16
0.077
ECS of lymph node
1.083
0.52–3.07
0.601
EGFR overexpression
5.882
1.07–32.31
0.041
Her-2 overexpression
1.083
0.36–3.26
0.888
a
CI=confidence interval.
. Lymph node metastasis (P=0.024) and overexpression of EGFR (P=0.041) were the only
independent variables associated with poor survival, with a risk ratio of 4.22 for
lymph node metastasis (95% CI=1.21–14.74), and 5.88 for EGFR overexpression (95% CI=1.07–32.31).
The Kaplan–Meier overall survival curves related to EGFR overexpression are shown
in the Figure 2
Figure 2
Kaplan–Meier overall survival curves according to EGFR overexpression.
.
DISCUSSION
In our study, we found EGFR and Her-2 to be differentially expressed in oral SCC.
Consistent with other reports, we found that both EGFR and Her-2 were overexpressed
in a subset of oral cancer patients (58 and 41%, respectively). However, compared
to Her-2, EGFR overexpression was more significant in terms of the expression level
(3.5-fold vs 1.5-fold). Although these two molecules were coexpressed in some patients,
this was not a statistically significant association (P=0.245). A high level of EGFR,
but not of Her-2, was strongly associated with tumour aggressiveness and poor survival.
Although some of our findings are consistent with other reports, we noted above the
conflicting data produced by various authors. These differences may be due to differences
in assay techniques. For example, most of the investigators used the immunohistochemistry
method to examine protein expressions (Storkel et al, 1993; Xia et al, 1999; Bei et
al, 2001; Khan et al, 2002), while we used the ELISA method to quantitatively analyse
EGFR and Her-2 protein levels. The advantage of the immunohistochemistry method is
the precise localisation of the protein molecules in cells. However, this method reported
that data determined by microscopic examination may be influenced by the subjective
assessment through different individuals. Although the ELISA technique is less used
in clinical study, this method is also widely accepted to determine protein expression
levels. Examples include using this technique in the studies of breast cancer, cervical
cancer, lung cancer as well as head and neck cancer (Christensen et al, 1995; Pfeiffer
et al, 1998; Contreras et al, 2002; Widschwendter et al, 2002). The key advantage
of the ELISA method is to provide a quantitative result with relatively less bias.
When comparing these two methods, Pfeiffer et al (1998) have reported that significant
correlation was found in the quantification of EGFR or Her-2 levels between ELISA
and immunohistochemical methods studied in the lung and in breast cancers. These studies
suggest that comparable evaluation results may be obtained by using these two assay
techniques.
Patient sampling is another variable between other studies and ours. Most investigators
examine protein expressions between different tumour tissues (but no grossly normal
counterpart tissues to compare) (Storkel et al, 1993; Xia et al, 1999; Bei et al,
2001; Khan et al, 2002). We analyse EGFR and Her-2 protein levels in the paired grossly
normal mucosa and cancer tissues obtained from the same patients. These results will
provide clearer information regarding the protein level changes after cellular transformation.
Additionally, most other reports have evaluated populations in the West (Xia et al,
1999; Werkmeister et al, 2000; Bei et al, 2001), whereas ours focused on Southeast
Asians. Since both carcinogen exposure (including betel quid chewing) and possible
genetic predisposition vary between different geographic areas, the reported differences
in EGFR or Her-2 expression may reflect different oral carcinogenic pathways in different
populations.
In the upper aerodigestive tract, significant exposure of the mucosa surface to the
same carcinogens or stimulants (such as alcohol or cigarettes) may lead to a multitude
of somatic changes that are susceptible to the development of multiple primary cancers.
This phenomenon is called ‘field cancerisation’ (Slaughter et al, 1953). Recent molecular
studies have shown that genetical alterations could be found in different areas of
histological normal mucosa. Examples are mutations of the p53 tumour suppressor gene
and deletions on the short arm of chromosome 3 (Li et al, 1994; Van Dyke et al, 1994).
In our grossly normal mucosa tissues, which provide an internal control for comparing
them with cancer tissues in the same patient, they may not represent true normal samples,
particularly in patients who drink, smoke, and chew betel quids. However, in our present
study, there is no statistical difference of the protein expression levels of EGFR
and Her-2 between the normal tissues from patients with or without exposure to tobacco,
alcohol, betel quid, or the combined exposure (data not shown). These results suggest
that the ‘filed’ effect of EGFR and Her-2 molecules on the surrounding oral normal
mucosa is minimum. Apparently, together with multiple injuries and cellular genetic
mutations on a specific tissue, a process described as ‘multistep carcinogenesis’
will eventually transform the cell into malignant cancer (Vogelstein et al, 1998).
For a molecule to be a good candidate as a target for anticancer therapy, several
criteria must be fulfilled. First, the protein should be overexpressed in cancer tissues
compared to normal tissues. Second, overexpression of the protein should be associated
with a poor prognosis, which suggests that manipulation of the protein may result
in alteration of the prognosis. In this study, we found that the membrane protein
EGFR had both these characteristics. Our results do not support similar targeting
of the Her-2 protein, even though it is commonly overexpressed in oral SCC. Recently,
targeting of EGFR as a molecular adjuvant therapy has been clinically tried in head
and neck cancer (Shin et al, 2001; Robert et al, 2002). This study provides a fundamental
knowledge base, suggesting that targeting this molecule might be useful in betel quid-associated
oral cancers (Shin et al, 2001).