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Original Article
The association of topoisomerase 2α expression with prognosis in
surgically resected non-small cell lung cancer (NSCLC) patients
12nd Chest Diseases Clinic, 2Pathology Department, Ataturk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey
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Abstract
Background: Topoisomerase 2α (Topo 2α) is a nuclear enzyme that alters the topology of DNA. It’s essential for normal chromosome segregation during cellular division. We aimed to investigate the association of Topo 2α expression with
clinical, pathological parameters and prognosis in surgically resected non-small cell lung cancer (NSCLC) patients.
Methods: The study is comprised of 100 surgically resected NSCLC (squamous cell carcinoma in 50 patients, adenocarcinoma in 50 patients). The paraffin embedded tumor sections were retrieved for expression of Topo 2α. Nuclear and cytoplasmic expression of Topo 2α was determined by immunohistochemistry. Clinical, pathological data and survival of patients were determined from the hospital files. Median follow-up time was 35 (range, 4-120) months. Results: Nuclear and cytoplasmic expression of Topo 2α was positive in 41 (41%) and 66 (66%) patients, respectively. There was no significant association between nuclear or cytoplasmic expression of Topo 2α and age, gender, smoking history. While nuclear expression was significantly increased in squamous cell carcinoma (P=0.008), OR (95% CI): 3.01 (1.31-6.92), cytoplasmic expression wasn’t different. Both nuclear and cytoplasmic expression didn’t show any association with tumor diameter, pathological stage, tumor differentiation and relapse. There was no significant association between nuclear or cytoplasmic expression of Topo 2α and survival. Tumor diameter (P=0.031) and metastasis to N2 lymph nodes (P=0.005) were independent prognostic factors. Conclusions: There was no association between Topo 2α expression and prognosis in surgically resected NSCLC patients. Nuclear expression of Topo 2α was significantly higher in patients with squamous cell carcinoma. Key words
Non-small cell lung cancer; topoisomerase 2α; prognosis
J Thorac Dis 2012;4(4):352-357. DOI: 10.3978/j.issn.2072-1439.2012.08.04 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Introduction
Lung cancer is a group of heterogeneous clinical entities
with common molecular and cellular origins, but different
accumulated genetic mutations with different clinical behaviors
and prognoses (1). The outcome of a lung cancer patient depends
on a variety of variables defined as prognostic factors (2). The
recurrence rates of surgically resected non-small cell lung cancer
(NSCLC) patients are highly variable changing between 20%
to 85% (3). The principal factors related to the recurrence are
tumor stage, histology, localization, adequacy of mediastinal
dissection and administration of adjuvant chemotherapy. There
are also new and promising molecular/biologic markers that
are not utilized routinely for determination of prognosis such
as regulators of cellular growth (kRAS, EGFR, RB), of the
metastatic cascade (TPA, Cyclin-D1, cathepsin) and of apoptosis
(p53, bcl-2) (1-4).
Topoisomerase 2 (Topo 2) is a nuclear enzyme that alters
the topology of DNA and is essential for normal chromosome
segregation at mitosis. In mammalian cells, there are two isoforms
as Topo 2α and Topo 2β. Topo 2α is considered a specific marker
of cell proliferation in both normal and neoplastic tissues (5).
It is also a target for some chemotherapeutic agents in clinical
use (6). Previous clinical studies have shown that Topo 2α expression reflects several biologic behaviours in human cancers
such as lung carcinomas (7,8). In a small study by Guinee et al.,
Topo 2α expression was higher in frequency in SCLC compared
with NSCLC (8). In a study by Dingemans et al., high expression
of Topo 2α was predictive of worse survival and high expression
of Topo 2β predictive of lower chemotherapy response rates in
patients with small cell lung cancer (SCLC) (9).
