Download Expression Level of Valosin-Containing Protein

5558 Vol. 10, 5558 –5565, August 15, 2004
Clinical Cancer Research
Expression Level of Valosin-Containing Protein (p97) Is Associated
with Prognosis of Esophageal Carcinoma
Shinji Yamamoto,1 Yasuhiko Tomita,2
Yoshihiko Hoshida,2 Norishige Iizuka,2
Shinya Kidogami,2 Hiroshi Miyata,1
Shuji Takiguchi,1 Yoshiyuki Fujiwara,1
Takushi Yasuda,1 Masahiko Yano,1
Shoji Nakamori,1 Masato Sakon,1
Morito Monden,1 and Katsuyuki Aozasa2
Departments of 1Surgery and Clinical Oncology and 2Pathology,
Osaka University Graduate School of Medicine, Osaka, Japan
ABSTRACT
Purpose: Esophageal squamous cell carcinoma (ESCC)
frequently shows a poor prognosis because of the occurrence
of systemic metastasis, mainly via lymphatic vessels. Valosin-containing protein (VCP) has been shown to be associated with antiapoptotic function and metastasis via activation of the nuclear factor-␬B signaling pathway. In the
present study, we examined the association of VCP with the
recurrence and prognosis of ESCC.
Experimental Design: VCP expression in 156 ESCC
patients [139 males and 17 females; age range, 38 – 82 (median, 60) years] was analyzed by immunohistochemistry.
Staining intensity in tumor cells was categorized as weaker
(level 1) or equal to or stronger (level 2) than that in endothelial cells. The correlation of VCP expression between the
mRNA and protein levels was examined in 12 patients.
Results: Fifty-seven (37.3%) cases showed level 1 and 96
(62.7%) level 2 VCP expression. Quantitative reverse transcription-PCR analysis revealed greater VCP mRNA expression in level 2 (n ⴝ 6) than level 1 cases (n ⴝ 6; P <
0.05). ESCC with level 2 expression showed higher rates of
lymph node metastasis (P < 0.01) and deep tumor invasion
(P < 0.01), and poorer disease-free and overall survival
rates (P < 0.001 for both analyses) than ESCC with level 1
expression. Multivariate analysis revealed that VCP expression level is an independent prognosticator for disease-free
and overall survival. Furthermore, VCP level was an indicator for disease-free survival in the early (pT1) and the
advanced (pT2–pT4) stage groups.
Received 5/12/03; revised 5/12/04; accepted 5/18/04.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Yasuhiko Tomita, Department of Pathology
(C3), Osaka University Graduate School of Medicine, 2-2 Yamadaoka,
Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3711; Fax: 81-6-68793719; E-mail: [email protected].
Conclusion: This study demonstrated the prognostic
significance of VCP expression in ESCC.
INTRODUCTION
There is remarkable geographic variation in both the incidence and the constituents of esophageal cancer. In Asian countries, where esophageal cancer is common, more than 90% of
esophageal cancers are squamous cell carcinomas (1–3). In
Western countries, where the incidence of esophageal cancer is
relatively low, the incidence rate of esophageal adenocarcinoma
has been increasing in the last few decades, whereas that of
esophageal squamous cell carcinoma (ESCC) remains constant
(4, 5). As a result, ESCC constitutes 50 – 60% of all esophageal
cancers in Western countries at present (4, 5).
ESCC shows a poor prognosis because of the occurrence of
systemic metastasis, mainly via lymphatic vessels (1– 6). In
Japan, 9991 patients died from ESCC in 1999, accounting for
3.44% of all cancer deaths (2). Detection of ESCC at an early
stage is possible with the use of X-ray and endoscopic examinations (7, 8), but there might be occult micrometastases at the
time of surgery even in such cases (9, 10). In this respect, an
assessment of metastatic potential is important to establish appropriate therapeutic modalities for ESCC.
