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Analytical Cellular Pathology
Volume 2014, Article ID 352925, 9 pages
Research Article
Expression of EGFR and Molecules Downstream to PI3K/Akt,
Raf-1-MEK-1-MAP (Erk1/2), and JAK (STAT3) Pathways in
Invasive Lung Adenocarcinomas Resected at a Single Institution
Alba Fabiola Torres,1 Cleto Nogueira,1 Juliana Magalhaes,1 Igor Santos Costa,1
Alessa Aragao,2 Antero Gomes Neto,3 Filadelfia Martins,4 and Fabio Tavora1,2
Department of Investigative Pathology, Argos Laboratories, 60175-047 Fortaleza, CE, Brazil
Department of Pathology, Messejana Heart and Lung Hospital, Rua Frei Cirilo 4290, 60846-190 Fortaleza, CE, Brazil
Department of Thoracic Surgery, Messejana Heart and Lung Hospital, 60846-190 Fortaleza, CE, Brazil
Department of Pulmonology, Messejana Heart and Lung Hospital, 60846-190 Fortaleza, CE, Brazil
Correspondence should be addressed to Fabio Tavora;
Received 20 August 2014; Revised 30 November 2014; Accepted 1 December 2014
Copyright © 2014 Alba Fabiola Torres et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Therapies targeting EGFR are effective in treating tumors that harbor molecular alterations; however, there is heterogeneity in longterm response to these therapies. We retrospectively analyzed protein expression of EGFR, Stat3, phospho-Akt, and phospho-Erk1/2
by immunohistochemistry in a series of resected cases from a single institution, correlated with clinicopathological variables. There
were 96 patients, with the majority of cases being of low stage tumors (17 pT1a, 23 pT1b, 30 pT2a, and 18 pT2b). Histologic subtypes
were 45 acinar predominant, 2 cribriform, 25 solid, 7 papillary, 11 lepidic, and 4 mucinous tumors. The EGFR score was higher
in tumors with vascular invasion ( = 0.013), in solid and cribriform acinar histology, and in high stage tumors ( = 0.006 and
 = 0.01). EGFR was more likely overexpressed in solid compared to lepidic tumors ( = 0.02). Acinar tumors had the highest
rate of ERK1/2 positivity (19%). There was a strong correlation among positivity for ERCC1 and other markers, including STAT3
( = 0.003), Akt ( = 0.02), and ERK1/ERK2 ( = 0.0005). Expression of molecules downstream to EGFR varied from 12% to
31% of tumors; however, the expression did not directly correlate to EGFR expression, which may suggest activation of the cascades
through different pathways. The correlation of protein expression and the new lung adenocarcinoma classification may help in the
understanding of activated pathways of each tumor type, which may act in the oncogenesis and drug resistance of these tumors.
1. Introduction
Lung cancer is the leading cause of cancer death worldwide.
Most lung patients present with advanced stage disease
and are not candidates for curative surgery; however, genetargeted therapies offer hope for prolonged survival in
advanced disease [1, 2]. A contemporary revolution in the
treatment of nonsmall cell lung cancer (NSCLC) centers on
biomarkers that predict who are more likely to respond to
specific targeted therapies [3, 4]. These biomarkers are typically assessed by protein overexpression evaluated by immunohistochemistry, gene copy number evaluated by in situ
hybridization, or specific mutations studied by a variety of
PCR methods. These studies have been used mainly as predictors of response to the EGFR tyrosine kinase inhibitors (TKIs)
[5, 6].
Until now, the major focus has been identification of
EGFR mutated adenocarcinomas that respond to TKI. While
therapies targeting EGFR and, more recently, ALK are effective in treating tumors that harbor molecular alterations,
there is heterogeneity in results due to the complexity of
signaling pathways involved both in oncogenesis and in
acquired resistance. Therefore, analyses of other molecular
signatures and profiles in protein expression may be critical
in understanding the development in effective new drugs and
combinations [7–11].
