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Romanian Journal of Morphology and Embryology 2009, 50(2):217–221
ORIGINAL PAPER
Immunohistochemical expression and
significance of epidermal growth factor
receptor (EGFR) in breast cancer
ADRIANA MECHE1), ANCA MARIA CÎMPEAN2), M. RAICA2)
1)
Department of Pathology, County Hospital of Arad
2)
Department of Histology and Molecular Pathology,
“Victor Babeş” University of Medicine and Pharmacy, Timisoara
Abstract
Breast cancer is a heterogeneous disease that includes several molecular types, characterized by the expression profile of sex hormone
receptors, HER2 protein, cytokeratin 5, p53, and Bcl-2. EGFR is an additional marker predominantly expressed by basal-like carcinoma,
but its significance in the other types is not completely understood. The aim of this study was to analyze the immunohistochemical
expression of EGFR and its relationships with other factors of prognosis. There were investigated benign lesions and 84 cases with
invasive breast carcinoma that were submitted first to the molecular classification. Next, we performed the staining for EGFR and two
patterns of the final product of reaction were described. EGFR expression was found in 41.66% of the cases with basal-like carcinoma, in
50% of the cases with luminal B carcinoma, and in 21.42% of the cases with HER2 overexpression. A significant correlation was found
between EGFR expression and degree of differentiation and distant metastasis. No significant correlation was found with the lymph node
status, excepting for the basal-like carcinoma in which an inverse correlation was noticed. Our results suggest that EGFR expression by
tumor cells of the breast cancer defines a specific subset of tumors with poor prognosis and potential resistance to the adjuvant therapy.
Keywords: breast cancer, molecular classification, epidermal growth factor receptor (EGFR), immunohistochemistry,
prognosis.
Introduction
Breast cancer is nowadays the most frequent malignnant tumor in females. Moreover, despite progresses
made in the field of early detection and therapy, morbidity
and specific mortality continue to increase. An important event in the therapy of breast cancer was the
introduction of targeted therapy, based on the hormone
receptors and HER2 protein expression. Unexpectedly,
it was shown that hormone therapy does not influence
the prognosis in long-term follow-up studies. On the
other hand, resistance to the therapy with trastuzumab
was already reported, and it is thought to be the
consequence of PTEN gene loss. Therefore, there is a
strong need to identify new therapeutic targets [1, 2].
A significant improvement in this field was the
recent introduction of the new molecular classification
that recognizes five different types of breast carcinoma:
basal-like, HER2, luminal A and B, and unclassified
[3, 4]. The main goal of this classification was to clarify
which are the molecular markers of prognosis and to
show the potential response to adjuvant therapy in
individual cases. The five molecular types of breast
cancer were demonstrated by gene profile analysis and
immunohistochemistry was shown to be a good and
accurate surrogate to define a particular type [5, 6].
In the panel of markers used to perform a molecular
classification of breast cancers are included estrogen
receptors (ER), progesterone receptors (PR), HER2,
cytokeratin 5/6, epidermal growth factor receptor
(EGFR), p53, and Bcl-2. From these markers, EGFR
plays an important role in defining basal-like carcinoma,
and previous studies suggested that this receptor could
be a target for specific inhibitors [7].
EGFR is a member of the ErbB family of receptors,
and its stimulation by endogenous ligands (epidermal
growth factor or transforming growth factor-alpha)
results in activation of intracellular tyrosine kinase,
therefore, leads to the inhibition of apoptosis, activation
of cell proliferation, and increases the metastatic
potential [8]. Based on these properties, EGFR was
investigated in many human malignant tumors and it is
now regarded as a potential target for cancer therapy
[9–11]. In breast cancer EGFR expression was found
mainly in basal-like carcinoma, but many reports already
signaled out positive cases associated with HER2 or
luminal types [12, 13]. It was shown that EGFR plays a
crucial role not only in the molecular diagnosis of breast
cancer, but also induces resistance to chemotherapy and
radiation treatment, and therefore, is a marker of poor
prognosis and survival [14, 15].
