Keywords
esophageal neoplasms - lymphatic metastasis - surgical procedures - metastasis-associated
gene 1
Introduction
Esophageal carcinoma is one of the most common malignant tumors worldwide, and its
incidence is very high in China. Surgical resection is the primary treatment but its
effect nowhere near the level required, with the overall 5-year survival rate of only
30–50%. To improve the overall cure rate of patients with esophageal cancer, it should
be emphasized that we should not only remove the tumor completely but also take targeted
measures as postoperative adjuvant treatment. However, after complete resection of
esophageal squamous cell carcinoma, patients whether should adopt adjuvant therapy
is still controversial. In 2009, NCCN esophageal cancer treatment guidelines pointed
out that patients of esophageal squamous cell carcinoma after complete resection do
not require postoperative adjuvant therapy. Our previous reports indicated that even
if patients with esophageal squamous cell carcinoma achieved a complete resection,
there were still some patients with relapse, metastasis,[1]
[2] and further study found that postoperative radiotherapy can reduce local recurrence
rate.[3] Accordingly, we cannot fully agree with the views of NCCN.
Except for clinical and pathological indicators, such as TNM staging, a new molecular
biomarker which can identify the patients with poor prognosis has great clinical value
as the indicator of that whether a postoperative adjuvant therapy is needed. Metastasis
associated gene 1 (MTA1) was discovered recently as a tumor invasion and metastasis-related
gene whose overexpression showed positive correlation with tumor invasion and metastasis.[4] We previously found that MTA1 protein overexpression of pN0 patients with midthoracic
esophageal squamous cell carcinoma was related to poor prognosis.[5] To further explore the relationship between MTA1 overexpression and the prognosis
of patients, the retrospective analysis of esophageal squamous cell carcinoma of different
stages patients with MTA1 overexpression, and prognosis factors of multiple regression
analysis, aimed at exploring the value of MTA1 in forecasting the prognosis after
complete resection in patients with mid thoracic esophageal cancer.
Materials and Methods
Materials
From January 2002 to January 2005, we have analyzed retrospectively the midthoracic
esophageal squamous cell carcinoma patients who underwent complete resection (R0 resection)
with intact clinical information. The specimens consisted of 130 male and 44 female
patients aged 38 to 76 years (average 56.7) ([Table 1]), including 100 patients who underwent Ivor-Lewis esophagectomy and other 74 patients
underwent esophagectomy through the left chest with esophagogastrostomy above the
aortic arch (Left chest incision). Preoperative radiotherapy or chemotherapy was not
taken to all cases. The tumor location and the TNM classification were determined
according to the International Union Against Cancer (UICC) in 1997; and the two-field
lymph node section criteria was based on the lymph node introduction from the American
Joint Committee on Cancer (AJCC)/UICC.