Previous studies proposed the prognostic significance of
several clinicopathologic factors for ESCC, such as tumor size,
age at diagnosis, and primary site (5, 6). Among them, depth of
cancer invasion in the esophageal wall and lymph node metastasis are the main factors for ESCC recurrence (5, 6, 11), and
these two factors together with distant metastasis have been
included in the pathological Tumor-Node-Metastasis (pTNM)
staging for ESCC (12). However, the prognosis for ESCC, even
in cases of early-stage disease (pT1), is heterogeneous, and a
strategy to establish appropriate therapeutic modalities for each
patient has yet to be formulated.
To reinforce the prognostic utility of the pTNM classification, several biological indices, such as aberrant expression or
mutation of the tumor suppressor p53, adhesion molecule Ecadherin, and cell-cycle-related molecules such as p27, have
been proposed (3, 13, 14). However, the utility of these factors
for the prediction of prognosis in ESCC is controversial (15).
Recently we identified the gene encoding valosin-containing protein (VCP; also known as p97) as associated with metastatic potential in a murine osteosarcoma cell line using the
mRNA subtraction technique (16). VCP, a member of the superfamily ATPases associated with various cellular activities, is
known to be involved in the ubiquitin-dependent proteasome
degradation pathway (17), which works in both the up-regulation of cell proliferation and the down-regulation of cell death in
human cancer cells (18). These findings suggest that the expression level of VCP could be used to predict the metastasis and
prognosis of patients with cancers.
In the present study, we analyzed VCP expression in 156
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Clinical Cancer Research 5559
patients with ESCC and evaluated its correlation with clinicopathologic features and survival. In parallel, we examined expression of the tumor suppressor p53 and the proliferative
activity of tumor cells shown by Ki-67 immunohistochemistry
and evaluated the correlation between VCP expression and these
markers. All of the procedures in the present study were approved by the Ethics Committee, Osaka University Graduate
School of Medicine.
PATIENTS AND METHODS
Patients. For the present study, 156 patients who had
undergone surgery for ESCC at the Gastroenterological Surgery
Division, Osaka University Hospital during the period from July
1989 to February 2002 were selected. There were 140 males and
16 females (age range, 38 – 82 years; median, 60 years). An
endoscopic examination of esophageal lesions was performed,
and a histologic diagnosis of ESCC was made based on biopsy
specimens obtained before surgery. Preoperative diagnostic examinations, including esophagography, computed tomography,
and ultrasound, were performed for the purpose of clinical
staging. All patients underwent curative surgery for ESCC. The
types of surgery used were subtotal esophagectomy with lymph
node resection in 148 patients, partial esophagectomy in 6, and
endoscopic mucosal resection in 2. The resected esophagus was
examined macroscopically to determine the location and size of
the tumor. Tumors were located in the cervical esophagus in 10,
the upper third of the thorax in 7, the middle third of the thorax
in 80, the lower third of the thorax in 48, and the abdominal
esophagus in 11 patients. The size of the main tumor ranged
from 2 to 100 mm (median, 44 mm). Samples obtained from the
esophageal lesions were fixed in 10% formalin and processed
routinely for paraffin embedding; the number of specimens
prepared for histologic examination per case ranged from 6 to
46. Histologic sections cut 4-␮m thick were stained with hematoxylin and eosin and by immunoperoxidase procedures (the
avidin-biotin complex method). Histologic sections were reviewed by one of the authors (Y. H.) to define the depth and
mode (expansive or infiltrative) of cancer invasion in the esophagus, lymph node metastasis, and histologic subtype of ESCC
based on the criteria of the Japanese Esophagus Society (19).
Tumor stages were defined based on the pTNM classification
(12).