Activation of the EGFR results in activation of downstream signaling pathways, including the Ras-Raf-MKK
extracellular signal regulated kinase (ERK) and lipid kinase
phosphatidylinositol 3-kinase/Akt pathways [8, 12]. The activation of the downstream molecules Akt and the signal
transduction and activator of transcription (STAT), which
promotes cell survival, may be responsible for resistance
to apoptosis induced by therapies [13]. Another pathway
that may act in the inhibition of apoptosis is the ERK1/2
pathway [11, 14, 15]. Furthermore, enzyme repair crosscomplementation group 1 (ERCC1) also plays a key role in
excision repair enzyme pathways and its expression has been
suggested to correlate with survival in lung carcinomas, as
well as predicting sensitivity to platinum-based chemotherapy [16–19].
Recently, the International Association for the Study of
Lung Cancer (IASLC), the American Thoracic Society (ATS),
and the European Respiratory Society (ERS) have proposed a
new subclassification of lung adenocarcinomas [20].
Herein, we studied a series of resected lung adenocarcinomas from a single institution reclassifying them with the
new proposed ERS/ATS Classification [21, 22]. Then, we correlated the histologic subtypes with the expression of EGFR,
Stat3, phospho-Akt, and phospho-Erk1/2, which are downstream signaling molecules that mediate the biological
actions, and investigated the expression with clinicopathological variables. The main objectives of the study were to
(1) evaluate the types of adenocarcinomas in a contemporary
series using the new proposed classification, (2) evaluate
expression of proteins related to the EGF oncogenic pathways, and (3) study the expression of these proteins in relation
to established clinical and pathological characteristics.
2. Material and Methods
We conducted a retrospective review of pulmonary adenocarcinomas that were resected between 2009 and 2013 at the
Messejana Heart and Lung Hospital, a reference center for
lung cancer from Northeastern Brazil. All resected specimens
were formalin fixed and stained with hematoxylin and eosin
in the routine manner. The average number of slides from
each case we reviewed in our study was 6.7 (range: 1–26).
Cases were reviewed by two independent pathologists
with experience in pulmonary pathology and were classified
according to the recent proposed classification of adenocarcinomas by the International Association for the Study of Lung
Cancer/American Thoracic Society/European Respiratory
Society [22–24]. The morphologic patterns, predominant
histologic subtypes (lepidic, acinar, papillary, micropapillary,
cribriform, and solid), presence of lymphovascular invasion,
pleural invasion, and other clinical data were recorded.
Percentage of solid and lepidic components was assessed
semiquantitatively at 10% increments and recorded for each
tumor. Tumors were divided into two groups, one of highgrade histology (predominant solid and cribriform tumors)
and others. Sarcomatoid carcinomas, adenosquamous carcinomas, and preinvasive or minimally invasive lesions according to the aforementioned classification (atypical adenomatous hyperplasia, adenocarcinoma in situ, and minimally
invasive adenocarcinoma) were not included in the study.
High-grade histology (predominant solid or acinar with
prominent cribriform gland formation) was compared with
the other tumors and variables were compared [25, 26].
Analytical Cellular Pathology
Clinicopathological characteristics including final pathologic stage, sex, and age were also recorded. In 22 of 96 cases,
information about EGFR mutation status was available, and,
in 39 of 96 cases, information on KRAS gene status was available and, while not being the main objective of this study, this
data was also recorded.
Immunohistochemical studies were performed in one
paraffin block chosen with a large percentage of tumor in
every case. Antibodies for biomarkers related to EGFR, Raf-1MEK-1-MAP, and JAK proteins were commercially obtained.
These included EGFR (1 : 100, clone 31G7, Invitrogen,
USA), STAT3 (1 : 400, polyclonal, Spring Bioscience, USA),
phospho-Akt (1 : 100, 98H9L8, Invitrogen, USA), phosphoERK1/2 (1 : 100, polyclonal, Invitrogen, USA), and ERCC1
(1 : 100, clone 8F1, DBS, USA). Dilutions were determined by
standard titration methods for optimal staining (strongest
signal with least background staining). Methods followed
standard procedures. Briefly, sections were deparaffinized
in xylene, hydrated in graded ethanol, rinsed in distilled
water, and placed in Citrate buffer, 10 mM, pH 6.0 with steam
for antigen retrieval. The immunostaining was done with
Novolink Max Polymer Detection System (Leica Biosystems,
UK) as per manufacturer’s recommendation.
