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doi:10.1111/jgh.12498
GASTROENTEROLOGY
Key words
esophageal squamous cell carcinoma,
lymphatic metastatic recurrence, stathmin-1.
Accepted for publication 12 December 2013.
Correspondence
Professor Zhou Wang, Department of
Thoracic Surgery, Provincial Hospital Affiliated
to Shandong University, Jinan, Shandong
250021, China. Email:
wz620226@hotmail.com
Conflict of interest: The authors declare that
they have no competing interests.
Abstract
Background and Aim: Common patterns of the operative failure after Ivor-Lewis
esophagectomy in esophageal squamous cell carcinoma (ESCC) patients are locoregional
lymph node metastasis. It is clinically significant to investigate the biological markers to
predict the subset of patients who are at higher risk of lymphatic metastatic recurrence. Our
research aimed to investigate the association between the Stathmin (STMN-1) gene expression and lymphatic metastatic recurrence in pN0 ESCC patients after surgery.
Methods: One hundred seventy-four patients who suffered from mid-thoracic ESCC and
completely resected with Ivor-Lewis esophagectomy were enrolled in our study. The entire
patients were restricted to pN0 ESCC. Tissue specimens were examined for STMN-1
expression levels by immunohistochemistry and Western blotting methods. The correlation
of STMN-1 levels with clinicopathological variables, prognosis, and metastatic potential
was analyzed.
Results: One hundred patients had STMN-1 protein overexpression (57.47%), and the
patients with overexpression were accompanied by significantly higher rate of lymphatic
metastatic recurrence as compared with patients who had low STMN-1 expression
(P = 0.003). Multivariable Cox regression analysis revealed that the STMN-1 protein
expression and T classification were independent factors to predict the lymphatic metastatic recurrence (P = 0.007, P = 0.000, respectively).
Conclusions: Even pN0 ESCC are a potential to lymphatic metastatic recurrence.
Stathmin overexpression can be used as a marker to identify those patients who are at high
risk for lymphatic metastatic recurrence in pN0 ESCC after an Ivor-Lewis esophagectomy.
Introduction
Esophageal squamous cell carcinoma (ESCC) is the leading cause
of cancer death worldwide. More than half of global esophageal
cancers occur in China, which is one of the areas showing the
highest incidence rate of ESCC, a major histological type of
esophageal cancer.1 Surgery is the best option for curing patients in
the early stages of this disease. It remains the superior therapeutic
modality for control in patients with locally advanced disease.
Despite advances in treatment, the benefits of surgical resection
combined with chemotherapy or radiotherapy is not satisfactory.
The prognosis of ESCC patients still remains poor. More than half
of all ESCC patients die from tumor relapse or metastasis. Some
reports show that the main reason for the failure of these operations is the lymphatic metastatic recurrence. Therefore, to improve
the long-term survival rate of esophageal cancer, the key is to
control lymph node metastatic recurrence. According to the
National Comprehensive Cancer Network (NCCN) guidelines,
patients with ESCC who undergo complete resections are not
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J Akhtar et al.
Table 1 Correlations of stathmin gene expression and lymphatic metastatic recurrence with clinicopathological characteristics in esophageal
squamous cell carcinoma patients
Characteristics
Age
< 50
50
Gender
Male
Female
Tumor size
< 3 cm
35 cm
> 5 cm
Differentiation
Low
Mid-high
T status
T1
T2
T3
STMN-1 overexpression
Yes
No
No. of patients
n = 174
Low (n = 74)
STMN-1 expression
High (n = 100)
37
137
16
58
21
79
134
40
58
16
76
24
58
68
48
27
31
16
31
37
32
49
125
20
54
29
71
12
70
92
6
33
35
6
37
57
Recurrence rate
0.921
0.649
16 (43.2%)
61 (44.5%)
0.712
0.110
63 (47.0%)
14 (35.0%)
0.006
0.013
16 (27.6%)
34 (50.0%)
27 (56.3%)
0.775
0.171
24 (49.0%)
53 (42.4%)
0.016
100
74
0.000
3 (25.0%)
18 (25.7%)
56 (60.9%)
0.003
52 (52.0%)
25 (33.8%)
Methods
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J Akhtar et al.