The immunohistochemical expression of EGFR is not
restricted to the basal-like carcinoma. Its expression was
also found with low rates in luminal and HER2 types,
but its significance in these conditions is still elusive, and
even not taken into account by definition of these types.
The purpose of this study was to evaluate the expression
of EGFR by the five molecular subclasses of breast
cancer, and to demonstrate the correlation with clinical
and pathological conventional factor of prognosis.
Adriana Meche et al.
218
Material and Methods
lymph node status and metastasis were available in all
cases with carcinoma.
Specimens and primary processing
Immunohistochemistry
There were investigated specimens of mammary
gland taken by open surgery from normal tissue at
distance from the tumor (n = 4), fibroadenoma (n = 5),
atypical ductal hyperplasia (n = 4), ductal carcinoma in
situ (n = 9) and invasive carcinoma (n = 84).
In patients with invasive carcinoma, in situ lesions
were found in 15 cases. Lymph node metastases were
found in 49 from 84 cases, and distant metastases in
three cases.
All specimens were formalin-fixed paraffin
embedded, and step section 5 µm thick were performed
from each block. Initial sections were stained with the
routine Hematoxylin–Eosin method for the pathological
diagnosis and grade. Information about tumor stage,
Additional
slides
were
prepared
for
immunohistochemistry and the first purpose was to
classify cases with carcinoma according to the
molecular classification, based on the expression of ER,
PR, HER2, cytokeratin 5, p53, and Bcl-2. Next, we
addressed to the expression of EGFR in the molecular
types in order to evaluate its diagnostic and prognostic
value. All reagents used in the present study were from
DakoCytomation (Glostrup, Denmark). Details on the
immunohistochemical technique, regarding antigen
retrieval, antibodies, dilution and working system are
shown in Table 1. For all methods, the chromogen was
3,3’-diamino-benzidine and nuclei were stained with
Lillie’s modified Hematoxylin.
Table 1 – Details on the immunohistochemical technique used in the study
1D5
Pgr636
MIB1
Rabbit
polyclonal
DO7
RTU
RTU
RTU
RTU
Incubation
Working system/
time
Chromogen
MW, 30’ citrate buffer pH 6
30’, RT
LSAB+/HRP, DAB
MW, 30’ citrate buffer pH 6
30’, RT
LSAB+/HRP, DAB
MW, 30’ citrate buffer pH 6
30’, RT
LSAB+/HRP, DAB
MW, 30’, antigen retrieval
HercepTest Visualization
30’, RT
solution HercepTest
reagent, DAB
MW, 30’ citrate buffer pH 6
30’, RT
LSAB+/HRP, DAB
124
RTU
MW, 30’ citrate buffer pH 6
30’, RT
8. Cytokeratin 5/6
10. Cytokeratin 18
D5/16 B4
DC10
1:80
1:25
MW, 30’ citrate buffer pH 6
MW, 30’ citrate buffer pH 6
30’, RT
30’, RT
12. EGFR
Polyclonal RTU
MW, 30’ citrate buffer pH 6
30’, RT
No.
Antibody
1. Estrogen receptor
2. Progesterone receptor
3. Ki67
4. HER2/neu
5. p53
Clone Dilution
6. Bcl-2
RTU
Antigen retrieval
Expression
Nuclear
Nuclear
Nuclear
Membrane
pattern
Nuclear
Nuclear,
LSAB+/HRP, DAB
cytoplasmic
LSAB+/HRP, DAB
Cytoplasmic
LSAB+/HRP, DAB
Cytoplasmic
EGFR PharmDx
Membrane,
visualization reagent, DAB cytoplasmic
Legend: RTU – ready-to-use, MW – microwave, RT – room temperature, LSAB – labeled streptavidin biotin, DAB – diaminobenzidine.
Evaluation of immunohistochemical reactions
Examination was performed with the microscope
Eclipse 80i Nikon and images captured as JPEG format
were evaluated with Lucia G microscopic analysis
soft.
