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Molecular Oncology, Markers, Clinical Correlates |
Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been reported that cancer development depends on a variety of physiological processes, such as carcinogenesis at the cell oncogene level, proliferation, and tumor growth and progression. Tumor angiogenesis is necessary for tumor growth as the means of supplying the oxygen and nutrients, and it is necessary for tumor progression because it increases the opportunity for tumor cells to enter the circulation (5) . Liotta et al. (6) have demonstrated that greater numbers of tumor vessels increase the opportunity for tumor cells to enter the circulation, and Nagy et al. (7) have shown that newly formed capillaries are more easily penetrated than mature vessels. Folkman et al. (8, 9, 10) have already demonstrated a relationship between tumor angiogenesis and tumor growth and a new advanced strategy for cancer therapy based on antitumor angiogenesis.
Several growth factors with angiogenic activity have been described (11) . VEGF is one of the angiogenic factors highly specific for endothelium, and it also functions as a vascular permeability factor (12 , 13) . VEGF has been reported to be secreted by various carcinomas (14) . Many earlier papers documented a positive correlation between tumor MVD and tumor aggressiveness in squamous cell carcinoma (15, 16, 17) , and a recent paper reported a positive association among VEGF expression, tumor MVD, and tumor aggressiveness (18, 19, 20, 21) . For these reasons, VEGF is thought to be an important factor in tumor angiogenesis.
However, no clear clinical studies on squamous cell carcinoma of the esophagus and VEGF expression have ever been reported. Accordingly, to clarify the prognostic significance and relationship between common clinicopathological factors and VEGF expression in esophageal squamous cell carcinoma, we retrospectively examined 117 primary thoracic esophageal carcinomas by immunohistochemical staining, and we investigated correlations among VEGF expression, tumor MVD, and clinical characteristics.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 2
summarize the clinicopathological background factors of the patients,
and the pathological examinations were performed according to the
Guidelines for the Clinical and Pathological Studies on Carcinoma of
the Esophagus of the Japanese Society for Esophageal Diseases
(22)
. Fifty-three
pT1b–pT3 patients who underwent
esophagectomy with thoracotomy and did not receive postoperative
adjuvant therapy were selected for life table analysis because their
background factors were the same.
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Immunohistochemical Staining.
Ten-µm sections were made from 10%-formalin-fixed, paraffin-embedded
blocks and mounted on slides. The blocks were selected from the most
invasive area of the carcinoma according to the pathology report. A
labeled streptavidin biotin (LSAB) kit (DAKO, Glostrup, Denmark) was
used for immunohistochemical staining. DAB was used as the
chromogen, and Mayer’s hematoxylin as the counterstain.
Determination of VEGF Expression.
Sections were deparaffinized and rehydrated and then digested in 10
µg/ml pepsin in 0.01 N HCl buffer (pH 2.5) for 30 min at
room temperature. The tissue sections were covered with 3%
H2O2 for 5 min and
incubated for 5 min in BSA to suppress nonspecific IgG binding. As
primary antibody, sections were incubated with a 1:50 dilution of
monoclonal antihuman VEGF antibody (IBL Inc., Gunma, Japan) in PBS at
room temperature for 60 min. The slides were reacted with Link Antibody
for 10 min at room temperature and with horseradish
peroxidase-conjugated streptavidin for 10 min at room temperature. As
chromogen, the slides were stained with 0.025% DAB and 0.003%
H2O2 in 0.05 M
Tris-HCl buffer for 6 min, and they were then lightly
counterstained with 10% Mayer’s hematoxylin.
Cases were considered as VEGF-positive if more than 80% of the cancer cells manifested cytoplasmic positivity.
Determination of MVD.
Sections were deparaffinized and rehydrated and then digested with
0.1% tripsin for 15 min at 37°C. As a primary antibody, sections
were incubated with a 1:200 dilution of polyclonal antihuman von
Willebrand factor antibody (DAKO, Glostrup, Denmark) in PBS at room
temperature for 15 min. The slides were then reacted with Link Antibody
for 10 min at room temperature followed by horseradish
peroxidase-conjugated streptavidin for 10 min at room temperature. As
chromogen, the slides were stained by 0.025% DAB and 0.003%
H2O2 in 0.05 M Tris-HCl
buffer for 10 min, and they were then lightly counterstained with 10%
Mayer’s hematoxylin.
As a parameter of tumor angiogenesis, after scanning the "vascular hot spot" in the tissues adjacent to the cancer at x10, microvessel counts were performed at x200 by using a calibrated grid (0.689 mm2/field). Any brown-stained vessel or endothelial cell that was clearly separate from the microvessels was considered a vessel and counted. An average of 20 fields per section were scanned by two investigators without previous knowledge of the outcome of the patients or any other patient data. Whenever there was a difference between two investigators of more than 20 counts in the average number for each section, the data were discarded. Microvessel densities (number of microvessels per mm2) were calculated from these microvessel counts.
Cases with a calculated density of more than 60/mm2 were considered to have a high MVD.
Statistical Analysis.