block for 10 min. Antigen retrieval was performed using microwaves in 10 mM of citrate buffer for 15 min and processed
using immunohistochemical streptavidin peroxidase conjugate
method. Rabbit anti-STMN-1 polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). As for the negative
control, the primary antibody was replaced by the phosphatebuffered saline (PBS). All sections were examined by two independent pathologists who were blinded to the clinical data. The
immunohistochemical score (IHS) was calculated by combining
the proportion score (percentage of positive stained cells) with the
staining intensity score. The proportion score ranged from 0 to 4,
as follows: 0 (< 5%), 1 (524%), 2 (2549%), 3 (5074%), and
4 ( 75%). The staining intensity was scored as follows: 0 (negative), 1(weak), 2 (moderate), and 3(strong). The proportion score
and staining intensities score were then multiplied to generate
the IHS for each case. The case with IHS 4 was considered to be
positive expression (same as our previous study).7
Protein extraction and Western blotting. We prepared tissue extracts in RIPA lysis buffer (25 mM Tris pH 7.5, 1%
Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 150 mM
NaCl) containing a cocktail of protease inhibitors (Sigma, St.
Louis, MO, USA). Protein concentrations were determined using
the Bradford method. The Bio-Rad protein assay reagent (Bio-Rad,
Bio-Rad Laboratories, Richmond, CA, USA) and 2040 mg of
protein mixed with loading buffer was loaded per lane, separated by
12% sodium dodecylsulfate -polyacrylamide gel electrophoresis
(SDS-PAGE). Proteins were transferred to polyvinylidene fluoride
(PVDF) membrane filters (Millipore, Billerica, MA, USA). Nonspecific binding was blocked by incubation in PBS containing 0.1%
Tween 20 (PBS-T) and 5% skim milk. PVDF membranes were
blocked with 5% dry milk for 1 h at 4C. Membranes were
incubated in STMN-1 primary antibody (1:1000) overnight at
4C. The membranes were then incubated with the corresponding
secondary antibody (1:2000, horseradish peroxidase-conjugated
anti-rabbit) in TBST-5% nonfat milk for 1 h at room temperature,
and the immunoreactive bands were visualized using EZ ECL
Chemiluminescence Detection Kit for HRP (Biological Industries
Ltd, Kibbutz Beit Haemek, Israel). Images were acquired using
the LAS3000 Imager (Fujifilm, Fuji, Edison, NJ, USA).
Membranes were reprobed for beta-actin as a loading control.
rate was analyzed with the logrank test. P < 0.05 was considered
statistically significant. Risk factors of lymph node metastasis after
surgery were analyzed with Cox regression model. All statistical
analysis was performed using IBM SPSS version 21.0 (IBM
Corp., Armonk, NY, USA).
Results
Tumor metastatic recurrence and prognosis. Of the
enrolled one hundred seventy four patients, 98 patients (56.32%)
experienced the recurrence and metastatic disease. The median
disease free interval was 25.5 1.1 months (Fig. 1). The mode of
first recurrence of the tumor included lymphatic metastatic recurrence in 54 cases, hematogenous metastasis in 21 cases, lymphatic
metastatic recurrence and blood born metastasis in 23 cases. Lymphatic metastatic recurrence in pN0 Esophageal Squamous cell
Carcinoma Patients after Ivor-Lewis Esophagectomy is shown in
Figure 2. The 5-year survival probability in this group of patients
is shown in survival curves (Fig. 3).
STMN-1 protein expression analysis in tumor
tissue specimen. Immunohistochemical analysis of 174
ESCC specimens revealed that 100 patients (57.47%) showed
STMN-1 protein overexpression, as shown in Figure 4b,c.