Evaluation of the immunohistochemical studies was done
using by semiquantitative analyses for EGFR results using
the H-score previously described. In summary, this method
assigns a continuous score of 0–300, based on the percentage
of positive cells multiplied by the staining intensity (0, 1, 2,
and 3) according to different staining intensity visualized at
different magnifications [27]. Evaluation of the remainder of
the antibodies considered a case positive if at least 10% of
the tumor had intense positivity in the cell site characteristic
of each antibody. A case was considered positive only if the
stain was present in the predominant growth pattern. STAT3
expression is granular cytoplasmic; ERK1/2 and Akt are both
nuclear (more specific) and cytoplasmic, while ERCC1 is
Comparisons of means were performed using Student’s test and of multiple categories were performed using ANOVA
means table with Fisher’s post hoc testing. Nonparametric
comparison was performed using Mann-Whitney comparison means testing. Statistical analysis was performed using
SAS software (Cary, NC).
3. Results
3.1. Clinical Data. There were 96 patients in the study, including 41 men (mean age 65 ± 10) and 55 women (62 ± 12). All
“tumors were from” were resected specimens: 91 cases were
lobectomies (3 bilobectomies) and 5 pneumonectomies; 55
from the right side, 34 from the left. All were adenocarcinomas diagnosed in “resections”. The majority of cases were low
stage tumors (17 pT1a, 23 pT1b, 30 pT2a, and 18 pT2b) with
only 8 cases with the final pathological stage pT3 and none
pT4. There were 15 patients with pN1 disease and 1 patient
with pN2 with no correlation between nodal status and pT
stage. Available in 62 of 96 cases, information about smoking
history elicited a positive history in 71% of cases, with no
Analytical Cellular Pathology
Table 1: Clinicopathological characteristics of cases.
Number of patients (%)
<60 yo
>60 yo
Pathological T stage
Pathological N stage
Tumor differentiation
Well differentiated
Moderately differentiated
Poorly differentiated
Histologic subtypes
Acinar cribriform
41 (57)
55 (42)
17 (18)
23 (24)
30 (31)
18 (19)
8 (8)
80 (80)
15 (16)
1 (1)
58 (61)
26 (27)
11 (11)
45 (46)
2 (2)
25 (26)
7 (7)
11 (11)
4 (4)
difference in age, pathological stage, and histologic type
between smokers and nonsmokers.
The clinicopathological data are summarized on Table 1.
3.2. Histology. There were 58 well-differentiated adenocarcinomas, 26 moderately differentiated adenocarcinomas, and 11
poorly differentiated adenocarcinomas. Histologic subtypes
encompassed 45 acinar predominant adenocarcinomas, 2
acinar tumors with prominent cribriform growth pattern,
25 solid predominant adenocarcinomas, 7 papillary adenocarcinomas, 11 lepidic predominant adenocarcinomas, and 4
mucinous/colloid tumors (Figure 1). Seventy-six cases (79%)
had multiple patterns, and 20 were of the pure type. No
tumor was predominantly micropapillary. Two cases were
predominantly cribriform and were considered to be of highgrade histology along with predominantly solid tumors.
Thirty-five cases (38.3%) had ≥10% lepidic component,
generally in the periphery of the mass (Figure 1) and 43
(44%) had ≥10% solid component. The percentage of solid
component correlated with advanced T stage ( = 0.2) and
did not correlate with age or sex. Both solid and papillary
tumors were more likely to harbor lymphovascular invasion
( = 0.04), and predominant solid tumors also correlated
with the presence of pleural invasion, independently of tumor
size on multivariate analyses ( = 0.005). High-grade
tumor histology correlated with pleural invasion ( = 0.02),
vascular invasion ( = 0.01), and tumor size ( = 0.01) but
did not correlate with nodal metastasis (pN stage) or age.
3.3. Immunohistochemistry. There were 46 cases positive for
TTF-1, most often present in acinar predominant tumors ( =
0.05). No mucinous tumors were positive for TTF-1.
