Keywords
intestinal adenocarcinoma - immunohistochemistry - prognosis
Introduction
Gall bladder cancer (GBC) is the most common cancer of the biliary tract and ranks
sixth among the gastrointestinal (GI) cancers.[1] It displays wide geographical and ethnical variations, with highest incidence being
reported in the Mapuche people of Chile, followed by India, Eastern Asia and some
Eastern and Central European countries.[2] It is associated with poor prognosis and low survival, attributed to the disease
being in an advanced stage at presentation.[1]
[2]
[3] In advanced diseases, the median overall survival is less than 12 months even with
palliative treatment.[4] In Chile and India, GBC occurs predominantly in females with gallstones, whereas
in Eastern Asia, it is also equally common in men, and the association with gallstone
being much weaker. Aflatoxin B1, which is found in the improperly stored foods in
rural areas, has also been implicated to play a role in triggering the inflammation.
Pancreaticobiliary maljunction (PBM) (union of the common bile duct with the main
pancreatic duct above the sphincter of Oddi) is yet another established risk factor.
Increased expression of EGFR and HER-2/NEU has been noted in GBCs.
Several studies have been performed around the globe, examining the expression of
these markers by these carcinomas.[5]
[6]
[7] EGFR is a protein kinase receptor which is involved in signal transduction, affecting
various cellular activities such as metabolism, transcription, cell cycle progression,
apoptosis and differentiation. Overexpression of receptors, gene amplification, and
the loss of inhibitory signals are among the various mechanisms of increased EGFR
activation, which results in phosphorylation of intracellular substrates downstream,
leading to subsequent activation of mitotic pathways.[8] HER-2/NEU, a protein mostly present at the surface epithelium of large and septal
bile ducts, is encoded by ERBB2 gene in humans. Overexpression of this gene product,
which occurs in about one-fourth to two-thirds of the biliary tract carcinomas, may
be used as a phenotypic marker for neoplastic transformation with a poor prognosis.[9]
In the present study, we assessed the expression of EGFR and HER-2/NEU in GBC, correlated
the findings with the clinical parameters and histological tumor types, and evaluated
the role of EGFR and HER-2/NEU as a prognostic marker and a likely indicator for targeted
therapy of GBC.
Materials and Methods
It was a cross-sectional observational study performed in the Department of Pathology
in collaboration with the Department of Surgery after obtaining the approval from
the Institutional Ethical Committee. A total of 30 resected specimens of patients
with GBC were selected over a study period of 24 months from November 2017 to November
2019. After processing the tissues, as per the standard procedure, hematoxylin and
eosin stained sections were subjected to histopathological evaluation. Following the
confirmation of GBC, histological grades and tumor subtypes were assigned ([Figs. 1]
[2]
[3]–[4]). Immunohistochemistry (IHC) was performed for EGFR and HER-2/NEU in all cases.
Four microns-thick sections were prepared from the formalin-fixed and paraffin-embedded
tissue samples. Subsequently, they were stained with antibody against EGFR mouse monoclonal
antibody Dako Clone: H 11 Lot no. AN7810616B, dilution: 1:100 for positive control
of EGFR, normal endometrial tissue was utilized, and negative control was achieved
by the omission of primary antibody in EGFR. Similarly, prepared sections were stained
with antibody against HER-2 (polyclonal rabbit anti-human antibody directed against
c-erbB-2 oncoprotein, Dako: Lot no. 20067288). For positive control of HER-2, breast
carcinoma tissue was chosen, and negative control was achieved by the omission of
primary antibody in HER-2.
Fig. 1 Photomicrograph showing intestinal variety of adenocarcinoma (×400) hematoxylin and
eosin (H&E).
Fig. 2 Photomicrograph showing intestinal variety of adenocarcinoma (×400) hematoxylin and
eosin (H&E).
Fig. 3 Photomicrograph showing mucinous variety of adenocarcinoma (×100) hematoxylin and
eosin (H&E).
Fig. 4 Photomicrograph showing mucinous variety of adenocarcinoma (×400) hematoxylin and
eosin (H&E).
