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Vol. 2, 1679-1684, October 1996 Clinical Cancer Research 1679
3 The abbreviations used are: VEGF, vascular endothelial growth factor;
bFGF, basic fibroblast growth factor.
Significance of Vessel Count and Vascular Endothelial Growth
Factor and Its Receptor (KDR) in Intestinal-type
Gastric Cancer’
Yutaka Takahashi, Karen R. Cleary,
Masayoshi Mai, Yasuhiko Kitadai,
Corazon D. Bucana, and Lee M. Ellis2
Departments of Cell Biology [Y. T., Y. K., C. D. B., L. M. E.],Surgical Oncology [Y. T., L. M. E.], and Pathology [K. R. C.], The
University of Texas M. D. Anderson Cancer Center, Houston, Texas
77030, and Department of Surgery, Cancer Research Institute,
Kanazawa University, Kanazawa, Japan [Y. T., M. M.l
ABSTRACT
Angiogenesis is essential for tumor growth and metas-
tasis and depends on the production of angiogenic factors by
host and/or tumor cells. The role of angiogenesis and angio-
genic factor expression in intestinal- and diffuse-type gastric
cancer are undefined. Archival specimens of 51 intestinal-
type and 38 diffuse-type human gastric carcinomas were
examined for tumor vessel counts, angiogenic factor expres-
sion, and the presence or absence of angiogenic factor re-
ceptors on tumor endothebium using antibodies against vas-
cubar endotheliab growth factor (VEGF) and its receptors
(KDR andflt-1), basic fibroblast growth factor (bFGF) and
its receptors (bek andflg), and factor VIII (endotheliab cells).
Vessel count and VEGF and bFGF expression were higher
in intestinal-type than in diffuse-type gastric cancers (P
0.01, P < 0.001, and P < 0.001, respectively). Similarly,
vessel count and VEGF expression were higher in patients
with liver metastasis than in patients with peritoneal dis-
semination (P = 0.003 and P = 0.01, respectively). Vessel
count correlated with VEGF expression and the presence of
endothelial KDR in intestinal-type gastric cancer (P 0.003
and P = 0.02, respectively) but not diffuse-type gastric
cancer. Vessel count, VEGF expression, and presence of
endothebial KDR increased with increasing stage of disease
in intestinal-type gastric cancer but not diffuse-type gastric
cancer. The expression of bFGF and its receptors did not
correlate with vessel count in either cancer type These
findings suggest that the pattern of metastasis in intestinal-
type gastric cancer is angiogenesis dependent. The correla-
tion of VEGF expression and its endothelial receptor with
vessel count and stage of disease suggests that VEGF is at
Received 2/29/96; revised 7/1/96; accepted 7/3/96.I This work was supported in part by American Cancer Society Career
Development Award 94-21 (to L. M. E.).2 To whom requests for reprints should be addressed, at Department of
Surgical Oncology, Box 106, The University of Texas M. D. Anderson
Cancer Center, 15 15 Holcombe Boulevard, Houston, TX 77030. Phone:
(713) 792-6926; Fax: (713) 792-0722.
least one of the factors responsible for the induction of
angiogenesis in intestinal-type gastric cancer.
INTRODUCTION
Gastric cancer prognosis is dependent on both pathological
tumor type and stage of disease (1, 2). The intestinal type of
gastric cancer tends to be exophytic, metastasizing to the liver
by hematogenous dissemination. In contrast, the diffuse type of
gastric cancer is more invasive, with predominantly peritoneab
dissemination. The factors responsible for liver metastasis and
peritoneal dissemination have not yet been identified.
Angiogenesis is essential for tumor growth and metastasis
and depends on the production of angiogenic factors by host
and/or tumor cells (3). Increased vascularity may allow not only
an increase in tumor growth but also a greater chance for
hematogenous metastasis (4). Weidner et a!. (5) showed a
correlation between the incidence of metastasis and microvessel
count in invasive breast carcinomas. Similar studies have con-
firmed this finding in other malignancies, including lung cancer
(6), prostate cancer (7), melanoma (8), cervical cancer (9), and
colon cancer ( I 0).
Angiogenesis is not a passive process and is driven by the
production of tumor and/or host-derived angiogenic factors. Of
the known angiogenic factors, two well-characterized peptides,
VEGF3 (1 1, 12) and bFGF (13, 14), have been shown to induce
angiogenesis in rodent tumor models. The receptors for these
factors have been characterized and demonstrated on tumor
endothelium (15-18). Previous studies from our laboratory have
demonstrated the importance of these factors in human colon
cancer metastasis (10, 19).
