Open Journal of Pathology, 2012, 2, 120-126
Published Online October 2012 (http://www.SciRP.org/journal/ojpathology)
http://dx.doi.org/10.4236/ojpathology.2012.24022
Overexpression of Her1 (EGFR) in Gastric Cancer: A
Saudi Regional Population Based Study*
Ayesha Ahmed1#, Dalal M. Al Tamimi1,2
1Anatomic Pa t h ology, Universi ty of Dammam, Damma m, KSA; 2King Fahd Hospital of the University, Khobar, KSA.
Received June 26th, 2012; revised July 30th, 2012; accepted August 14th, 2012
ABSTRACT
Background: Gastric cancer is one of the commonest malignant tumor worldwide. Its treatment remains a challenge for
physicians. Epidermal growth factor receptor (EGFR) inhibitors have played a significant role in the management of
solid malignancies including colorectal cancer. In this study we aimed to determine EGFR expression in gastric ad eno-
carcinoma by standardized immunohistochemistry in a Saudi regional population based cohort and also to evaluate
Ki-67 proliferating index and p-53 mutation status. Materials and Methods: Gastric carcinoma (GC) cases comprising
surgical resection specimens an d endoscopic biopsies, were selected, from the patho logy archives of King Fahd Hospi-
tal of the University of Dammam (KFHU), spanning a time period of 6 years. The histological GC type was delineated
according to Laurens classification and immunohistochemical (IHC) protein analysis for EGFR, Ki-67 and p-53 was
carried out. Results: 42 cases of gastric GC were analyzed and EGFR overexpression was demonstrated in 4.76% of
cases. Out of these 2.38% had membranous and the remaining demonstrated predominantly cytoplasmic along with
focal membranous positivity. Ki-67 proliferatio n index ranged from moderate to high and and p-53 mutation statu s was
negative in these cases. Conclusion: Low EGFR expressivity could be reflective of regional variation in cancer charac-
teristics. The study also highlig hts the inadequacy of the currently employed gastric EGFR interpretation criterion s and
stresses on development of standardized and uniform EGFR evaluation protocols tailored for gastric needs.
Keywords: Gastric Cancer; EGFR Overexpression; Immunocytochemistry; Protein Analysis
1. Introduction
Gastric cancer is one of the commonest malignant tumor
worldwide and [1-3]. It ranks fourth amongst the most
commonly diagnosed cancer, with more than 1,300,000
cases diagnosed yearly [4,5]. The estimated incidence of
gastric cancer in the United States was 21,500 in 2008 [6 ]
and a cumulative mortality of esophageal and gastric
cancer being approximately 1,100,000 [5], which em-
phasizes the global challenge in dealing with these dis-
eases. This cancer epitomizes the concept of geographi-
cal variation in prevalence distribution with an incid ence
wise changing and shifting epidemiological trends, and
currently East Asia makes up for a significant proportion
of new cases [7]. Japan ranks the highest with increasing
incidence also seen in many develop ing countries in Asia,
Africa and Latin America [8].
Gastric cancer treatment remains a challenge for phy-
sicians [9]. Advanced gastric cancer is associated with
poor prognosis, with the resultant, post diagnostic mean
survival rate being approximately 10 to 11 months [4].
Targeted therapies based on the evaluation and analysis
of the status of target genes [10,11] have been the recent
addition in gastric cancer treatment. Molecular therapies
constitute monoclonal antibodies or small molecule in-
hibitors targeting either growth factors or growth factor
receptor kinases. Anti-HER2 humanized monoclonal
antibody Trastuzumab has been proved to improve pa-
tients prognostics in HER2-positive gastric carcinoma
[12]. Epidermal growth factor receptor (EGFR) inhibitors
have played a significant role in the management of solid
malignancies including colorectal cancer. Currently, there
are four EGFR inhibitors approved by the FDA including
two small molecule tyrosine kinase inhibitors (erlotinib
and gefitinib) and two monoclonal antibodies (cetuximab
and panitumumab) [13-17]. In gastric carcinoma EGFR
is a new field, unraveling new therapeutic options for
patients. Recapitulating the pattern of drug-diagnostic
Herceptin/HercepTest, patients trialed and evaluated for
anti EGFR drugs need to fulfill the prerequisite of IHC
evidence of EGFR positive expression. This not only
augments the pathologist role in therapy selection but
also stresses on their responsibility of providing accurate
and reproducible EGFR analysis results [18]. St an da rd iz a-
*We declare that we have no financial or non-financial competing interests.