The 
2 tests were used to evaluate differences
in background factors between patient groups. The cumulative survival
rates for patient groups were calculated by the Kaplan-Meier method and
compared by using the Cox-Mantel test and the generalized Wilcoxon
test. The influence of each variable on survival was assessed by the
Cox proportional-hazards regression model. Statistical significance was
defined as P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and 36 (31%) of the 117
cases were evaluated as VEGF-positive. The patients were divided into
two groups: a VEGF-positive group and a VEGF-negative group (Tables 1
and 2)
. There were no significant differences in clinicopathological
background factors between the two groups according to the results of
the 2 analysis.
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). Because
no metastatic regional lymph nodes were detected at surgery in
pT1a cases, and all of the pT4 cases
underwent palliative resection, these cases were excluded. Recurrence
developed in 26 of the 81 patients who underwent curative resection.
The recurrence rate was 33.3% (10 of 30 patients) in the VEGF-positive
group and 31.4% (16 of 51 patients) in the VEGF-negative group. There
was no significant difference between these two groups in the
recurrence rate, and no correlation was observed between VEGF
expression and the type of postoperative recurrence.
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]. MVD was
59.4 ± 24.7 (mean ± SE)/mm2 in the
VEGF-positive cases (n = 34) and 47.9 ±
3.8/mm2 in the VEGF-negative cases
(n = 61; Fig. 4
). MVD in
the patients with VEGF-positive tumors was higher than in those with
VEGF-negative tumors, but the difference was not significant
(P = 0.08).
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2 test. (Table 2)
. The 4-year survival rates were 32.8% in the VEGF-positive group
and 53.3% in the VEGF-negative group and significantly lower in the
patients with VEGF-positive tumors (P = 0.04; Fig. 5
).
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. In the pT1b group
(n = 16), the 4-year survival rates of the patients
with VEGF-positive tumors and VEGF-negative tumors were 66.7%
(n = 7) and 83.3% (n = 9),
respectively; they were not significantly different. It was difficult
to analyze the survival rates of the patients in the
pT2 group because of the small number of cases
(n = 8). In the pT3 group
(n = 29), the 3-year survival rates of the patients
with VEGF-positive tumors and VEGF-negative tumors were 0%
(n = 9) and 87.5% (n = 20),
respectively, and survival was significantly poorer in the patients
with VEGF-positive tumors than in those with VEGF-negative tumors
(P = 0.0002).
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. In the pN0
group (n = 16), the 4-year survival rates of the
patients with VEGF-positive tumors and VEGF-negative tumors were 100%
(n = 4) and 75.0% (n = 12),
respectively, and the survival rates between these two groups was not
statistically significant. In the pN1 group
(n = 37), the 3-year survival rates of the patients
with VEGF-positive tumors and VEGF-negative tumors were 13.4%
(n = 14) and 61.0% (n = 23),
respectively, and survival was significantly poorer in the patients
with VEGF-positive tumors than in those with VEGF-negative tumors
(P = 0.006).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was no correlation in esophageal cancer between VEGF expression and the type of postoperative recurrence. However, the average number of metastatic lymph nodes detected at surgery in the patients with VEGF-positive tumors was significantly higher than in those with VEGF-negative tumors. It has been reported that large numbers of metastatic lymph nodes decrease the postoperative survival rate in squamous cell carcinoma of the esophagus (23 , 24) . Maeda et al. (20) have reported that expression of VEGF in gastric cancer is significantly associated with the presence of lymph node metastasis at the time of surgery and with postoperative recurrence in the liver.
Why is VEGF expression correlated with the number of metastatic lymph nodes? VEGF is a selective mitogen for vascular endothelial cells, and it directly stimulates neovascularization and increases microvascular permeability (12 , 13) . The leaky state of microvessels may cause extravasation of tissue metalloproteinase and promote cancer cell invasion into the circulation (7 , 25) . The first hypothesis is that the cancer cells in the circulation directly reach the regional lymph nodes via the vessels that supply them, the second hypothesis is that they reach them via blood vessel-lymph vessel junctions (26) , and the third hypothesis is that VEGF may directly affect lymph vessels as well as blood vessels and cause direct invasion of cancer cells into the lymph circulation because lymph vessels and blood vessels are known to have similar structures. However, no studies have ever demonstrated that VEGF directly affects lymph vessels.
The incidence of pT2–4 cases among high-MVD tumors was significantly higher than among low-MVD tumors, and this finding is highly consistent with the tumor angiogenesis theory. The increases in new capillaries that converge on the tumor supply it with more oxygen and nutrients and accelerate tumor growth. Presumably, the growth of the tumor accelerates when it invades the submucosa, which is richer in capillaries than the lamina propria.