The expression of ER and PR was evaluated on the
base of Allred score, and overexpression of HER2
protein was estimated on the base of the conventional
system indicated by the manufacturer and applied as
HercepTest.
Staining for cytokeratin 5, cytokeratin 18, Bcl-2 and
EGFR were considered positive only if a minimum of
15% of definite tumor cells show positive reaction.
EGFR scoring was based on the pattern of the final
reaction product, intensity and percentage of positive
tumor cells, and graded from 0 (negative) to +3 (strong
positive reaction).
External control slides
Slides provided by the manufacturer were tested
together with human specimens and the intensity of the
reaction was interpreted according the control.
Statistical analysis
Statistical analysis was performed with SPSS13.0
soft, and included Chi square and Student tests, p<0.05
being considered as significant.
Results
The reaction for EGFR was negative in all cases of
normal breast tissue, atypical hyperplasia and
fibroadenoma. DCIS as isolated lesion was found in
nine cases, and from these, three showed weak positive
reaction in tumor cells, restricted to the cell membrane.
In DCIS, the reaction was heterogeneous and the
average showed between 15 and 25% of positive tumor
cells.
In the control slides (Figure 1) the positive reaction
was restricted to the membrane of tumor cell line isolated
from an EGFR-positive human breast carcinoma.
We next investigated 84 cases with invasive
carcinoma, and from them, there were 78 ductal
invasive carcinomas, two lobular invasive carcinomas,
one papillary carcinoma and three mucinous
carcinomas. According to the grading system accepted
by WHO, there were 21 cases with G1, 45 cases with
G2, and 18 cases with G3. In all these cases it was
applied the panel of antibodies necessary to perform the
molecular classification.
Based on the tandem expression of ER, PR, HER2,
cytokeratin 5, cytokeratin 18, p53 and Bcl-2, we found a
specific molecular profile for basal-like carcinoma in
12 (14.29%) cases, luminal A in 44 (52.38%), luminal B
in four (4.76%), HER2 type in 14 (16.66%) (Figure 2),
and unclassified in 10 (11.9%) cases.
Immunohistochemical expression and significance of epidermal growth factor receptor (EGFR) in breast cancer
The immunohistochemical evaluation of EGFR
expression showed two patterns (Figure 3, a and b).
The first, found in the majority of positive cases, is
characterized by a membrane-restricted pattern of the
final product of reaction. The second that we called
‘aberrant expression’, was found only in two cases, and
showed diffuse cytoplasmic pattern without membrane
enhancement.
The distribution of EGFR positive reaction in the
five molecular types of breast cancer was the following:
basal-cell carcinoma was positive in five (41.66%) from
12 cases, luminal A in none from 22, luminal B in two
(50%) from four cases, HER2 in three (21.42%) from
14 cases, and unclassified in none. Our findings clearly
demonstrate that EGFR expression is not found in type
luminal A and unclassified breast cancer.
219
Figure 1 – EGFR-positive control slide, ×400.
(a)
(b)
(c)
(d)
(e)
(f)
Figure 2 – Immunohistochemical profile of HER2 type: ER negative (a), PR negative (b), HER2 +3 (c), EGFR
positive (d), cytokeratin 5 (e), and cytokeratin 18 positive (f), ×400.
220
Adriana Meche et al.
(a)
(b)
Figure 3 – Immunohistochemical expression of EGFR: membrane pattern (a), diffuse cytoplasmic pattern (b), ×400.
No correlation was found between the expression of
EGFR, tumor stage, pathological form and degree of
differentiation. From the overall 11 positive cases,
nine were G3 and two were moderately differentiated.
A significant correlation was found between the
expression of EGFR with distant metastases (all three
cases were positive in the primary) (p<0.00032).
Regarding the correlation between EGFR expression
and lymph node metastases, a significant inverse
correlation was found only in cases with basal-like
carcinoma (p<0.001).
Discussion
EGFR expression was demonstrated by many
authors to be a useful marker in the molecular
classification and a good predictor of prognosis in breast
cancer [16–18]. Moreover, detection of EGFR on the
surface on circulating tumor cells derived from
individuals with metastatic breast cancer seems to be
useful to stratify patients for anti-EGFR therapy [19].