Table 1
The correlations between MTA1 protein overexpression and clinicopathologic factors
|
Characteristics
|
Numbers
|
5-year survival (%)
|
P
[*]
|
MTA1(−)
|
MTA1(+)
|
χ2
|
P**
|
|
Gender
|
|
|
0.928
|
|
|
0.11
|
0.732
|
|
Male
|
130
|
34.6
|
|
70
|
60
|
|
|
|
Female
|
44
|
31.8
|
|
25
|
19
|
|
|
|
Age (y)
|
|
|
0.931
|
|
|
0.21
|
0.644
|
|
≤0
|
38
|
34.2
|
|
22
|
16
|
|
|
|
>0
|
136
|
33.8
|
|
73
|
63
|
|
|
|
Size of tumor
|
|
|
0.096
|
|
|
2.74
|
0.253
|
|
<3 cm
|
41
|
51.2
|
|
27
|
14
|
|
|
|
3–5 cm
|
70
|
30.0
|
|
36
|
34
|
|
|
|
>5 cm
|
63
|
27.0
|
|
32
|
31
|
|
|
|
Operation
|
|
|
0.754
|
|
|
0.64
|
0.424
|
|
Ivor-Lewis
|
100
|
34.0
|
|
52
|
48
|
|
|
|
Left chest incision
|
74
|
33.8
|
|
43
|
31
|
|
|
|
TNM staging
|
|
|
0.000
|
|
|
5.93
|
0.052
|
|
Stage I (T1N0M0)
|
10
|
80.0
|
|
8
|
2
|
|
|
|
Stage II (T2,3N0M0)
(T1,2N1M0)
|
95
|
37.9
|
|
56
|
39
|
|
|
|
Stage III (T3N1M0)
|
69
|
21.7
|
|
31
|
38
|
|
|
|
T status
|
|
|
0.017
|
|
|
8.17
|
0.017
|
|
T1
|
12
|
66.7
|
|
9
|
3
|
|
|
|
T2
|
47
|
40.0
|
|
32
|
15
|
|
|
|
T3
|
115
|
27.8
|
|
54
|
61
|
|
|
|
N status
|
|
|
0.000
|
|
|
4.15
|
0.042
|
|
Yes
|
91
|
22.0
|
|
43
|
48
|
|
|
|
No
|
83
|
47.0
|
|
52
|
31
|
|
|
|
Loss of weight
|
|
|
0.482
|
|
|
0.70
|
0.042
|
|
Yes
|
24
|
29.2
|
|
15
|
9
|
|
|
|
No
|
150
|
34.7
|
|
80
|
70
|
|
|
|
Differentiation
|
|
|
0.366
|
|
|
0.00
|
0.967
|
|
Well/moderately
|
139
|
35.3
|
|
76
|
63
|
|
|
|
Poorly
|
35
|
28.6
|
|
19
|
16
|
|
|
|
MTA1 overexpression
|
|
|
0.000
|
|
|
|
|
|
Yes
|
79
|
19.0
|
|
|
|
|
|
|
No
|
95
|
46.3
|
|
|
|
|
|
* Log-rank test; ** χ2 test.
The patients with serious postoperative complications and perioperative mortality
were not enrolled in the study. After 5 years, follow-up data were obtained by telephone
or mail from the patients or his or her family. The last check on follow-up of all
patients was performed in June 2010. They were evaluated by clinical history, physical
examination, laboratory analysis, barium esophagram, computed tomography, ultrasound
examination, and fiberoptic esophagoscopy if necessary. The median follow-up period
was 47 months.
Surgical Procedure
Ivor-Lewis esophagectomy: With a right anterolateral thoracotomy, the chest was entered through the fourth
intercostal space. The azygos vein arch was cut off, and the esophagus was dissected
from esophagogastric junction to the apex of the chest. When the tumor invaded significantly
outside, the thoracic duct was routinely ligated above the diaphragm. Then an upper
midline abdominal incision was made, and the abdomen was explored. During the mobilization
of the stomach, the right gastroepiploic vessels and arcades must be preserved. The
left gastric artery was cut off at its origin. Subsequently, the hiatus was enlarged
and the stomach was carried to the right chest. An end-to-side esophagogastric mechanical
anastomosis was performed in the apex of the chest. 2R, right upper paratracheal nodes;
3P, posterior mediastinal nodes; 4R, right lower paratracheal nodes; 7, subcarinal
nodes; 8M, middle paraesophageal lymphnodes; 9, pulmonary ligament nodes; 16, paracardial
nodes; 17, left gastric nodes were dissected.