Because of the unique growth characteristics and meta-
static patterns of the intestinal and diffuse types of gastric
cancers, we hypothesized that the intestinal type of gastric
cancer is more angiogenesis dependent than the diffuse type of
gastric cancer. Therefore, the purpose of this study was to
determine whether there are differences in vessel count and
expression of VEGF and bFGF between the intestinal and
diffuse types of gastric cancers. We also sought to determine
whether specific angiogenic factors are associated with vessel
count and if the receptors for these factors are simultaneously
present on endothelial cells.
PATIENTS AND METHODS
Patients and Tumor Specimens. Paraffin-embedded tu-
mor specimens from 89 randomly selected patients with gastric
carcinomas who had undergone surgery at the Cancer Research
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Table 1 Clinical characteristics of gastric cancer patients
Intestinal type
(n = 51)
39-79 (59.2)
36/15
Diffuse type
(n 38)
33-68 (53.5)19/19
Total(n 89)
33-79 (59.6)55/34
1680 Angiogenesis in Gastric Cancer
Age (mean), yrMale/femaleStage of disease”
1 15 9 242 12 10 22
3 9 6 154 15 13 28
Distant metastasis
Liver 15 0 15Peritoneum 1 9 10
Other 2 1 3
a Stage 1: T1, N0,1, M0; or T2, N0, M0. Stage 2: T1, N2, M0; T2, N1,
M0; or T3, N0, lvi,,,. Stage 3: T2, N2, M,�; T3, N1,2, M0; or T4, N0,1, M,,�.
Stage 4: T�, N�, M1; or T4, N2, M0. T1, tumor confined to laminapropria, submucosa. T2, tumor confined to muscularis propria, subse-
rosa. T3, tumor penetrates serosa. T4, tumor invades adjacent structures.N1, positive perigastric lymph node �3 cm from primary. N2, positive
lymph node >3 cm from primary, along left gastric, common hepatic,splenic, or celiac arteries. M,,�, no distant metastasis. M1, distant metas-
tasis.
Institute (Kanazawa University, Kanazawa, Japan) were studied.
The pathology reports and clinical histories at the time of
surgery were reviewed to verify the accuracy of staging. All
slides were reevaluated and classified according to the Lauren
classification of gastric cancer by one pathologist (K. R. C.; Ref.
20). After all available H&E-stained specimen sections were
reviewed, one representative paraffin block from each case was
selected for further study.
The patients ranged in age from 33 to 79 years (mean, 59.6
years); 55 were men and 34 were women. According to the Lauren
classification (20), 51 patients had intestinal-type tumors and 38
had diffuse-type tumors. Patients with mixed-type tumors were not
included in this study. Patient characteristics, tumor stages, and
patterns of metastasis are shown in Table 1. Tumor staging was
done according to the general rules for gastric cancer study (21);
stage distributions were similar in the two groups.
Immunohistochemicab Staining. Consecutive 4-p�m
sections were cut from each study block. Sections were
immunostained for VEGF, bFGF, factor VIII (specific for
endothelial cells), receptors for VEGF (KDR and fit-i), and
receptors for bFGF (bek and fig). Immunohistochemical
staining was performed by the immunoperoxidase technique
following predigestion and trypsinization. Antibodies used
were a rabbit polyclonal antibody (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA) at a 1:200 dilution for VEGF; a rabbit
polyclonal antibody (Sigma Chemical Co., St. Louis, MO) at
a 1:30 dilution for bFGF; a rabbit polyclonal antibody (Dako
Co., Carpinteria, CA) at a 1:250 dilution for factor VIII; a
rabbit polyclonal antibody (Santa Cruz Biotechnology) at a
1 : 100 dilution for KDR; a rabbit polyclonal antibody (Santa
Cruz Biotechnology) at a 1:200 dilution for fit-i ; a rabbit
polyclonal antibody (Santa Cruz Biotechnology) at a 1:200
dilution for bek; and a rabbit polycbonal antibody (Santa Cruz
Biotechnology) at a 1 :200 dilution for fig. To determine the
specificity of the antibody, we tested the antibody against the
two proteins known to have significant homology: placenta
growth factor (>50% homology) and platelet-derived growth
factor (-20% homology). By Western blot analysis, the
antibody for VEGF detected only VEGF, and there was no
cross-reactivity with placenta growth factor or platelet-de-
rived growth factor (data not shown).
For positive controls, tissue from a colon cancer known to
express VEGF was stained for VEGF, tissue from a bladder
tumor was stained for bFGF, umbilical vein tissue was stained
for KDR and fit-i, and normal liver tissue was stained for bek
and fig. Negative controls were done using nonspecific !gG as
the primary antibody.