After surgery, all patients underwent laboratory examinations such as routine peripheral blood cell counts and measurement of the serum squamous cell carcinoma antigen levels every
1– 6 months, and chest roentgenography, ultrasonography of the
liver, a computerized tomographic scan of the thorax and abdomen, and an endoscopic examination of the remaining esophagus at intervals of 6 –12 months. Pre- and postoperative chemotherapy and radiotherapy were performed in 47 patients at high
risk for tumor recurrence, i.e., presence of lymph node metastasis, tumor with a diameter larger than 10 cm, and tumor
invasion beyond the muscularis propria. The types of adjuvant
therapies were as follows: preoperative chemotherapy in 21
cases, radiotherapy in 1 case, and combined chemo- and radiotherapy in 20 cases; postoperative chemotherapy in 1 case,
radiotherapy in 1 case, and combined chemo- and radiotherapy
in 2 cases; and combined preoperative chemo- and radiotherapy
and postoperative chemotherapy in 1 case. The radiation dose
ranged from 40 to 60 Gy. The chemotherapeutic agents used
were 5-fluorouracil and cisplatinum. The follow-up period for
survivors ranged from 1 to 96 months (median, 52.0 months).
Immunohistochemistry. The immunoperoxidase procedure (avidin-biotin complex method) was performed with
paraffin-embedded sections; one representative section per case
was selected. Briefly, antigen retrieval was performed by heating the sections in a 10 mM citrate buffer for 5 min. Mouse
monoclonal anti-VCP (p97) antibody (PROGEN Biotechnik,
Heidelberg, Germany), anti-p53 monoclonal antibody (Dako
Cytomation A/S, Copenhagen, Denmark), and anti-Ki-67 (clone
MIB 1) monoclonal antibody (Dako Cytomation A/S) were used
as the primary antibodies at dilutions of 1:3000, 1:100, and
1:100, respectively. Sections were lightly counterstained with
methyl green. For negative controls, nonimmunized mouse IgG
serum (Vector Laboratories, Burlingame, CA) was used as the
primary antibody, and uniformly gave negative results. Stained
sections were evaluated in a blinded manner without previous
knowledge of the clinicopathologic parameters. Endothelial
cells showed positive staining with a constant intensity; thus the
staining intensity in the cytoplasm of the tumor cells was shown
in comparison with that of endothelial cells and categorized as
follows: weaker (level 1 VCP expression) or equal to or stronger
than (level 2) that in endothelial cells. Cases showing combined
level 1 and 2 staining in different areas of the tumor were
classified as having level 2 expression. For p53 and Ki-67
immunohistochemistry, cells showing intranuclear staining
were judged positive. The p53- or Ki-67-positive cells among
200 tumor cells were counted, and the percentage was taken as
the p53 and Ki-67 labeling index. Cases were divided into two
groups: for p53, level 1, p53 labeling index ⬍ 10%; level 2, p53
labeling index ⱖ 10%; for Ki-67, level 1, Ki-67 labeling index ⬍ 20%; level 2, Ki-67 labeling index ⱖ 20%.
Quantitative Reverse Transcription-PCR Analysis of
VCP Expression. Quantitative reverse transcription-PCR was
performed on fresh-frozen samples from 12 patients with ESCC
as described previously (20). Briefly, 10 ␮g of DNase I-treated
total RNA were used for reverse transcription with Superscript
III (Invitrogen, Carlsbad, CA). An aliquot representing 100 ng
of input RNA was amplified by quantitative real-time PCR
using the TaqMan PCR Reagent Kit and Assay-on-Demand
Gene Expression Products (Applied Biosystems, Foster City,
CA; Refs. 21, 22). RNA extracted from a noncancerous lesion in
one patient was used as a standard. After reverse transcription,
standard cDNA was serially diluted to obtain five standard
solutions for use in PCR to generate the reference curve. The
relative amount of cDNA in each sample was measured by
interpolation using the standard curve (22), and then the relative
ratio of VCP to ␤-actin expression was calculated for each
ESCC sample.