Left chest incision: Left posterolateral thoracotomy incision through the sixth intercostal space. The
thoracic esophagus was liberated to the apical pleura. When the tumor invasion obviously
extended outside the esophagus, the thoracic duct was routinely ligated above the
diaphragm. The greater and lesser curvatures of the stomach were liberated through
the radial incision of the diaphragm. The right gastroepiploic vessels and arcades
should be preserved, but the left gastric artery and vein should be ligated at the
origin. The stomach was then pulled to the chest above the aortic arch, and the mechanical
anastomosis was performed within the left apex of the chest. The fields of lymph nodes
dissection were as follows: 4L, left lower paratracheal nodes; 7, subcarinal nodes;
8M, middle paraesophageal lymphnodes; 9, pulmonary ligament nodes; 16, paracardial
nodes; 17, left gastric nodes. And 2L, left upper paratracheal nodes were dissected
selectively.
This paper has no further analysis of postoperative adjuvant therapy because postoperative
chemotherapy programs and chemotherapy cycle were not unified, and also due to the
lack of balance and comparability.
MTA1 Detection
Anti-MTA1 goat polyclonal antibody was raised against a peptide mapping at the C-terminus
of MTA1 of human origin (sc-9446, Santa Cruz Biochemistry, Santa Cruz, California,
United States). Immunohistochemical staining for MTA1 protein was performed using
the avidin–biotin peroxidase complex method with 3,30-diaminobenzidine as a chromogen
using an LSAB kit (Dako, Carpenteria, California, United States). Slides were deparaffinized
and rehydrated with xylene and graded alcohol. The slides were then incubated in 3%
hydrogen peroxide for 10 minutes to inactivate the endogenous peroxidase. Optimal
antigen retrieval was performed in citrate buffer (pH = 6.0) for 10 minutes with a
steam oven to enhance the immunoreactivity. The primary antibody against MTA1 was
used at a dilution of 1:100. Subsequently, the secondary biotinylated antibody and
avidin-biotin complex reagent were applied, and the slides were counterstained. The
positive cells of the MTA1 protein staining was the brown particles appeared in the
nucleus, and with the positive outcome assessed by the percentages of the positive
cells. For MTA1 protein assessment, immunoreactivity was evaluated using a semiquantitative
scoring system for both staining intensity (0, negative staining; 1, weak staining;
2, moderate staining; 3, intense staining) and percentage of positively stained cancer
cells (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, C76%). The final staining score
was the sum of the scores of staining intensity and percentage of positive cells,
and was further graded as follows: (-), 0 to 1; (+), 2 to 3; (++), 4 to 5; (++ + ),
6 to 7. Tumors with final staining score ≥4 were defined as overexpressing MTA1 protein[6] ([Fig. 1]). All sections were judged by two pathologists together in blind principle and the
agreement was reached by negotiation when inconsistent cases emerged.
Fig. 1 MTA1 protein expression in esophageal carcinoma. (A) Well differentiated; (B) medium
differentiated; (C) poorly differentiated (IHC ×200).
Statistical Methods
All statistics analyses were performed with SPSS 10.0 statistical software. The Kaplan–Meier
method was used to calculate the survival rate, and the log-rank test was performed
to identify the difference of survival. Cox regression multivariate analysis was performed
to judge the independent prognostic factors.
Results
The overall 5-year survival rates of 174 patients was 33.9%, and the rates of stage
I, stage II and stage III was 80.0, 37.9, and 21.7%, respectively (survival curve
depicted in [Fig. 2]).
Fig. 2 Kaplan-Meier survival curves for 174 patients.
Of the 174 patients in this study, recurrence was recognized in 89 patients (51.1%)
in the first 3 years after operation. The distribution of the sites of tumor recurrence
is shown in [Table 2]; 51 patients (26.5%) developed a locoregional recurrence; 38 patients (22.4%) developed
a hematogenous recurrence, including 8 patients (5.1%) with simultaneous locoregional
and hematogenous recurrence.