In this study, we aimed to investigate the association of
Topo 2α expression with clinical (age, gender, smoking history,
administration of adjuvant chemotherapy), pathological
parameters (tumor histology, stage, tumor diameter, involvement
of lymph nodes, differentiation) and prognosis in surgically
resected NSCLC patients.
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Material and methods
Patients
This study is approved by the local ethic comittee of Ataturk
Chest Diseases and Chest Surgery Education and Research
Hospital. Informed consent waived for this retrospective study.
Surgically resected tumor specimens from 100 randomly selected
NSCLC patients who were not treated with preoperative
chemotherapy were studied. Hospital files were reviewed for
clinical data. There were 91 male and 9 female patients with a
mean age of 59.7±10.5 years (range, 37-78 years). All patients
underwent lobectomy (82 patients) or pneumonectomy (18
patients) with hilar and mediastinal lymph node dissection.
Pathological stage was determined according to the 7th edition
of TNM staging system (10). Histopathological diagnosis
was squamous cell carcinoma and adenocarcinoma in 50
patients each. There were 5 patients with stage 1a, 28 patients
with stage 1b, 21 patients with stage 2a, 21 patients with stage
2b, 25 patients with stage 3a. The clinical and pathological
characteristics of patients are seen in Table 1. Twenty-nine
patients received adjuvant cisplatin based chemotherapy
regimens, 15 patients adjuvant radiotherapy and 6 patients both
adjuvant chemotherapy and radiotherapy.
Nine patients who died due to postoperative complications
or toxicities due to adjuvant chemotherapies were excluded from
survival analysis. Median follow up time was 35 [4-120] months.
There were 49 (53.8%) relapses during the follow up period.
Mean relapse time was 35.5±25.7 [3-120] months.
Immunohistochemistry
All slides of the patients were reviewed. Representative blocks
were selected for immunohistochemistry. Tissue samples were
fixed in 10% buffered formalin embedded in parafin and cut
at 6 μm for immunohistochemistry. Sections were dewaxed in
xylene substitute (ThermoScientific) and hydrated with a graded
series of ethanol concentrations and water.
Immunostaining of Topo 2α was performed using the
streptavidin-biotin complex kit (ThermoScientific, Fremon,
USA). Sections were incubated with primary antibody solution
for Topo 2α Ab-4 (ThermoScientific) at a dilution of 1:20 for
30 minutes at room temperature. Diaminobenzidine was used
as the chromogen. After incubation, the chromogen specimens
were counterstained with Haris hematoxylin and coverslipped.
Samples of tonsil tissues were used as positive control. Negative
controls were performed by substituting without primary
antibody.
The intensity of Topo 2α immunostaining was evaluated
by light microscopy (Labsphot-2; Nikon, Tokyo, Japan). The
expression of Topo 2α was evaluated as nuclear and cytoplasmic.
More than 10% of nuclear positive cells were considered as
nuclear positivity. Cytoplasmic immunoreactivity was evaluated
based on the percentage of positive tumor cells and scored as
negative: no cytoplasmic Topo 2α staining, weak (1+): 0-30%
staining, moderate (2+): 31-60% staining, and intense (3+):
more than 60% staining.
Statistical analysis
Statistical analysis was performed using SPSS for windows
release 11.5 package program. Univariate Logistic Regression
Analysis was performed to analize the relation between
expression of nucleer or cytoplasmic expression of Topo 2α
and clinical and pathological findings. Survival curves were
computed by using method of Kaplan Meier. In order to evaluate
the independent prognostic relevance of nuclear and cytoplasmic
expression of Topo 2α, we performed multivariate analysis
using Cox Regression model. A value of P<0.05 was accepted as
statistically significant.
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Results
Nuclear and cytoplasmic expression of Topo 2α in tumor tissues
Nuclear expression of Topo 2α was positive in 41 (41%) and
negative in 59 (59%) specimens. Cytoplasmic expression of
Topo 2α was positive in 66 (66%) and negative in 34 (34%)
specimens. Cytoplasmic expression was weak in 33, moderate
in 13 and intense in 20 specimens. Figure 1 demonstrates
intranuclear expression of Topo 2α in an adenocarcinoma and
intracytoplasmic expression of Topo 2α in a squamous cell
carcinoma respectively.