#Corresponding author.

Overexpression of Her1 (EGFR) in Gastric Cancer: A Saudi Regional Population Based Study 121
tion of EGFR immunoreactivity expression criteria in
gastric cancer is undergoing trials and tribulations and
requires to be tailored precisely for the endogenous gas-
tric needs as has been accomplished for Her-2 in gastric
cancer.
In our study we aimed to determine EGFR expression
in gastric adenocarcinoma by standardized immunohis-
tochemistry in a Saudi regional population based cohort
and simultaneous evaluation of Ki-67 proliferating index
and p-53 mutation status.
2. Materials and Methods
2.1. Specimen Selection and Clinicopathological
Parameters
Gastric carcinoma cases comprising surgical resection
specimens and endoscopic biopsies, were selected, from
the pathology archives of King Fahd Hospital of the
University (KFHU), spanning a time period of 6 years,
under the approved protocols of the research and ethical
committee of University of Dammam. Patient consent
was waived due to loss of follow up for old cases. Only
gastric adenocarcinomas with availability of representa-
tive blocks and sufficient tissue material to perform the
required histopatholgical procedures were selected. Gas-
tric neoplasia besides adenocarcinoma, metastatic cancer
of the stomach from another organ and metastatic tumor
of gastric origin without concomitant histological mat eria l
from primary tumor in the stomach were excluded from
the study. The current study comprises 42 cases of ga stric
adenocarcinoma
2.2. Histological Classification of Gastric Cancer
The histological GC type was delineated according to
Laurens classification, this classification divides adeno-
carcinomas into two types: intestinal (consisting of well
formed tubules) and diffuse (diffuse tumor infiltration
without well formed tubules, frequently with signet ring
cells) [19].
2.3. Immunohistochemistry
Immunohistochemical staining using the labeled strepta-
vidin-biotin (LSAB) method with 3,3’-diaminobenzidine
(DAB) as a chromagen was performed for EGFR
(HER1), p-53 and Ki-67 on 4 µm thick paraffin sections
cut from conventional blocks. The staining was per-
formed concurrently in a Ventana Benchmark automated
immunostainer according to the manufacturer’s instruct-
tions (Ventana Medical Systems Inc., Strasbourg).
Sources and dilutions of the primary antibodies used in
the study are listed in Table 1. The immunostained sec-
tions were examined under a light microscope and evalu-
ated manually by 2 pathologists (AA and DT). Any in
Table 1. Sources and dilutions of primary antibodies used
in the study.
Antibody Clone Manufacturer Dilution
EGFR 5B7 Ventana Prediluted
Ki-67 MIB-1 Dako Prediluted
p-53 DO-7 Dako Prediluted
EGFR = epidermal growth factor receptor.
terpretational discrepancies were resolved under a dou-
ble-headed microscope.
2.4. Evaluation of Immunostaining
EGFR: Both membranous and cytoplasmic staining
were considered for evaluation. The membranous posi-
tivity for EGFR (mEGFR) was evaluated in the follow-
ing manner: 0, no discernible staining or background
type staining; 1+, equivocal discontinuous membrane
staining; 2+, unequivocal membrane staining with mod-
erate intensity; and 3+, strong and complete plasma
membrane staining. More than 10% of the cells were
required to meet the criteria for EGFR analysis. Scores of
2+ and 3+ staining levels were considered to be EGFR
overexpression [9]. For positive cytoplasmic EGFR
(cEGFR) staining, an intense homogenous staining of the
cytoplasm was necessary. There was no cEGFR without
membrane stainin [20].
Ki-67: Positive staining was defined as positive nu-
clear staining. Cytoplasmic staining was considered ne-
gative. The percentage of positive nuclei was expressed
as a “Ki-67 labeling index” which is the percent of cells
expressing Ki-67 determined by counting 1000 cell s/s li de .