Interpretation of Immunostaining
The interpretation of staining of EGFR and HER-2/NEU was reported as percentage of
positive cells and intensity of staining. Cell membrane staining was used to assess
positivity for EGFR and HER-2/NEU. EGFR intensity was scored from 0 to 3+ and the
threshold for positivity was +1 staining intensity in 10% of tumor cells. ([Figs. 5]
[6]
[7]
[8]
[9]–[10]).
Fig. 5 Photomicrograph showing strong membranous staining pattern of EGFR in poorly differentiated
carcinoma (×100).
Fig. 6 Photomicrograph showing strong membranous staining pattern of EGFR in poorly differentiated
carcinoma (×400).
Fig. 7 Photomicrograph showing strong membranous staining pattern of HER-2/NEU in well-differentiated
carcinoma (×100).
Fig. 8 Photomicrograph showing strong membranous staining pattern of HER-2/NEU in well-differentiated
carcinoma (×400).
Fig. 9 Photomicrograph showing EGFR moderate staining in mucinous carcinoma of gall bladder
(×400).
Fig. 10 Photomicrograph showing HER-2/NEU mild staining in poorly differentiated carcinoma
of gall bladder (×400).
The quantification of EGFR immunostaining was determined as shown in [Table 1].[10]
Table 1
Quantification of EGFR immunostaining
|
EGFR score
|
Positive cells
|
Staining intensity
|
|
0
|
< 10%
|
Faint/none
|
|
1+
|
> 10%
|
Weak
|
|
2+
|
≥ 10%
|
Moderate
|
|
3+
|
≥ 10%
|
Strong
|
The staining pattern for HER-2 was determined as shown in [Table 2]
[11]
Table 2
Interpretation of HER-2 immunostaining
|
Result
|
Criteria
|
|
Score 0
|
No staining observed.
|
|
Score 1+
|
Faint membrane staining in > 10% of tumor cells in part of cell membrane.
|
|
Score 2+
|
Weak to moderate incomplete membrane staining in over 1% of tumor cells.
|
|
Score 3+
|
Strong complete membrane staining in over 1% of tumor cells.
|
Statistical Analysis
All data were thoroughly tabulated on Microsoft Excel worksheet. Mean values with
standard deviation were calculated for quantitative variables, whereas proportions
represented qualitative variables. The Chi-square test was conducted to assess the
correlation between the clinicopathological parameters and the IHC results. Statistical
analyses were performed using SPSS software version 20.0 (IBM Inc.). Two-tailed p < 0.05 was considered as statistically significant.
Results
In our series of 30 patients, 21 (70%) were females and 9 (30%) were males. Age ranged
from 23 to 75 years, with mean age being 52.33 years. Fourteen (46.66%) presented
with pain in the abdomen and 16 (53.33%) presented with jaundice. On radiological
evaluation, 19 (63.33%) cases showed GB mass, 8 (26.66%) showed intraluminal mass,
and 3 (10%) showed diffuse intramural thickening. Cholelithiasis was associated in
19 (63.33%) cases. Regarding the histological tumor type, 13 (45.35%) had intestinal
type of adenocarcinoma, 7 (25.33%) had mucinous carcinoma, 7 (25.33%) had biliary
carcinoma, 2 (6.66%) had poorly cohesive carcinoma, and 1 (3.33%) had adenosquamous
carcinoma. Lymphovascular invasion was seen in 11 (36.66%) and perineural invasion
in 18 (60%) patients.
With regard to the hormone receptor status, EGFR was positive in 28 (93.33%) of patients,
out of which 5 (17.85%) were assigned with score 1, 14 (50%) with score 2, and 9 (32.15%)
with score 3. HER-2/NEU was found to be positive in 17 (56.66%) patients, out of which
9 (52.94%) were assigned with score 1, 6 (35.29%) with score 2, and 2 (11.77%) with
a score of 3. The correlations between EGFR expression and clinicopathologic parameters
are summarized in [Table 3]. No significant correlation found between age group and EGFR expression, but a statistically
significant correlation was found between histological tumor type (p = 0.000) and EGFR expression as well as between cholelithiasis (p = 0.033) and EGFR expression.