Statistical Analysis. Statistical analyses were performed
with JMP software (SAS Institute Inc, Cary, NC). Correlation
between the expression level of VCP determined by quantitative
reverse transcription-PCR and immunohistochemistry was evaluated by a one-way ANOVA test. The ␹2 test and Fisher’s exact
probability test were used to analyze the correlation between
VCP expression as assessed immunohistochemically and the
clinicopathologic features of ESCC. The Kaplan–Meier method
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5560 VCP Expression in ESCC
Fig. 1 A and B, valosin-containing protein (VCP)
level 1 esophageal squamous cell carcinoma of the
well-differentiated subtype. Tumor cells exhibited
weak VCP staining in the cytoplasm. C and D,
VCP level 2 esophageal squamous cell carcinoma
of the well-differentiated subtype. Tumor cells exhibited strong cytoplasmic VCP staining (original
magnification, ⫻50). E, endothelial cells show
strong cytoplasmic VCP staining. (original magnification, ⫻150). H and E, hematoxylin and eosin; VCP, VCP immunohistochemistry.
was used to calculate survival rates (23), and differences in
survival curves were evaluated with the log-rank test. Cox’s
proportional hazards regression model with stepwise comparisons was used to analyze the independent prognostic factors
(24). P values ⬍0.05 were considered statistically significant.
staining were evaluated for VCP expression. Cancer cells in 57
(37.2%) and 54 (35.3%) cases showed constant level 1 and level
2 VCP expression, respectively, in all areas of the specimens
(Fig. 1). Tumor cells in 42 cases showed combined level 1 and
RESULTS
Histologic Findings. Histologically, 44 tumors were
well differentiated, 50 were moderately differentiated, and 61
were poorly differentiated squamous cell carcinomas. Tumor
cells invaded the mucosa or submucosa (pT1) in 38 patients,
muscularis propria or subadventitia (pT2) in 35, adventitia (pT3)
in 74, and adjacent organs (pT4) in 8.
Patient Outcome. The 5-year disease-free and overall
survival rates were 47.3% and 49.0%, respectively. Seventyfive patients showed tumor recurrence; recurrence was local
in 18 patients, in the lymph nodes in 39, in the liver in 17, and
in other organs in 14 patients. Sixty-six patients died from
their tumors.
VCP Expression in ESCC. Three (1.9%) of 156 sections that did not show endothelial staining were regarded as
having poor antigen preservation and were excluded from further analyses. The remaining 153 cases showing endothelial
Fig. 2 Relative ratio of valosin-containing protein (VCP) to ␤-actin
mRNA expression in esophageal squamous cell carcinoma (ESCC) with
level 1 and 2 VCP expression. All but two cases of level 2 ESCC
showed higher ratios than cases of level 1 ESCC (P ⬍ 0.05). Bars, SD.
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Clinical Cancer Research 5561
Table 1
Relationship between valosin-containing protein expression and clinicopathologic factors in 153 patients with esophageal
squamous cell carcinoma
Clinicopathologic features
Mean (SD) age (yrs)
Gender, n (%)
Male
Female
Tumor location, n (%)
Cervix
Upper third of thorax
Middle third of thorax
Lower third of thorax
Abdomen
Mean (SD) tumor size (mm)
Histologic differentiation, n (%)
Well-differentiated