The percentage of positive cells was scored as follows:
less than 10% = low proliferative activity, 10% - 40% =
moderate proliferative activity, and more than 40% =
high proliferative activity [21].
P53: Positive staining was defined as positive nuclear
staining. Cytoplasmic staining was considered negative.
Tumors were considered focally positive when unequivo-
cal staining was present in 10% - 50% of tumor cells, and
as diffusely positive when more than 50% of the tumor
cells were positive [22].
2.5. Statistical Analysis
Data was entered into SPSS windows. Frequencies were
calculated using descriptive statistics for categ orical v ari-
ables.
3. Results
Out of a total of 42 cases of gastric cancer retrieved, 38
specimens were biopsies and 4 were partial gastrectomies.
Male to female ratio was 25:9 and median age of the p a-

Overexpression of Her1 (EGFR) in Gastric Cancer: A Saudi Regional Population Based Study 122
tients was 67 years (extreme 88 - 42 years). Diffuse type
gastric cancer was seen in 23 (54.76%) and Intestinal
type in 19 (45.23%) of cases. EGFR expression pattern is
shown in Table 2 and Figure 1. Ki-67 proliferation in-
dex and p-53 mutation status is given in Tables 3 and 4
respectively. In cases showing EGFR overexpression (n
= 2) p-53 mutation status was negative and Ki-67 ranged
from moderate to low.
The Figure 2 shows H & E staining in gastric carci-
noma cases while Figure 3 shows the respective immu-
nohistochemical panels.
4. Discussion
We demonstrated a low EGFR overexpression by pro-
tein analysis, comprising a mere 4.76% of 2+ intensity
and none with 3+ immunoreactivity. Out of these 2.38%
had membranous and the remaining demonstrated pre-
dominantly cytoplasmic along with focal membranous
positivity. This low positivity and pattern of expression
arouses several discussable issues. Firstly whether this
expressivity could be part of the concept of ethnic varia-
Table 2. EGFR overexpression in gastric cancer (n = 42).
Staining intensity N % age
0 34 80.95
1+ 6 14.28
2+ 2 4.76
3+ 0 0
EGFR
1+(negative)
2+(moderate lypositi ve )
3+(strongl ypos itive)
Figure 1. EGFR overexpression in gastric cancer (n = 42).
Table 3. Ki-67 Proliferation Index in gastric cancer (n = 42).
Proliferative activity N % age
Low 6 14.28
Moderate 14 33.33
High 22 52.38
Table 4. p-53 mutation status in gastric cancer (n = 42).
p-53 status N % age
Negative 22 52.38
Focal 10 23.80
Diffuse 10 23.80
tion in cancer distribution or could it be merely reflective
of the marked variations in the intrinsic protein accentu-
ating potential by various commercially available kits.
Also they grey zone of moderate cytoplasmic positivity
needs to be stressed on as regards whether these tumors
be trialed for further genetic evaluation and selected for
anti-EGFR therapy, the sensitivity and long term prog-
nostic benefits of which are currently being debated.
Both of the cases were endoscopic biopsies and 10% of
tumor showing positivity was the cut off point. Is this
criterion applicable only for the gastrectomy specimens
or needs to be downgraded as in Her-2neu positivity,
where presence of just 5 positive cells now justifies in-
stitution of targeted therapy in small biopsies?
Regarding tumor related ethnic variation a tremendous
heterogeneity is seen in terms of epidemiology, tumor
histology and expression of molecular markers in gastric
cancer [23]. EGFR overexpression has been documented
to show marked ethnic variations. Mammano et al. de-
tected EGFR protein expression in only 6% of the cases,
and concluded in their study that EGFR protein expres-
sion is low and specific EGFR gene mutations are very
rare or absent in gastric adenocarcinoma [24]. A some
what similar expression pattern is also reported by Take-
hana et al., who, in their series of 413 gastric carcinomas,
found negative EGFR protein expression in 89.6%, low
level in 8.2% and high level in 2.2% of cases [25], Lee et
al., who found no EGFR gene mutations in 185 gastric
adenocarcinomas in a series of Korean patients [26], and
by Mimori et al., who found a silent mutation in exon 20
in only 5 of 39 (5.1%) Japanese patients [27]. These
studies are in agreement with our study that also shows a
very low EGFR expressivity but in contrast with multiple
studies documenting a much higher prevalence pattern.