Table 2
Site of recurrence in 89 patients
|
Site of recurrence
|
Number of patients (%)
|
|
Locoregional recurrence
|
51/89 (57.3)
|
|
Mediastinal node
|
30/51 (58.8)
|
|
Cervical/supraclavicular node
|
9/51 (17.6)
|
|
Multiple nodes[a]
|
9/51 (17.6)
|
|
Abdominal node
|
3/51 (5.9)
|
|
Hematogenous recurrence
|
30/89 (33.7)
|
|
Liver
|
13/30 (43.3)
|
|
Bone
|
6/30 (20.0)
|
|
Lung
|
5/30 (16.7)
|
|
Multiple organs[b]
|
4/30 (13.3)
|
|
Brain
|
1/30 (3.3)
|
|
Pleura
|
1/30 (3.3)
|
|
Locoregional and hematogenous[c]
|
8/89 (8.9)
|
a Mediastinal and cervical node recurrence in six patients; mediastinal and abdominal
node recurrence in three patients.
b Liver and bone recurrence in three patients; liver and brain recurrence in one patient.
c Liver recurrence in four patients; lung recurrence in three patients; bone recurrence
in one patient.
MAT1 Overexpression
Of all the 174 esophageal cancer specimens, MTA1 protein overexpression was present
in 79 (45.4%) specimens. The overexpression rates of MTA1 protein in stage I, stage
II and stage III were 20.0% (2/10), 41.1% (39/95), and 55.0% (38/69), respectively.
The difference of MTA1 protein overexpression between them was not statistically significant
(χ2 = 5.9, p = 0.052). The overexpression rates of MTA1 protein in T1, T2, and T3 were 25.0% (3/12),
31.9% (15/47), and 53.0% (61/115), respectively. The difference of MTA1 protein overexpression
between them was statistically significant (χ2 = 8.1, p = 0.017). The overexpression rates of MTA1 protein in patients with lymph node metastasis
and without metastasis were 52.7% (48/91) and 37.3% (31/83), respectively. It was
statistically significant between them (χ2 = 4.1, p = 0.042).
The Relationship between MTA1 Overexpression and Prognosis
In this group specimens, 79 cases of MTA1 protein overexpression in patients with
5-year survival rates were 19.0%, 95 patients without MTA1 overexpression with 5-year
survival rates were 46.3%, difference between them was statistically significant (χ2 = 14.3, p = 0.000, [Fig. 3]).
Fig. 3 Kaplan-Meier survival curves for patients with and without MTA1 expression.
In the similar TNM stage because of different MTA1 overexpression, there were also
differences in the prognosis of patients. Stage II patients, with or without MTA1
protein overexpression in 5-year survival rates were 25.6 and 47.4%, the difference
between the two groups was statistically significant (χ2 = 4.7, p = 0.031); stage III patients, with or without MTA1 protein overexpression in the
5-year survival rates were 10.5 and 35.5%, the difference between them was statistically
significant (χ2 = 4.1, p = 0.042); however, stage I patients, with or without MTA1 protein overexpression
in 5-year survival rates were 50.0 and 87.5%, the difference between the two groups
was not statistically significant (χ2 = 1.8, p = 0.176).
Different TNM stages of patients have different prognosis due to different MTA1 overexpression.
T2 patients with or without MTA1 protein overexpression in 5-year survival rates were
20.0 and 50.0%, the difference between them was statistically significant (χ2 = 5.0, p = 0.025); T3 patients with or without MTA1 protein overexpression in 5-year survival
rates were 18.0 and 48.9%, difference between the two groups was statistically significant
(χ2 = 3.9, p = 0.048); but T1 patients with or without MTA1 protein overexpression in 5-year survival
rates were 33.3 and 37.8%, the difference between them was statistically significant
(χ2 = 3.5, p = 0.061).
In patients with and without lymph node metastasis because of different reason of
MTA1 overexpression, there were differences in the prognosis of patients also. In
pN0 patients with or without MTA1 protein overexpression in 5-year survival rates
were 29.0 and 57.7%, respectively, the difference between them was statistically significant
(χ2 = 6.8, p = 0.009); pN1 patients with or without MTA1 protein overexpression in 5-year survival
rates were 12.5 and 32.6%, the difference between them was statistically significant
(χ2 = 4.5, p = 0.032).