Figure 1 Intranuclear expression of topoisomerase 2α in an adenocarcinoma (A), Intracytoplasmic expression of topoisomerase 2α in a squamous cell carcinoma (B) (Topo 2α ×200).
The association of Topo 2α expression with clinical and
pathological characteristics
There was not any significant association between nuclear or cytoplasmic expression of Topo 2α and patient age, gender, smoking
history (P>0.05). While cytoplasmic expression of Topo 2α was not
different, nuclear expression was significantly increased in squamous
cell carcinoma (P=0.008), OR (95% CI): 3.01 (1.31-6.92). Both
nuclear and cytoplasmic expression of Topo 2α did not show
any association with tumor diameter, pathological stage, tumor
differentiation and presence of relapse.
The association of Topo 2α expression and survival
The median overall survival was 34 [4-120] months. At the end
of study 47 (51.6%) patients were dead and 44 (48.4%) patients
were alive. There wasn’t any significant association between
nuclear or cytoplasmic expression of Topo 2α and survival
(P>0.05).
Multivariate analysis related to survival using Cox regression
is shown in Table 2. Tumor diameter (P=0.031) and involvement
of N2 lymph nodes (P=0.005) were independent prognostic
factors. They were related with poor prognosis.
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Discussion
Topo 2 is an essential nuclear enzyme that catalyzes the changes
in the topology of DNA. It plays a critical role during mitosis for
chromosome condensation and segregation, in both neoplastic
and nonneoplastic cells (5). There are two isoforms of Topo 2
in mammalian cells: Topo 2α and Topo 2β. In the experimental
studies on cell cycles, Topo 2α expression was undetectable
until the cells reached to late S phase, peaked in G2-M phase
and decreased after mitosis. Topo 2β expression was constant
through the cell cycle (11). Studies including specimens of lung
carcinoma patients revealed that Topo 2α gene expression was
significantly higher in tumor tissues compared to normal tissues.
But Topo 2β gene expression was not different between tumor
tissues and normal lung tissues (7,12,13). Therefore Topo 2α
expression is a specific marker of cell proliferation and is thought
to be related to poor prognosis.
There are several studies reporting that high expression of
Topo 2α was associated with poor prognosis in lung cancer
patients (9,14). In a study including tumor samples derived from
93 previously untreated SCLC patients, survival was shorter
in patients with extensive disease, poorer performance status,
and in patients whose tumors expressed high Topo 2α and Ki67
levels. High Topo 2β expression was found to be predictive for
lower chemotherapy response rates. The authors concluded that
immunohistochemical assessment of these markers in diagnostic
biopsies may give important prognostic information and may
help selecting patients in the worse prognostic categories for
novel therapeutic agents (9). Topo 2α was also studied as a
drug resistance marker in advanced NSCLC patients. There
wasn’t any relation between Topo 2α expression and response to
chemotherapy. But they observed a shorter survival in patients
with high Topo 2α levels. They explained the shorter survival
rate with higher Topo 2α expression, higher proliferation rate
and increased aggressiveness (14). In the present study, since we
studied patients who had a curative surgery, our hypothesis was
“Higher Topo 2α expression might be related to early recurrence
or in other words poorer survival”. But we couldn’t find a relation
between Topo 2α expression and survival.
Studies investigating the biological difference between
NSCLC and SCLC observed a lower Topo 2α expression in
NSCLC compared to SCLC patients (7,8,15). This stituation can be an explanation for the difference in chemosensitivity. Higher
Topo 2α expressing tumors are more chemosensitive than lower
Topo 2α expressing tumors. In a study of Giaccone et al., higher
expression of Topo 2α was correlated not only with sensitivity to
Topo 2α inhibitors, but also with sensitivity to other classes of
chemotherapeutic agents. They postulated that Topo 2α might
be an essential component of a common pathway of cell death
which is triggered by multiple or all antineoplastic agents (16).