(a) (c)
(
b
)(d)
Figure 2. Gastric Carcinoma H & E. (a) Diffuse gastric
carcinoma H & E ×20; (b) Diffuse gastric carcinoma H & E
×40; (c) Intestinal type gastric carcinoma H & E ×20; (d)
Intestinal type gastric carcinoma H & E ×40.

Overexpression of Her1 (EGFR) in Gastric Cancer: A Saudi Regional Population Based Study 123
(d (i)
(e) (j)
(
b
(g
(c) (h)
(a) (f)
Figure 3. Gastric carcinomas with panel of IHC. (a) Diffuse
gastric cancer ×40 H & E; (b) EGFR ×20 2+ (Cyto + memb);
(c) EGFR ×40; (d) Ki-67 moderate; (e) p-53 negative; (f)
Intestinal Type gastric cancer ×40 H & E; (g) EGFR ×20 2+
(cyto); (h) EGFR ×40; (i) Ki-67 high; (j) p-53 negative.
Liang et al. documented 41% of EGFR expression by
IHC in a subset of Chinese patients; with 16% also re-
vealing FISH positivity [9 ]. In a study by Galizia et al. in
an Italian population with an aim to correlate EGFR
positivity by IHC with disease recurrence and su rvival in
univariate and multivariate analyses, forty-four percent
(36 cases) of gastric cancers were EGFR positive [28].
These multiple studies high light the concept that specific
EGFR mutations show a wide regional and ethnic varia-
tion in gastric adenocarcinoma, a fact substantiated in
other tumors also, as demonstrated by Paez et al., who
found a significant difference between the mutation rate
of EGFR gene in non small cell lung carcinoma(NSCLC)
from USA (2%) and from Japan (26%) [29].
The low EGFR expressivity pattern may also be at-
tributed the type of kit employed for immunostaining. In
a comparative study conducted by Lee et al. in NSCLC
different intensities of staining were discerned by differ-
ent commercially available kits. EGFR protein overex-
pression was observed in 56% of tumors with Zymed
EGFR kit, in 51% with Dako EGFR pharmDx kit, in 5%
with Dako and in 18% with Novocastra. Both Zymed and
Dako pharmDx kit were more sensitive than the Dako
test (clone H11) and Novocastra clone EGFR 113. They
concluded that EGFR protein overexpression rate varied
from 4% to 72% according to different antibody clones
and histologic subtypes, and EGFR protein expression
detected by Zymed and Dako pharmDx was signif icantly
associated with a high EGFR gene copy number [30].
We had employed Ventanna kit for our study. Our pat-
tern of expression could be attributed to the different
intrinsic sensitivity of the protein highlighting potential
of the kit. More extensive comparative studies regarding
standardization and harmonization of the different meth-
odologies employed for EGFR protein expression, need
to be carried out so such marked variations, if existent
with our logistics, may not get manifested.
Regarding membranous pattern of immunoreactivity,
in our study 2.38% of cases had a heterogenous pattern
of membranous staining, with mostly 2+ pattern but with
scattered individual cells showing intense complete
membranous 3+ pattern of expression. These cells how-
ever did not fulfil the 10% criteria to accord 3+ to the
case as a whole. The EGFR IHC interpretation criteria in
gastric cancer is essentially the same as in breast malign-
nancies. Does it need to be questioned, modified and
downgraded as in Her-2 neu testing in gastric cancer
where after validation in TOGA trial [31], the criterion is
less strict and 10% cut-off rule applies to resection
specimen whereas in biopsies any group of at least 5 tu-
mour cells showing immunoreactivity satisfies the posi-
tivity standard and in addition [31] the completeness of
membrane staining is not a “condition sine qua non” [12].
In EGFR IHC interpreting protocol, no such downgrda-
tion and revision has been implemented. The intratu-
moral heterogeniety, more significant in small endo-
scopic biopsies mandates modification of expressivity

Overexpression of Her1 (EGFR) in Gastric Cancer: A Saudi Regional Population Based Study 124
criterions and need them to be divergent from those ac-
cepted and validated for breast cancers.