Table 3
Correlation between EGFR expression and clinicopathological parameters
|
Prognostic markers
|
EGFR expression
|
p-Value
|
|
n
|
3+
|
2+
|
1+
|
0
|
|
(n = 9)
|
(n = 18)
|
(n = 1)
|
(n = 2)
|
|
Age group
|
21–40
|
4
|
1
|
3
|
0
|
0
|
0.168
|
|
41–60
|
19
|
8
|
10
|
1
|
0
|
|
|
61–80
|
7
|
0
|
5
|
0
|
2
|
|
|
Sex
|
Female
|
21
|
6
|
13
|
0
|
2
|
0.350
|
|
Male
|
9
|
3
|
5
|
1
|
0
|
|
|
Histological tumor type:
|
|
|
|
|
|
|
|
Intestinal
|
13
|
2
|
11
|
0
|
0
|
0.000
|
|
Biliary
|
7
|
2
|
3
|
0
|
2
|
|
|
Mucinous
|
7
|
3
|
4
|
0
|
0
|
|
|
Poorly cohesive
|
2
|
2
|
0
|
0
|
0
|
|
|
Adenosquamous
|
1
|
0
|
0
|
1
|
0
|
|
|
Cholelithiasis
|
Present
|
20
|
4
|
15
|
1
|
0
|
0.033
|
|
Absent
|
10
|
5
|
3
|
0
|
2
|
|
The correlations between HER-2/NEU expression and clinicopathological parameters are
summarized in [Table 4]
. No significant correlation was found between HER-2 expr-ession and age group and
between HER-2/NEU and sex. Cholelithiasis and HER-2 expression also showed no significant
correlation. However, there was a statistically significant correlation between HER-2
expression and histological tumor type (p = 0.02). There was a statistically significant (p = 0.000) correlation between EGFR and HER-2/NEU expression too ([Table 5]).
Table 4
Correlation between HER-2/NEU expression and clinicopathological parameters
|
Prognostic parameters
|
HER-2/NEU expression
|
|
p-Value
|
|
n
|
3+
|
2+
|
1+
|
0
|
|
(n = 3)
|
(n = 9)
|
(n = 10)
|
(n = 8)
|
|
Age group
|
21–40
|
4
|
1
|
1
|
1
|
1
|
0.428
|
|
41–60
|
19
|
0
|
7
|
5
|
7
|
|
|
61–80
|
7
|
2
|
1
|
4
|
0
|
|
|
Sex
|
Female
|
21
|
3
|
5
|
4
|
9
|
0.674
|
|
Male
|
9
|
0
|
3
|
2
|
4
|
|
|
|
|
|
|
|
|
|
|
Histological tumor type:
|
|
|
|
|
|
|
|
Intestinal
|
13
|
1
|
7
|
3
|
2
|
0.021
|
|
Biliary
|
7
|
2
|
2
|
1
|
2
|
|
|
Mucinous
|
7
|
0
|
0
|
6
|
1
|
|
|
Poorly cohesive
|
2
|
0
|
0
|
0
|
2
|
|
|
Adenosquamous
|
1
|
0
|
0
|
0
|
1
|
|
|
Cholelithiasis
|
Present
|
20
|
1
|
8
|
7
|
4
|
0.208
|
|
Absent
|
10
|
2
|
1
|
3
|
4
|
|
Table 5
Correlation between EGFR and HER-2/NEU expression
|
EGFR
|
HER-2/NEU expression
|
p-Value
|
|
0
|
1+
|
2+
|
3+
|
|
(n = 13)
|
(n = 9)
|
(n = 6)
|
(n = 2)
|
|
0 (n = 2)
|
0
|
0
|
0
|
2
|
0.000
|
|
1+ (n = 5)
|
1
|
0
|
0
|
0
|
|
|
2+ (n = 14)
|
1
|
7
|
9
|
1
|
|
|
3+ (n = 9)
|
6
|
3
|
0
|
0
|
|
Discussion
In our study, the median age of presentation of GBC was 52.3 years. Similar results
were observed in other studies.[12]
[13]
[14] In yet another study conducted by Chijiiwa et al, perineural invasion was found
in 43% of cases and lymphovascular invasion in 68% of cases.[15] However, we found perineural invasion in 60% and lymphovascular invasion in 36.66%
of patients, which was also nearly supportive to the previous studies.