Moderately differentiated
Poorly differentiated
Pattern of tumor growth, n (%)
Expansive
Intermediate
Infiltrative
Vascular invasion, n (%)
Absent
Present
Lymphatic invasion, n (%)
Absent
Present
Lymph node metastasis, n (%)
Absent
Present
Depth of tumor invasion, n (%)
pT1
pT2
pT3
pT4
Adjuvant therapy, n (%)
Not performed
Chemotherapy alone
Radiotherapy alone
Combined chemo- and radiotherapy
Types of surgery, n (%)
Subtotal esophagectomy
Cervical partial esophagectomy
Abdominal partial esophagectomy
Endoscopic mucosal resection
p53 labeling index, n (%)
Level 1 (⬍10%)
Level 2 (ⱖ10%)
Ki-67 labeling index, n (%)
Level 1 (⬍20%)
Level 2 (ⱖ20%)
Patients with
VCP level 1
expression
Patients with
VCP level 2
expression
P
61.1 ⫾ 7.4
60.4 ⫾ 7.3
NS
137
16
49 (35.8)
8 (50.0)
88 (64.2)
8 (50.0)
NS
10
7
78
47
11
5 (50.0)
0 (0.0)
35 (44.9)
14 (29.8)
3 (27.3)
46.1 ⫾ 24.3
5 (50.0)
7 (100)
43 (55.1)
33 (70.2)
8 (72.7)
42.4 ⫾ 20.9
NS
43
50
60
17 (39.5)
23 (46.0)
17 (28.3)
26 (60.5)
27 (54.0)
43 (71.7)
NS
34
90
29
15 (44.1)
36 (40.0)
6 (20.7)
19 (55.9)
54 (60.0)
23 (79.3)
NS
113
40
43 (38.1)
14 (35.0)
70 (61.9)
26 (65.0)
NS
54
99
23 (42.6)
34 (34.3)
31 (57.4)
65 (65.7)
NS
50
103
26 (52.0)
31 (30.1)
24 (48.0)
72 (69.9)
⬍0.01
37
35
73
8
19 (51.4)
16 (45.7)
20 (27.4)
2 (25.0)
18 (48.6)
19 (54.3)
53 (72.6)
6 (75.0)
⬍0.05*
⬍0.01†
107
22
2
22
44 (41.1)
9 (40.9)
0 (0.0)
4 (18.2)
63 (58.9)
13 (59.1)
2 (100)
18 (81.8)
NS
145
3
3
2
52 (35.9)
2 (66.7)
2 (66.7)
1 (50.0)
93 (64.1)
1 (33.3)
1 (33.3)
1 (50.0)
NS
65
88
28 (43.1)
29 (33.0)
37 (56.9)
59 (67.0)
NS
85
68
33 (38.8)
24 (35.3)
52 (61.2)
44 (64.7)
NS
Total no.
of patients
NS
Abbreviations: VCP, valosin-containing protein; NS, not significant.
* pT1 vs. pT2–pT4.
† pT1 and pT2 vs. pT3 and pT4.
2 staining. In total, 96 cases (62.7%) were regarded as having
level 2 VCP expression. Nontumorous esophageal mucosa constantly showed level 1 expression.
Quantitative reverse transcription-PCR analysis was performed on six ESSC patients with level 1 and six with level 2
expression. The relative ratios of VCP to ␤-actin expression in
cases with level 1 and 2 expression were 0.4 ⫾ 0.3 and 1.7 ⫾
1.4 (mean ⫾ SD), respectively (P ⬍ 0.05; Fig. 2).
The correlation between VCP expression and clinical
factors is listed in Table 1. Compared with ESCC with level
1 expression, cases with level 2 showed significantly higher
frequencies of the following: presence of lymph node metastasis, 31 of 57 (54.4%) versus 72 of 96 (75.0%); and deep
invasion (pT3 and pT4), 22 of 57 (38.5%) versus 59 of 96
(61.5%). We observed no correlation between VCP staining
intensity and p53 labeling index or Ki-67 labeling index
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5562 VCP Expression in ESCC
Table 2 Correlation between valosin-containing protein expression
and recurrence of esophageal squamous cell carcinoma
No. of patients (%)
Pattern of tumor
recurrence
Total no.
of patients
Total cases
Local recurrence
Distant metastasis
Lymph node
Liver
Other organs
75
18
64
39
17
14
VCP level 1 VCP level 2
expression
expression
(n ⫽ 57)
(n ⫽ 96)
16 (28.1)
1 (1.8)
16 (25.0)
11 (19.3)
3 (5.3)
3 (5.3)
59 (61.5)
17 (17.7)
48 (50.0)
28 (29.2)
14 (14.6)
11 (11.5)
P
⬍0.001
⬍0.01
⬍0.01
⬍0.05
⬍0.05
NS
Abbreviations: VCP, valosin-containing protein; NS, not significant.