As far as cytoplasmic pattern of expression is con-
cerned, 2.38% of cases in our study revealed, diffuse
moderate cytoplasmic staining with focal membranous
accentuation. Although marked, diffuse cytoplasmic st ai n-
ing has been considered by some studies [20] but no well
defined protocols have been delineated and inclusion of
these cases for further genetic evaluation and incyto-
plasmic positivity needs to be standardized and vali-
dated as recent studies have proved EGFR to act as a
cytoplasmic/nuclear shuttling transcription factor [32]
with its activation and subsequent nuclear translocaliza-
tion, leading to regulation of gene expression and media-
tion of specific cellular p rocesses [33,34]. This is d istinct
from EGFR mediated transduction of mitogenic signals
through activating multiple signaling cascades [35].
Could tracking into this cytoplasmic positivity unmask a
potential valid anti EGFR th erapy candidate, yet need s to
be ascertained. EGFR inspite of being a promising target
in cancer therapy, yet failed to acquire definitive, sensi-
tive potential accurate molecular predictors of sensitivity
to EGFR inhibitors for patients having gastro-esophageal
cancers [36]. The dichotomous cytoplasmic and mem-
branous staining in other tumors holds prognostic sig-
nificance as reported in renal cell carcinoma [37] with its
association with worsening progression and prognosis
[38]. In gastric cancer, however, such confirmation yet
remains to be done.
Ki-67 proliferation index in our cases with EGFR im-
munoreactivity ranged from moderate to high, implicat-
ing these tumors to be aggressive. EGFR expression cor-
relates with disease recurrence and poorer survival [28].
This is substantiated by other studies as well in which
EGFR overexpression is associated with an aggressive
tumor and also with lymph node metastasis [39]. EGFR
dimerization and activation leads to downstream upregu-
lation of multiple processes that can result in cancer cell
proliferation, reduced apoptosis, tumor-induced angio-
genesis, and activation of invasion, dedifferentiation of
cancer cells and enhanced metastatic potential [40,41].
The role of p-53 as a prognostic factor in gastric can-
cer is controversial. One school of thought describes ex-
pression of EGFR and p-53 to have a negative correlation
with patients prognostics [42-44]. In a study on an arab
population the expression of p53 was found to correlate
with aggressive gastric cancer characteristics [45]. On
the other hand, studies have reported p53 to have no in-
fluence on prognosis [46 ,47 ]. Liu et al. demonstrated p53
nuclear reactivity in association with bax and c-myc and
with well-differentiated histology and with no prognostic
significance [48]. In our study the o verall p-53 reactivity
was found to be in 47.60% of cases with diffuse positiv-
ity in 23.80% and focal positivity also in 23.52% but in
both the EGFR expressing cases, it was non reactive. The
cases comprised equal percentage of diffuse and intesti-
nal types. Multiple genetic and epigenetic alterations
have been found to underlie gastric carcinogenesis with
different combinations becoming apparent in the two
histological types of gastric cancer. p-53 mutation, re-
duced p27 expression, cyclin E expression etc in the in-
testinal-type and LOH at chromosome 17p, mutation of
p53 and mutation or loss of E-cadherin to name some in
the poorly differentiated gastric cancers [49]. Although
p-53 mutation is seen to formulate an integral overlap-
ping component in both the types the lack of expression
in our cases could imply other molecular alterations be-
sides p-53 mutation to be more operative.
5. Conclusion
Low EGFR expressivity could be reflective of regional
variation in cancer characteristics. The study also high-
lights the inadequacy of the currently employed gastric
EGFR interpretation criterions and stresses on develop-
ment of standardized and uniform EGFR evaluation pro-
tocols tailored for gastric needs.
6. Acknowledgements
We acknowledge university of Dammam for providing
financial and other logistic support for carrying out the
research work. We also acknowledge services of Mrs
Khalda Al Johy, Mrs Zainab Al Najar, Mr Shakir Ahmad
and Mrs Maria Rosario Lazaro in conducting the labora-
tory work .
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