To date, many studies have demonstrated the correlation of biomarkers associated with
tumorigenesis and prognosis, which indicates that these markers may have complementary
roles in improving the diagnosis and predicting the prognosis of cancers.[16] The identification of the risk of mortality and disease recurrence in cancer patients
is critical for guiding surveillance and selecting adjunctive therapies. It has been
reported that EGFR and HER-2/NEU expression are related to clinicopathological parameters
in breast cancer and colonic cancer. Through our study, we found that EGFR and HER-2/NEU
are not related to age and gender in GBC. As found by Hadi et al in their study, cholelithiasis
is closely related to GBC.[17] We also found a significant correlation between EGFR expression in GBC and cholelithiasis.
Since the preceding years, the pathogenesis of GBC has become an important and a much
concerned phenomenon and often highlights the involvement of major proto-oncogenes
such as EGFR and HER-2/NEU.[18] In the management of GBC, several phase II trials have been performed, investigating
the role of tyrosine kinase inhibitors like Erlotinib.[19]
[20]
[21] Therefore in this study, we have attempted to identify the immune expression of
EGFR and HER-2 in 30 patients with GBC, assessing their correlation with the various
clinicopathological parameters to understand their role in targeted therapy and significance
in prognosis. Several studies from Europe, Asia and Australia have complemented this
work by examining the level of expression of EGFR in biliary tumors ([Table 6]). These studies demonstrated a consistent and significant overexpression of EGFR
in biliary tumors. In a study performed by Lee et al on IHC stains for EGFR on 13
GBC specimens from Australia, 100% of the GBC specimens were found to stain strongly
positive for EGFR.[22] In this context, in yet another study performed by Kaufman et al in a series of
16 patients, 15 (93.75%) were noted to overexpress EGFR. In the present study, EGFR
expression was found in 93.33% (28 patients), which is concordant to the previous
studies.[23]
Table 6
Comparative studies on EGFR expression in biliary cancer
|
Study
|
n
|
Immunoreactivity
|
|
Lee et al22
|
Gall bladder–13, biliary duct–7
|
100%, 86%
|
|
Zhou et al5
|
Gall bladder–41
|
71%
|
|
Kaufmann et al23
|
Gall bladder–16
|
93.57%
|
|
Shafizadeh N et al25
|
Gall bladder
|
80%
|
|
Present study
|
Gall bladder
|
93.33%
|
In a study conducted by Viswanath et al, they found that advanced biliary tract malignancies
show increased expression of EGFR.[24] Similarly, in the present study, we found that EGFR expression is more in poorly
differentiated advanced tumors.
Thus, our study revealed that EGFR is a prognostic marker of aggressiveness in GBC.
Various studies have shown that HER-2 protein is variably amplified in 16 to 64% of
GBCs.[25]
[26]
[27] In the present study, HER-2/NEU immunoreactivity was found to be positive in 56.66%
of GBC cases. However, in a study conducted by Javle et al, HER-2/NEU overexpression
was found in 8/9 (88.88%) of patients with GBC which, though of a much higher percentage
than our study, still supports our result.28 Through their study, they have also concluded that targeted therapy against HER-2/NEU
is a promising treatment strategy for GBC patients.
In a study conducted by Yoshida et al,[29] they found a significant patient population that can derive benefit from anti-HER-2
therapy by designing planned clinical trials based on preliminary IHC reports. HER-2
can be considered as a potential candidate for targeted therapy in GBC, as several
drugs are now available that can successfully inhibit HER-2, as in cases of breast
and gastric carcinoma. In a study conducted by Kiguchi et al, it was found that Lapatinib
(anti-HER-2 agent), when combined with Gemcitabine, had a synergistic antiproliferative
effect on a GBC cell line (TGBC1-TKB) in vitro.[30]
Therefore, in this study, we have attempted to identify the immune expression of EGFR
and HER-2 in 30 patients with GBC, assessing their correlation with the various clinicopathological
parameters to understand their role in targeted therapy and significance in prognosis.
Conclusion
Our study deals with the clinicopathological parameters and expression of RAS pathway
molecules like EGFR and HER-2/NEU in GBC. The present study revealed that these molecules
show significant expression in GBC, suggesting that significant interactions take
place among the different members of ErbB family during the process of tumorigenesis.
We analyzed the correlation of EGFR and HER-2/NEU expression in different histological
subtypes of GBC and also with the clinicopathological parameters. We identified a
significant subgroup of GBC cases in which targeted therapy may increase the survival
of patients.