(Table 1). The correlation between VCP expression and patterns of tumor recurrence is shown in Table 2. Patients with
level 2 ESCC showed higher frequencies of both local recurrence and distant metastasis than those with level 1 ESCC
(P ⬍ 0.01 for both analyses).
Patients with level 1 ESCC had better 5-year survival rates
than those with level 2 ESCC (disease-free, 65.9% versus
33.2%; overall, 68.7% versus 36.7%; P ⬍ 0.001 for both analyses; Table 3; Fig. 3). Univariate analysis revealed that tumor
size, pattern of tumor growth, depth of tumor invasion, vascular
invasion, lymphatic invasion, and lymph node metastasis were
Fig. 3 Disease-free (A) and overall (B) survival of patients with valosincontaining protein (VCP) expression level 1 and 2 esophageal squamous
cell carcinoma. The differences between the two groups are significant for
both disease-free and overall survival (P ⬍ 0.001 for both).
Table 3 Univariate analysis of clinicopathologic factors for disease-free and overall survival in 153 patients with esophageal
squamous cell carcinoma
Factors
VCP expression
Tumor size
Histologic differentiation
Pattern of tumor growth
Vascular invasion
Lymphatic invasion
Lymph node metastasis
Depth of tumor invasion
p53 labeling index
Ki-67 labeling index
Category
No. of
patients
5-Year disease-free
survival rate (%)
Level 1
Level 2
⬍40.0 mm
ⱖ40.0 mm
Well differentiated
Moderately differentiated
Poorly differentiated
Expansive
Intermediate
Infiltrative
Absent
Present
Absent
Present
Absent
Present
pT1
pT2
pT3
pT4
Level 1 (⬍10%)
Level 2 (ⱖ10%)
Level 1 (⬍20%)
Level 2 (ⱖ20%)
57
96
62
91
43
50
60
34
90
29
113
40
54
99
50
103
37
35
73
8
65
88
85
68
65.9
33.2
58.9
37.2
54.7
43.8
40.4
43.6
50.4
31.3
49.6
33.8
61.6
36.4
75.5
31.8
60.8
76.3
26.1
25.0
42.9
47.1
51.8
31.8
P
⬍0.001
⬍0.05
NS
⬍0.01*
⬍0.01
⬍0.001
⬍0.0001
⬍0.01†
⬍0.0001‡
⬍0.05§
NS
NS
5-Year overall
survival rate (%)
68.7
36.7
68.7
38.3
56.1
44.1
48.0
52.5
54.4
30.6
55.3
30.5
65.8
39.4
77.9
36.3
69.4
76.6
27.5
25.0
41.2
54.7
55.1
34.9
P
⬍0.001
⬍0.01
NS
⬍0.001*
⬍0.001
⬍0.001
⬍0.0001
⬍0.01†
⬍0.0001‡
⬍0.01§
NS
NS
Abbreviations: VCP, valosin-containing protein; NS, not significant.
* Expansive and intermediate vs. infiltrative.
† pT1 vs. pT2–pT4.
‡ pT1 and pT2 vs. pT3 and pT4.
§ pT1–pT3 vs. pT4.
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Table 4
Multivariate analysis of clinicopathologic factors for disease-free and overall survival of 153 patients with esophageal
squamous cell carcinoma
Factors
Disease-free survival
VCP expression
Vascular invasion
Lymph node metastasis
Depth of tumor invasion
Overall survival
VCP expression
Vascular invasion
Lymph node metastasis
Depth of tumor invasion
Category
Relative risk
95% confidence interval
␹2
Level 1
Level 2
Absent
Present
Absent
Present
pT1 and pT2
pT3 and pT4
1.41
1.08–1.90
6.57
⬍0.05
1.31
1.02–1.66
4.43
⬍0.05
1.71
1.26–2.43
13.20
⬍0.001
1.68
1.30–2.22
16.36
⬍0.001
Level 1
Level 2
Absent
Present
Absent
Present
pT1 and pT2
pT3 and pT4
1.45
1.09–1.98
6.65
⬍0.01
1.51
1.17–1.94
9.61
⬍0.01
1.74
1.25–2.57
12.05
⬍0.001
1.83
1.39–2.51
19.56
⬍0.0001
P
Abbreviation: VCP, valosin-containing protein.
significant factors for both disease-free and overall survival
(Table 3).