EGFR histologic score was performed in all tumors, 64
being negative and 32 being positive (cutoff above score of
200). Forty-six cases (48%) had a score of 0. Fourteen cases
(15%) had a score of 300. The EGFR score was higher in
tumors with vascular invasion ( = 0.013) and there was a
trend towards tumors with pleural invasion ( = 0.06). Highgrade histology and higher stage tumors (pT2 or pT3) also
had significantly higher EGFR score ( = 0.006 and  = 0.01
resp.), and there was no correlation with age and sex. Across
histologic subtypes, EGFR was more likely overexpressed in
solid compared to lepidic tumors ( = 0.02).
The STAT3, ERK1/2, and Akt data are summarized in
Table 2. STAT3 was positive in 29 tumors (32%), including
16 acinar, 1 cribriform, 2 lepidic, 2 papillary, and 8 solid type.
ERK1/2 was positive in 14 tumors (15.5%), including 8 acinar,
2 lepidic, 1 mucinous, and 3 solid type. Akt was positive in 12
tumors, which included 5 acinar, 1 lepidic, 1 mucinous, and 5
solid. The results are summarized in Figures 2 and 3.
There was no correlation among protein expression of
STAT3, Akt, and ERK1/2 with histologic subtype, clinical
characteristics, or tumor stage. Acinar tumors had the highest
rate of ERK1/2 positivity (19%). High-grade histology did not
correlate with any of the aforementioned epitopes.
ERCC1 positivity was found in 38 cases (39%) and
tumors were more likely lepidic or solid. No papillary tumor
was ERCC1 positive. There was a strong correlation among
positivity for ERCC1 and other markers, including STAT3
( = 0.003), Akt ( = 0.02), and ERK1/ERK2 ( = 0.0005).
Positive cases tended to present a lower EGFR score.
High expression of EGFR (score above 200) did not
correlate with positivity of AKT, ERK1/2, or STAT3 ( > 0.5
for all proteins). Cases that were positive for STAT3 had a
mean score of 123.4, while negative cases had a mean score
of 99.1, with the correlation not being statistically significant
( = 0.3). Cases that were positive for Akt and ERK1/2 had
lower EGFR score, with no statistical significance ( > 0.5).
4. Discussion
Lung adenocarcinomas are the most common histologic
subtype and are part of the larger group of nonsmall cell lung
cancer (NSCLC) that has been target of a recent evolution on
the chemotherapeutical regimens based on molecular profiling. While NSCLC patients may harbor EGFR mutations in
about 20% of the cases [28–30] and these tumors may respond
well to EGFR tyrosine kinase inhibition, the responses are
usually incomplete [31]. This phenomenon of drug resistance
or failure may be explained in part by the activation of different oncogenic pathways or de novo mutations that activate
downstream molecules and increase signal transduction. In
Analytical Cellular Pathology
Figure 1: Morphologic features of lung adenocarcinoma according to the predominant growth pattern: (a) acinar; (b) lepidic; (c) solid;
(d) papillary; (e) mucinous adenocarcinoma; (f) acinar tumor with prominent cribriform formation. All figures hematoxylin-eosin.
this scenario, the identification of overexpressed molecules in
different subtypes of lung cancer may help identify potential
targets for combined therapy. For example, combining AKT
inhibition with EGFR TKI may improve response in lung
cancer patients harboring EGFR mutations [32].
We set out to determine the expression of molecules
related or downstream to the EGFR receptor oncogenic
development in lung adenocarcinoma subtypes according to
the IASLC/ATS/ERS classification. The prognostic value of
this classification by itself has been validated in retrospective
studies, which include a previous study from our group [22,
33, 34]. To the best of our knowledge, the association between
these epitopes and the new morphologic subtypes has not
been hitherto investigated in this population. In addition,
there has not been a contemporary study on lung cancer
from South America and particularly Brazil, with emphasis
on the new morphological classification and its relation with
immunohistochemical expression outline. Protein expression
is aligned directly with cellular activity and the proteomic
profiling of tumors may help in the understanding of tumor
progression, resistance mechanisms, and metastasis [35].