We found two model of distribution of the final
product of reaction in breast cancer. One is the typical
described in the literature, with membrane-restricted
pattern. To the best of our knowledge, this paper
describes for the first time the aberrant expression, with
diffuse cytoplasmic pattern. The significance of this
model of distribution is not known and the number of
cases from this subgroup was to low (n = 2) for
statistical analysis.
Our results strongly suggest that EGFR-positive
reaction is restricted to the basal-like, HER2 and
luminal B-types of breast cancer. This finding confirms
previous data that showed the expression of EGFR in
HER2-type and in a reduced number of luminal types.
We found that EGFR expression in the later is restricted
to the luminal B-type, and this could support the
existence of a carcinoma arising from luminal cells that
cluster close to the basal-like carcinoma. A basoluminal type was already purposed but this entity is not
yet fully characterized and accepted. It can be
speculated that EGFR-positive luminal carcinoma is
more aggressive and is likely to believe that this cases
have a higher risk for metastases.
The expression of EGFR by a subset of HER2-type
breast cancer clearly defines a subtype that is also
ER-negative, and consequently, associated with worst
prognosis.
In terms of prognosis, it was shown that basal-like
carcinoma gives rise less frequently to lymph node
metastases, and more frequently to systemic metastases.
In our study, the expression of EGFR strongly correlates
with the presence of systemic metastases and an inverse
correlation was found with lymph node status.
Tyrosine kinase inhibitors are effective anti-cancer
therapies but resistance to these agents was already
reported and includes EGFR inhibitors [20]. Phosphorylation of EGFR was detected in triple-negative tumors
and was blocked by gefitinib treatment in vitro [21].
These results are promising in terms of clinical trials
with EGFR inhibitors and this may be extended to a
subset of metastatic HER2-positive cases that show
poor response to trastuzumab therapy [22]. Recently, in
a phase II trial with an EGFR inhibitor in metastatic breast
cancer it was shown that the combination between
erlotinib and bevacizumab (anti-vascular endothelial
growth factor antibody) had only a limited efficiency
[23, 24]. This is not a surprising result, because our
findings demonstrated that only a limited number of
cases with breast carcinoma (11 from 84) do express
EGFR. An anti-EGFR antibody, cetuximab, alone or in
combination with cisplatin, was effective in vitro on
breast carcinoma cells [25], but its clinical application
needs further validation. A high-rate of local recurrence
was observed in EGFR-positive cases [26]. This is in
accord with our findings regarding the significant correlation found between EGFR expression, grade of the
tumor and metastatic potential. Based on these data, the
immunohistochemical evaluation of EGFR expression
becomes a useful tool to perform characterization of
breast cancer and to define new therapeutic targets.
Conclusions
Our study demonstrated the immunohistochemical
expression of EGFR in 13.09% of the cases with
invasive breast carcinoma. No expression was found in
the normal mammary tissue, fibroadenoma and atypical
hyperplasia. EGFR is an important marker to stratify
patients with breast cancer according the molecular
classification. The expression of EGFR correlated with
the degree of differentiation, inversely with the lymph
Immunohistochemical expression and significance of epidermal growth factor receptor (EGFR) in breast cancer
node status only in basal-like carcinoma, and with
distant metastases. In a subset of patients with breast
cancer, EGFR could be considered an effective target
for specific therapy.
Acknowledgements
This work was supported by Grant 41054/2007/PNII
and Grant 96/2007/PNII of the Romanian Ministry of
Education and Research. The authors are grateful to
Diana Tătucu for her excellent technical assistance.
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Corresponding author
Marius Raica, Professor, MD, PhD, Department of Histology and Cytology, “Victor Babeş” University of
Medicine and Pharmacy, 2 Eftimie Murgu Square, 300041 Timişoara, Romania; Phone +40722–438 179,
Fax +40256–490 626, e-mail: [email protected]
Received: February 15th, 2009
Accepted: April 10th, 2009