Multivariate analysis with factors shown to be significant
in the univariate analysis revealed that VCP expression level in
tumor cells, depth of tumor invasion, vascular invasion, and
lymph node metastasis were independent prognostic factors for
both disease-free and overall survival (Table 4).
Prognostic Significance of VCP Expression in the
pTNM Classification. The prognostic significance of VCP
expression was further analyzed according to the pTNM classification (12). We found a significant difference in disease-free
and overall survival between patients with level 1 and 2 expression at the early stage of disease (pT1; P ⬍ 0.001 and P ⬍ 0.01,
respectively; Table 5; Fig. 4). Lymph node metastasis was found
in 4 (20.0%) of 20 level 1 patients and in 13 (72.2%) of 18 level
2 patients (P ⬍ 0.01). At the advanced stage (pT2–pT4), we
observed a significant difference between patients with level 1
and 2 expression for disease-free and overall survival (P ⬍ 0.05
for both analyses; Fig. 4).
DISCUSSION
The conventional TNM staging system for ESCC provides
useful information for the prediction of tumor recurrence and
patient survival (5, 6, 11). Recent advances in therapeutic modalities for ESCC, however, have yielded new problems to be
solved. For example, recently introduced reduction surgeries,
such as endoscopic mucosal resection and transhiatal esophagectomy, enable tumor excision without a severe influence on the
patient’s general condition (25–28); however, lymph node metastasis, observed at relatively high frequencies even in early
ESCC (29), becomes a risk factor when these techniques are
Table 5 Univariate analysis of clinicopathologic factors for disease-free and overall survival of 37 patients with esophageal squamous cell
carcinoma at early stage (pT1)
Factors
VCP expression
Tumor size
Histologic differentiation
Pattern of tumor growth
Vascular invasion
Lymphatic invasion
Lymph node metastasis
Category
No. of patients
5-Year disease-free
survival rate (%)
Level 1
Level 2
⬍40.0 mm
ⱖ40.0 mm
Well differentiated
Moderately differentiated
Poorly differentiated
Expansive
Intermediate
Infiltrative
Absent
Present
Absent
Present
Absent
Present
19
18
13
24
10
11
16
16
19
2
34
3
24
13
20
17
87.5
29.9
62.6
61.5
75.0
57.7
55.2
46.3
66.1
100
58.6
66.7
66.8
50.8
94.7
23.0
P
⬍0.001
NS
NS
NS
NS
NS
⬍0.001
5-Year overall
survival rate (%)
87.5
54.9
79.8
60.2
75.0
68.6
72.2
64.3
70.2
100
68.4
66.7
71.0
71.3
94.7
38.2
P
⬍0.01
NS
NS
NS
NS
NS
⬍0.01
Abbreviations: VCP, valosin-containing protein; NS, not significant.
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5564 VCP Expression in ESCC
Fig. 4 Disease-free and overall survival of patients with valosin-containing protein (VCP)
expression level 1 and 2 esophageal squamous cell carcinoma,
stage pT1 (A and B) or pT2
through pT4 (C and D). The differences were significant for
both disease-free and overall
survival between level 1 and 2
patients with stage pT1 (A, P ⬍
0.001; B, P ⬍ 0.01) or pT2
through pT4 (C and D, P ⬍
0.05) disease.
used. In contrast, the introduction of adjuvant chemo- and
radiotherapy has improved the prognosis of patients with ESCC,
particularly those with high potential for lymph node metastasis
(30, 31). Under these circumstances, preoperative assessment of
lymph node metastasis and the metastatic potential of ESCC is
essential for therapeutic decision-making.