In the current series, the majority of tumors were of acinar
subtype, followed by solid and lepidic variants, similar to
other contemporaneous studies with the new classification
[22, 33, 36]. Some other studies have tried stratifying invasive
adenocarcinoma subtypes in architectural categories related
Analytical Cellular Pathology
Table 2: Expression of EGFR, p-ERK1/2, p-STAT3, ERCC1, and Akt by immunohistochemistry, and clinicopathological characteristics in 96
patients with lung adenocarcinoma.
Below 60 years
Above 60 years
Greater than T1
Vascular invasion
Pleural invasion
High grade histology
EGFR mean  value
7 (7.61)
7 (7.61)
14 (15.3)
15 (16.4)
4 (4.3)
8 (8.6)
18 (20.4)
21 (23.8)
3 (3.2)
11 (11.9)
8 (8.7)
21 (23)
5 (5.3)
7 (7.5)
12 (13.6)
27 (30.6)
8 (8.7)
6 (6.5)
11 (12)
18 (19.7)
6 (6.4)
6 (6.4)
18 (20.4)
21 (23.8)
2 (2.2)
12 (13.3)
11 (12.3)
18 (20.2)
2 (2.2)
11 (12)
12 (13.9)
26 (30.2)
2 (2.2)
13 (13.9)
8 (8.9)
21 (23.6)
1 (1.1)
11 (12)
10 (11.6)
28 (32.5)
3 (3.2)
11 (11.9)
9 (9.8)
20 (21.9)
5 (5.3)
7 (7.5)
13 (14.7)
26 (29.5)
Predominantly solid or acinar cribriform tumors (high grade) versus other subtypes.
to the aggressiveness of the tumors and predictive behaviour.
Solid, micropapillary, and acinar tumors with prominent
cribriform features are considered high grade, acinar and
papillary are considered to be of intermediate grade, and predominantly lepidic tumors are considered to be of low-grade
behaviour [26, 34, 37, 38]. Herein, we divided the tumors into
high grade (solid predominant and cribriform) and others, to
evaluate differences in expression between these two groups.
The main purpose of the study was to assess EGFR
expression by immunohistochemistry and correlate with the
morphologic subtypes according to the new adenocarcinoma
classification. While currently only patients with tumors
harboring identified mutations in the tyrosine kinase regions
of the EGFR gene are candidates for targeted drugs, there is a
body of literature enforcing the importance of EGFR expression in these tumors. Particularly, high EGFR expression may
predict survival benefit from drugs such as cetuximab in
patients with advanced NSCLC [27]. Interestingly, our data
shows that, in this population, the EFGR mean score, assessed
using the H-score method previously reported, was significantly higher in tumors with vascular invasion and higher T
stage and in tumors with high-grade histology (mainly solid
subtype). Li et al. have demonstrated similar correlation of
aggressiveness with nodal status and overall status [12]. This
finding is interesting in light of a few studies that have
found that activating EGFR mutations is more common in
tumors with acinar or lepidic subtype [39–41]. These studies
have not, however, addressed EGFR expression by immunohistochemistry. The current study is in agreement with
a study by Sholl et al. who have demonstrated both greater
expression and EGFR gene amplification in solid tumors
compared to other subtypes [42].
Studies of excision repair cross-complementing gene 1
(ERCC1) expression have shown correlation with prognosis
and response to treatment gastric, pancreatic, cervical, and
lung adenocarcinomas [10, 17, 43–46]. Particularly in lung
adenocarcinomas, ERCC1 has been shown to be associated
with unchanged EGFR mutation status [16], to predict
response to cisplatin-based therapies and also to be of prognosis value by itself [19, 47, 48]. We have shown that there was
no difference in ERCC1 expression among various morphological subtypes and also no statistical difference in the
clinicopathological variables such as age, sex, tumor size,
vascular invasion, pleural invasion, and high-grade histology,
suggesting that its expression may be an independent prognostic variable, similar to other studies [49, 50].