In another study involving 103 squamous cell carcinomas of
the head and neck, higher Topo 2α expression was significantly
related to better response to chemotherapy, despite the cytotoxic
drugs used was not Topo 2α antagonists (17). In this study we
observed, 41% nuclear expression and 66% cytoplasmic expression
of Topo 2α. These ratios are high enough not to be ignored. Whereas
most NSCLC patients are resistant to Topo 2α drugs, it may be
possible to predict the minority of tumors which are sensitive.
Larger prospective studies comparing the chemotherapy
responses in Topo 2α expressing and not expressing NSCLC are
needed. In contrary to the study of Dingemans et al. (14), the
demonstration of higher chemosensitivity in higher Topo 2α
expressing tumors would be a milestone in managing NSCLC
patients.
There are a few studies investigating the difference of Topo
2α expression in the histological subtypes of NSCLC (13,18).
In these studies they analyse Topo 2α gene expression by
PCR method. In the study by Liu et al., Topo 2α gene expression
was significantly higher in squamous cell carcinomas than in
adenocarcinomas (18). Conversely to this study, Mirski et al. found
that Topo 2α levels were lower in squamous cell carcinomas than
in adenocarcinomas (13). In the present study we investigate the
intensity of immunostaining Topo 2α in tumor tissues. We found
a significantly higher nuclear expression of Topo 2α in patients
with squamous cell carcinoma.
There are also studies showing the association of Topo 2α
gene expression and tumor differentiation in breast carcinomas,
head and neck carcinomas and lung cancer (17-19). In a study
including surgically resected tumor specimens from 98 NSCLC
patients, Topo 2α gene expression was significantly higher in
moderately and poorly differentiated tumors compared to well
differentiated ones. The overexpression of Topo 2α gene was
associated with more aggressive carcinogenesis, accelerated
cell proliferation and tumor dedifferentiation. There wasn’t any
statistically significant relation between Topo 2α gene expression
and gender or pathological tumor stage (18). In our study we
couldn’t find a relation between Topo 2α expression and tumoral
differentiation.
There are a few studies investigating the association of Topo
2α expression with clinical parameters such as age and gender. As
in this study they did not find any relation (18,20). In this study
we didn’t find an association between Topo 2α expression and
smoking history. In study of Liu et al., Topo 2α expression was
higher in smokers (18). The reason for this difference may be
a relatively higher ratio of smoking history (90% present study,
63% Liu et al.) in the present study.
In conclusion; in this study we determined both the nuclear
and cytoplasmic expression of Topo 2α in tumor specimens of
surgically resected NSCLC patients by immunohistochemistry.
We investigated the association of both nuclear and cytoplasmic
expression of Topo 2α with clinical (age, gender, smoking
history, administration of adjuvant chemotherapy), pathological
parameters (tumor histology, stage, tumor diameter, involvement
of lymph nodes, differentiation) and prognosis. We couldn’t
find any association between Topo 2α expression and clinical
findings. Nuclear expression of Topo 2α was significantly
higher in patients with squamous cell carcinoma. There wasn’t
any association between Topo 2α expression and survival. As
expected tumor diameter and involvement of N2 lymph nodes
were independent prognostic parameters in multivariate analysis.
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Acknowledgements
Disclosure: The authors declare no conflict of interest.
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References
Cite this article as: Erguden HÇ, Koksal D, Demirag
F, Bayiz H, Mutluay N, Berktas B, Berkoglu M. The
association of topoisomerase 2α expression with
prognosis in surgically resected non-small cell lung
cancer (NSCLC) patients. J Thorac Dis 2012;4(4):352-
357. doi: 10.3978/j.issn.2072-1439.2012.08.04
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