According to the univariate analyses, the overexpression of MTA1 is correlative with
T and N classifications, and the multivariate analyses of these factors that may be
correlative with the MTA1 indicated that the T classification is the independent pathologic
risk factor of MTA1 overexpression ([Table 3]).
Table 3
Logistic regression analysis for MTA1 overexpression
|
Characteristics
|
B
|
Wald
|
P
|
OR
|
95% CI
|
|
Gender
|
0.037
|
0.010
|
0.920
|
1.038
|
0.502–2.147
|
|
Age
|
−0.088
|
0.052
|
0.820
|
0.916
|
0.431–1.949
|
|
Size of tumor
|
0.110
|
0.259
|
0.611
|
1.116
|
0.731–1.704
|
|
T status
|
0.634
|
4.820
|
0.028
|
1.885
|
1.070–3.321
|
|
N status
|
0.445
|
1.888
|
0.169
|
1.560
|
0.827–2.943
|
|
Differentiation
|
0.032
|
0.006
|
0.936
|
1.032
|
0.477–2.235
|
Abbreviations: B, regression coefficient; Wald, Wald value; OR, odds ratio; CI, confidence
interval.
Cox Regressive Analysis for Prognostic Risk Factor
The Cox regression analysis revealed ([Table 4]) that N status and MTA1 protein overexpression were independent prognostic factors.
Table 4
Cox regression analysis of the risk factor on esophageal cancer after esophagectomy
|
Characteristics
|
B
|
Wald
|
P
|
OR
|
95% CI
|
|
Gender
|
0.009
|
0.002
|
0.969
|
1.009
|
0.655–1.554
|
|
Age
|
0.106
|
0.214
|
0.644
|
1.112
|
0.709–1.743
|
|
Size of tumor
|
0.018
|
0.018
|
0.894
|
1.018
|
0.783–1.324
|
|
T status
|
0.230
|
1.613
|
0.204
|
1.259
|
0.883–1.795
|
|
N status
|
0.710
|
11.942
|
0.001
|
2.033
|
1.359–3.040
|
|
MTA1 overexpression
|
0.564
|
8.186
|
0.004
|
1.758
|
1.194–2.587
|
|
Operation
|
0.169
|
0.748
|
0.387
|
1.184
|
0.808–1.735
|
|
Differentiation
|
0.205
|
0.764
|
0.382
|
1.227
|
0.775–1.942
|
Abbreviation: CI, confidence interval.
Discussion
Middle third thoracic esophagus is the predilection site of esophageal squamous cell
carcinoma, and the tumors of this part are always accompanied with lymph nodes metastasis
in the mediastinum, abdominal cavity, and neck. There were ~40% of patients with lymph
node metastasis by immunohistochemical and molecular biological methods for further
examination even though they were diagnosed pN0 in the routine pathological examination
previously.[7] It seems that subtotal esophagectomy with three-field lymph nodes dissection is
an ideal surgical procedure for local control or even cure of the esophageal carcinoma.
Theoretically, the surgical procedure has many advantages, but its overall effect
for esophageal carcinoma patients is not so satisfactory. Many patients still die
of local recurrence and/or hematogenous metastasis. So this surgical procedure has
not been widely adopted at home and abroad, besides the serious surgical trauma and
latent complications cannot be ignored.[8]
[9]
[10] Currently, there are many different surgical procedures for midthoracic esophageal
carcinoma, and we always take lvor-Lewis esophagectomy and esophagectomy through the
left chest with esophagogastrostomy above the aortic arch.