In the present study, we examined the correlation of VCP
expression with the previously proposed prognosticators, p53
expression and proliferative activity shown by Ki-67 immunohistochemistry, and found no relationship between them, demonstrating that VCP expression is a prognosticator independent
of previously known factors.
The patient characteristics, such as gender, age, primary
site, and 5-year survival rate, in the present series were similar
to those in previous reports from Japan and other Asian countries where ESCC is common (1, 10, 15, 29). ESCC in Western
countries, where the disease is relatively uncommon, shares the
above-mentioned characteristics except that tumors are rather
frequent in the lower esophagus (5, 6). In addition, the uni- and
multivariate analyses in the present study showed the prognostic
significance of clinicopathologic factors such as size and depth
of the tumor and lymph node metastasis, as reported previously
from Western countries (5, 9) and Japan (10). These findings
indicate that the results obtained from the present series of cases
are applicable to ESCC in other counties.
VCP is involved in the ubiquitin/proteasome-dependent
protein degradation pathway, which plays an essential role in
controlling the levels of various cellular proteins and therefore
regulates basic cellular processes such as cell cycle progression,
signal transduction, and cell transformation (17). Notably, VCP
is involved in regulating the activation of nuclear factor-␬B (16,
17), a transcription factor whose activity is correlated with
antiapoptosis, cell proliferation, and invasion (32). It is therefore
postulated that VCP plays a crucial role in tumor invasion and
metastasis.
We examined VCP expression in 12 cases at the mRNA
and protein levels by quantitative reverse transcription-PCR and
immunohistochemical analyses, respectively, and found a clear
correlation between the two measurements. The present results,
together with our previous study on hepatocellular carcinoma
and prostate cancer (20, 33) and the study by Muller et al. (34)
on murine tissues, indicates the reliability of immunohistochemistry in the measurement of VCP expression.
Among the clinicopathologic factors examined, we observed a significant correlation between VCP expression and
depth of invasion and lymph node metastasis, indicating the
close association between VCP expression and growth and
invasiveness of ESCC. These findings are consistent with our
reports of the correlation of VCP overexpression with increased
metastatic potential of tumor cells in an experimental metastasis
model (16) and increased recurrence and poor prognosis of
hepatocellular carcinomas in a clinical analysis (20).
The uni- and multivariate analyses revealed that the VCP
expression level is an independent prognosticator for ESCC recur-
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 2004 American Association for Cancer
Research.
Clinical Cancer Research 5565
rence and patient survival. In addition, the assessment of VCP
expression is useful for predicting the recurrence of ESCC in
patients in both the early (pT1) and advanced (pT2–pT4) stages: the
5-year disease-free survival rates in patients with VCP level 1 and
2 were 87.5% and 29.9% at stage pT1 and 56.7% and 33.4% at
stages pT2 through pT4, respectively. The combination of pTNM
classification and VCP expression level in tumor cells is useful for
predicting the prognosis of patients with ESCC.
Grouping the cases with ESCC based on the present system
could be a good guide for choosing the various modalities of
adjuvant therapy. In patients with VCP level 1 ESCC in the
early stages, a favorable outcome could be expected. Lymph
node metastasis was observed in only 20% of these patients;
therefore, reduction surgeries might be considered, particularly
for those in poor general condition or with a small tumor. In
contrast, patients with level 2 ESCC and/or advanced stage
disease might have a dismal prognosis, and the introduction of
intensive adjuvant therapies for these patients might be justified.
In conclusion, VCP expression as determined by immunohistochemistry could be a new prognostic marker for ESCC. The
present study indicated that the stratification of ESCC patients
based on the disease stage and VCP expression level would be
valuable for predicting tumor recurrence and prognosis. This
system might provide a novel way to explore effective treatment
modalities for ESCC.
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Expression Level of Valosin-Containing Protein (p97) Is
Associated with Prognosis of Esophageal Carcinoma
Shinji Yamamoto, Yasuhiko Tomita, Yoshihiko Hoshida, et al.
Clin Cancer Res 2004;10:5558-5565.
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