We also analyzed the expression of the PI3K/AKT pathway by the expression of the phosphorylated AKT and
also the mitogen-activated protein kinase/extracellular signal
regulated kinase (MAPK/ERK) pathway by the expression
of the phosphorylated ERK1/ERK2 proteins. Activation of
AKT has been associated with disease progression in tumors
with wild-type EGFR, suggesting that it plays a role in
contributing to gefitinib resistance [9]. Other studies have
shown that ERK1/ERK2 expression is linked with increase in
cell proliferation and also correlates with advanced stage and
lymph node metastases [14, 15]. In our tumors, AKT positivity
correlated inversely with vascular invasion. This information
Analytical Cellular Pathology
Figure 2: Immunohistochemical findings of different markers in lung adenocarcinomas. (a) EGFR positive acinar predominant tumor
showing both distinct membranous and cytoplasm expression. (b) High power of EGFR positive solid predominant adenocarcinoma. (c) TTF1 positive acinar tumor. (d) High power of STAT3 positive tumor with intense cytoplasm positivity. (e) ERCC1 positive lepidic predominant
tumor, with intense nuclear positivity. (f) ERK1/2 expression in solid tumor showing both nuclear and cytoplasm reactivity.
may be important especially in small samples, given that most
patients present with unresectable tumors and no other tissue
may be available for protein or DNA analyses. Although this
series has a selection bias on resected specimens, analyzing a
large tumor volume is important in identifying these markers
that are of prognostic value. Although EGFR activation may
play a role in both ERK and AKT pathways stimulation, we
have found no correlation between EGFR mean score and
expression of activated ERK [51, 52]. This may suggest that
there are different oncogenic pathways in tumors with high
EGFR expression profiles, and this information may help
understand resistance mechanisms.
Signal transducer and activator of transcription (STAT)
3 is involved in cell proliferation and apoptosis. In lung adenocarcinomas, STAT3 is one of the most important signaling
mediators in both normal and EGFR-mutated tumors [53–
55]. It is persistently activated in about 50% of NSCLC tumors
and shows correlation with EGFR mutation status [56]. In one
study, using lung carcinoma cell line, EGFR inhibition did not
block STAT3 phosphorylation and growth arrest, suggesting
Analytical Cellular Pathology
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Conflict of Interests
lung tumors, information regarding tumor epitope expression may greatly contribute to the understanding of disease
recurrence and/or responsiveness to adjuvant therapy.
The research presented in this paper was partially funded by
CNPq, Grant no. 475724/2012-7 (Brazilian National Council
for Scientific and Technological Development).
Figure 3: Percentage of expression of predominant histologic
subtype of each immunohistochemical protein (EGFR, ERK1/2,
STAT3, Akt, and ERCC1). The EGFR cutoff for positivity was the
histologic score of 200.
that upstream pathways, such as JAK and IL6 receptor,
may play a role in activating STAT3 and contributing to
oncogenesis in lung tumors, even with EGFR activation [53].
In this series, expression of phosphorylated STAT3 was found
in all subtypes, except the mucinous variant, but there was no
correlation with clinicopathological variables or EGFR mean
expression. It is remarkable that mucinous tumors also did
not show TTF-1 expression. It has been postulated that the
JAK-STAT signaling pathway may be involved in the oxidative stress-induced decrease in expression of surfactant protein genes, such as TTF-1, which may play a role in oncogenic
of this specific morphologic subtype [57].
The current study has a few weaknesses. The main one
is the lack of mutational analyses. Since it has been known
that specific mutations in the 18–21 exons of the EGFR gene
predict response to targeted therapies, it would be of great
clinical value to compare the expression of each of the studied
proteins with genetic information.
The second weakness refers to lack of follow-up information. Given the recent retrospective review, information
regarding follow-up status is lacking. It would be of great
interest to also evaluate the expression of the aforementioned
markers with information regarding tumor progression of
We also acknowledge a selection bias in including only
resected tumors, but, to better evaluate the extent of protein
expression and morphologic subtypes, we chose not to
include unresectable tumors with the diagnoses made on
small biopsies or cytology specimens.
We have shown a series of resected cases of adenocarcinomas from a single institution and estimated the expression of
proteins that may be target for current or future drug development. In this particular population of low stage, resectable
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