The MVD of VEGF-positive carcinomas tended to be higher than the MVD of VEGF-negative carcinomas, and, thus, the MVD of tumors is closely related to their expression of VEGF. However, some cases were high-MVD despite being VEGF-negative, and some were low-MVD although they were VEGF-positive. These results suggest that VEGF may be one of the key angiogenic factors, and that it promotes tumor angiogenesis in esophageal carcinoma tissue, in the same way as previously described in other carcinomas. The two-compartment theory states that the VEGF secreted by tumor cells and vascular endothelial cells accelerates the tumor angiogenesis cycle (27) . Recently, Toi et al. have documented that expression of VEGF is closely associated with the promotion of angiogenesis in breast cancer (19) . Maeda et al. and Tanigawa et al. reported similar findings in gastric cancer (20 , 21) , Takahashi et al. in colon cancer (18) , and Mattern et al. in lung cancer (28) . We were unable to show a significant correlation between VEGF expression and MVD as these previous papers did, but this matter will be clarified as the number of cases increases.
In this study, we demonstrated for the first time that expression of VEGF predicts postoperative outcome in patients with squamous cell carcinoma of the esophagus, that is, that the outcome of patients with VEGF-positive tumors is significantly worse than that of patients with VEGF-negative tumors. Even when stratified according to pN and pT factors, the survival rates of the patients in the VEGF-positive group were found to be significantly poorer than in the VEGF-negative group in patients with pT3 and pN1 tumors. The reason for the significantly poorer outcome in these two more advanced-stage cancer groups may be due to the number of metastatic lymph nodes observed at surgery. As stated above, there was a strong positive correlation between the number of metastatic lymph nodes and VEGF-positive tumors. Therefore, by additionally investigating VEGF expression, we are more likely to predict the probability of lymph node metastasis and the prognosis of patients with more advanced stage esophageal squamous cell carcinoma. We also observed a significantly poorer outcome among the patients with high-MVD tumors than among those with low-MVD tumors. In the life table analysis with Cox’s proportional hazards model, VEGF expression had the second highest hazard ratio after the pN factor (which was previously found to be the most useful factor for predicting the outcome in esophageal squamous cell carcinoma), and MVD had the third highest. Thus, expression of VEGF and tumor MVD, which is the final result of the tumor angiogenesis cascade, are useful prognostic indicators in esophageal cancer, and they will supplement conventional clinicopathological factors in predicting prognoses. Toi et al. (19) have clearly documented that VEGF expression is an independent prognostic marker in breast cancer, Takahashi et al. and Kang et al. (18 , 29) showed a correlation between VEGF expression and prognosis in colon cancer, and Maeda et al. (20) found that VEGF is of prognostic value in gastric cancer. By contrast, Tanigawa et al. (21) reported no significant correlation between VEGF expression and prognosis in gastric cancer, but that MVD was a useful prognostic marker. Thus, almost all of the previous articles have stated that there is a correlation between VEGF expression and outcome in other cancer patients, and our study has yielded the same results for squamous cell carcinoma of the esophagus.
Because of recent evidence that the outcome of treatment for advanced squamous cell carcinoma of the esophagus has reached a plateau, interest is being focused on combination therapies, although little information is available on the selection of patients for adjuvant therapy. There have been no articles documenting a relationship between VEGF expression and chemosensitivity, but Albo et al. have documented a possible correlation between tumor MVD and chemosensitivity, that is, they found that tumors with higher MVD are more sensitive to chemotherapy (16) . Thus, the analysis of tumor MVD or VEGF expression in resected cancers may provide additional guidance in identifying the patients who require postoperative adjuvant therapy. Several kinds of antiangiogenesis molecules have recently been developed, and some clinical trials are in progress (30, 31, 32) . Thus, evaluating tumor MVD or VEGF expression in resected cancers will play an important role in selecting patients for antiangiogenesis therapy.
In conclusion, this retrospective study indicates that VEGF promotes lymph node metastasis in vivo and is a useful marker for predicting outcome in patients with esophageal squamous cell carcinoma. In the near future, it may be possible to perform tumor dormant therapy by using angiogenesis inhibitors in patients with squamous cell carcinoma of the esophagus based on the combined use of TNM classification and testing for VEGF expression and tumor MVD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid from the
Ministry of Education, Science, Sports and Culture, the Ministry of
Health and Welfare, and the Fund for Establishment of High-Tech
Research Centers in Private Universities. 
2 To whom requests for reprints should be
addressed, at the Department of Surgery, School of Medicine, Keio
University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone:
81-3-3353-1211, extension 2334; Fax: 81-3-3355-4707.
3 The abbreviations used are: TNM,
tumor-node-metastasis; pN, pathological node (category); pT,
pathological tumor (category); MVD, microvessel density; VEGF, vascular
endothelial growth factor; DAB, diaminobenzidine tetrahydrochloride.
4 pT1, tumor invades the lamina
propria or submucosa (pT1a, tumor invades the lamina
propria; pT1b, tumor invades the submucosa);
pT2, tumor invades the muscularis propria; pT3,
tumor invades the adventitia; pT4, tumor invades adjacent
structures.
5 pN0, no regional lymph node
metastasis; pN1, regional lymph node metastasis.
Received 7/22/99; revised 11/23/99; accepted 12/ 9/99.
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REFERENCES |
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,
P = 0.08.