STMN-1 protein expression was low or undetected in normal
esophageal tissue (Fig. 4a). In ESCC specimens, STMN-1 protein
expression was higher than in normal esophageal tissues.
Relationship between STMN-1 expression and
lymphatic metastatic recurrence. Based on the immunohistochemical analysis of STMN-1 protein expression, in 100
patients with STMN-1 protein overexpression, 52 (52.00%) of
our cases had lymphatic metastatic recurrence, while of 74 patients
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J Akhtar et al.
the lymphatic recurrence rate was higher than in patients with low
STMN-1 expression.
Based on both types of protein assay, the patients with higher
STMN-1 protein expression were accompanied by higher lymphatic metastatic recurrence.
Multivariate Cox regression analysis of lymphatic
metastatic recurrence. We performed multivariate Cox
regression analysis to identify variables that closely related to the
lymphatic metastatic recurrence in pN0 ESCC patients. Results
showed that tumor T stage and STMN-1 overexpression are independent factors of lymphatic metastatic recurrence in pN0 ESCC
(Table 2).
Discussion
ESCC is a malignant tumor with strong invasion and high frequency of lymph node metastasis. There are still about 40% of
them that are found to be lymph node micro-metastasis by IHC
and molecular biological methods.8 The long-term survival of
patients with mid-thoracic ESCC remains poor because of the high
incidence of lymph node metastases and early recurrence even
after curative surgery.911 The embryological structure of the
esophagus is different from other digestive tracts, especially in
presence of lymph-vessels in muscularis mucosae. Lymph node
metastasis of esophageal cancer can occur when the primary tumor
is very small. The lymphatic drainage of the esophagus is complex
with a rich lymphatic network, and lymph node metastasis may
present as regional metastasis, skipping metastasis, or distant
metastasis.1214 The middle thoracic ESCC has bidirectional metastasis trend, including upward spread to neck and upper mediastinum, and downward to thoracic lower mediastinum abdomen.15
All of the above likely contribute to the high rate of local and
regional lymph node recurrence after resection.
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J Akhtar et al.
Figure 4 Expression
analysis
of
the
stathmin (STMN-1) protein by immunohistochemistry in esophagus and esophageal squamous cell carcinoma (ESCC) specimens. (a)
Normal esophageal tissue shows no STMN-1
protein expression. Original magnification,
400. (b) Strong STMN-1 protein expression
in ESCC specimen, STMN-1 expression was
mainly localized within cytoplasm. Original
magnification, 400. (c) Moderate STMN-1
expression in ESCC specimen. Original magnification, 400. (d) Low STMN-1 expression
in ESCC specimen. Original magnification,
400.
A complete surgical resection is considered to be first-line treatment for individuals with localized ESCC1618 and remains the
superior therapeutic modality for patient with locally advanced
disease. On the basis of oncology, McKeown (three-incision)
esophagogastrectomy with three-field lymphadenectomy is
regarded as the best approach for mid-thoracic ESCC. However,
this procedure has been criticized because it is very invasive and
has a high incidence of complications.1922 Whether three-field
lymph node dissection should be performed in all patients with
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J Akhtar et al.
Table 2
surgery
Multivariate Cox regression analysis for risk factor of lymphatic metastatic recurrence in esophageal squamous cell carcinoma patients after
Risk factors
SE
Wald
d.f.
Sig.
Exp (B)
95.0% confidence
interval for exp (B)
Lower
Upper
Age
Gender
Size
T descriptor
Differentiation
STMN-1
0.269
0.468
0.112
0.924
0.235
0.661
0.287
0.308
0.171
0.250
0.257
0.247
0.878
2.306
0.426
13.671
0.837
7.160
1
1
1
1
1
1
0.349
0.129
0.514
0.000
0.360
0.007
1.309
1.597
1.118
2.519
1.265
1.936
0.745
0.873
0.799
1.544
0.765
1.193
2.297
2.921
1.565
4.112
2.093
3.14
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