Overexpression of Her1 (EGFR) in Gastric Cancer: A Saudi Regional Population Based Study

doi:10.4236/ojpathology.2012.24022

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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.

Email: #[email protected], dalaltam@ yahoo.com

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)

(

)(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)

(

(a) (f)

Figure 3. Gastric carcinomas with panel of IHC. (a) Diffuse

gastric cancer ×40 H & E; (b) EGFR ×20 2+ (Cyto + memb);

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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