We have analyzed the prognosis of the patients with midthoracic esophagus cancer undergone
Ivor-Lewis esophagectomy,[11]
[12] and found that the first tumor recurrence of 28.7% patients was mediastinal or cervical
lymph node metastasis within 3 years after the operation, the postoperative adjuvant
radiation therapy for mediastinum, bilateral supraclavicular fossa and the root of
the neck can significantly reduce the incidence of lymph node metastasis. Based on
the results of previous studies, we cannot fully agree with the view of NCCN: “the
postoperative adjuvant radiation therapy is not necessary for the complete resection
of esophageal squamous cell carcinoma patients.” In our opinion, middle third thoracic
esophageal carcinoma patients of high-risk tumor recurrence and metastasis, even though
have accepted complete resection, should adopt targeted auxiliary treatment measures.
Prognosis of esophageal cancer patients is always predicted by TNM staging in clinic
but sometimes lack of sensitivity. The prognosis prediction has great potential clinical
value, and therefore it will be of great significance to detect the molecular biology
signs, furthermore, identification of novel biomarker that could be utilized as a
possible therapeutic target or prognostic predictor may be employed as an adjunct
to the staging system and contribute to optimize treatment for esophageal cancer patients.
MTA1 gene encodes a protein with 703 amino acids, molecular mass of 79.4 kDa, its
amino acid sequence contains multiple tyrosine kinase, protein kinase C, and casein
kinase-2 phosphorylation sites.[4]
[13] The MTA1 protein is a component of the nucleosome remodeling and histone deacetylation
(NURD) complex, which is associated with ATP-dependent chromatin remodeling and histone
deacetylase activity. Metastasis-associated protein 1 functions in conjunction with
other components of NURD to mediate transcriptional repression as it facilitates the
association of repressor molecules with the chromatin.[13]
[14]
[15]
[16] MTA1 along with its protein product overexpression was closely associated with tumorigenesis
and aggressiveness of a wide range of human malignant tumors.[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] The study showed that MTA1 protein overexpression was common in early-stage NSCLC
and was significantly associated with tumor angiogenesis and poor survival.[6] Haili Qian's research showed that the MTA1 expression associates with the invasion
and migration of esophageal cancer cells in vitro.[25] In Y Toh's study, esophageal tumors overexpression of MTA1 mRNA showed significantly
higher frequencies of adventitial invasion and lymph node metastasis.[26] We have had studied the prognostic significance of the MTA1 protein overexpression
in pN0 esophageal cancer patients and found that overexpression of MTA1 protein is
an independent prognostic risk factors, patients with MTA1 protein overexpression
have shorter disease-free survival and lower 5-year survival rate. In this paper,
we studied the midthoracic esophageal cancer with different TNM staging and found
that there were significant differences in MTA1 protein overexpression of different
TNM classification of tumors. The patients with MTA1 protein overexpression have a
lower 5-year survival rate, patients of stage II and stage III with or without MTA1
protein overexpression have significant statistically differences in 5-year survival
rate; Cox regression analysis confirmed that MTA1 protein overexpression was an independent
adverse prognostic factor. Except for the analysis of the selected various prognostic
factors, the postoperative adjuvant radiotherapy and chemotherapy may affect the local
recurrence and long-term survival of cancer patients. This paper has no further analysis
of postoperative adjuvant therapy because the study of this group is retrospective
and postoperative adjuvant treatment programs are not unified, and it lacks balance
and comparability.
Given that there is no well-accepted standardized surgical method for midthoracic
esophageal cancer, lvor-Lewis esophagectomy and esophagectomy through the left chest
with esophagogastrostomy above the aortic arch could be an optional operation method
for midthoracic esophageal cancer. Even though these two methods can accomplish the
complete resection of tumor, the overall 5-year survival rate was just 33.9%, so the
targeted postoperative adjuvant treatment measure for patients is necessary. This
study showed that the patients with MTA1 protein overexpression have a lower 5-year
survival rate, we concluded that MTA1 gene overexpression can be used as a molecular
biological marker to predict the prognosis of middle esophageal squamous cell carcinoma,
and it is recommended that we should suggest the patients with MTA1 gene overexpression
to take positive postoperative adjuvant therapy.