Evaluation of Immunostaining and Vessel Counting.The intensity of staining for VEGF and bFGF was evaluated
blindly at the invasive edge and was confirmed by an image
analyzer using the Optimas software program (Bioscan, Ed-
monds, WA). Intensity of staining for VEGF and bFGF was
graded on a scale of 0 to 3 +, with 0 representing no detectable
stain and 3 + representing the strongest stain (Fig. I). The
presence or absence of KDR, fit-i, bek, andfig was evaluated on
both tumor endothelial cells and tumor epithelial cells.
Vessel count was assessed by light microscopy in areas of
the tumor containing the highest numbers of capillaries and
small venules at the invasive edge. The highly vascular areas
were identified by scanning tumor sections at low power ( X 40
and X 100). After the area of highest neovascularization was
identified, a vessel count was performed on a X 200 field (X 20
objective and X 10 ocular, 0.739 mm2 per field). As Weidner et
al. described (5), vessel lumens were not necessary for a struc-
ture to be defined as a vessel.
Statistical Analysis. Differences in vessel count and in
mean intensity ofVEGF and bFGF staining between intestinal-type
tumors and diffuse-type tumors were analyzed by Student’s t test.
Differences in mean vessel count between receptor-positive and
receptor-negative tumors were also analyzed by Student’s t test.
Correlations between stage of disease and vessel count, expression
of VEGF and bFGF and vessel count, and intensity of VEGF and
bFGF staining were examined by the Spearman rank correlation
coefficient. Differences in rates of positivity for the receptors
among subgroups were assessed by x2 analysis. All statistical
analyses were performed using Statworks statistical software
(Cricket Software, Inc., Philadelphia, PA), and differences were
deemed significant at the 95% confidence interval.
RESULTS
Vessel Count and VEGF and bFGF Expression in Tu-mors. The intensity of VEGF and bFGF staining was homo-
geneous within tumors, and there were no detectable “hot
spots.” Vessel count, VEGF expression, and bFGF expression
were all significantly higher in intestinal-type tumors than in
diffuse-type tumors (P = 0.01, P < 0.001, and P < 0.001,respectively; Table 2).
Vessel count (P = 0.003) and VEGF expression (P =
0.01) were significantly higher in patients with liver metas-
tasis than in patients with peritoneal dissemination. bFGF
expression did not differ between the two groups of patients.
Photomicrographs of representative intestinal-type and dif-
fuse-type gastric cancers stained for factor VIII, VEGF, and
bFGF are shown in Fig. 2.
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,, 1� ,
.�? �
A B
Clinical Cancer Research 1681
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+3
+2
+1
ctrl
Fig. 1 Subjective evaluation of staining intensity for VEGF in tumor cells at the invasive edge was done blindly. To confirm the validity of this
assessment, five representative specimens from each group were selected. Integrated absorbance of 12 distinct areas (5 p.m in diameter, indicated bycircles) within the tumor cell cytoplasm were obtained (A). The average intensity was found to correlate closely with the subjective findings, in that
tumors with a higher subjective score demonstrated a higher intensity by image analysis. In B, representative staining for VEGF is demonstrated for
each grade.
Table 2 Vessel count and staining intensity in prima ry tu mors fr om patients with gastric cane er, liver metastases, and peritoneal dissemination
Histological and metastatic types
Vessel count
(mean ± SE)
Staining intensity (mean ± SE)
VEGF bFGF
Intestinal type
Diffuse type
(1,
(1)
= 51)
= 38)
37.2 ± 34fl
26.1 ± 2.5P0�0l
2.2 ± 0.1 � 1.4 ± 0.fl
1.4 ± 0.1 ] P<0.OOl o.s ± O.l_J P<0.OOl
Primary gastric cancer with liver metastasis
Primary gastric cancer with peritoneal dissemination(a
(�i
= 15)
= 10)
54.9 ± 5.Ofl
26.8 ± 3.9_iP=0.003 2.3 ± 0.1 � 1.5 ± 0.2
1.6 ± 0.2 P0.01 11 #{247}0.2
Correlations between Stage of Disease and Vessel
Count, VEGF Expression, and bFGF Expression. As
shown in Fig. 3, vessel count correlated with stage of disease
in patients with intestinal-type tumors. Similarly, the inten-
sity of VEGF staining also correlated with stage of disease in
patients with intestinal-type tumors. In patients with diffuse-
type gastric tumors, there were no correlations among vessel
count, VEGF expression, and stage of disease. The intensity
of bFGF staining did not correlate with stage of disease in
either tumor type.
Correlations between Vessel Count and VEGF and
bFGF Expression. A correlation between tumor vessel count
and VEGF expression was observed in specimens from patients
with intestinal-type tumors (P 0.003) but not in specimens
from patients with diffuse-type tumors. There was no correlation
between vessel count and bFGF expression in either tumor type.
Positivity for KDR, fit-i, bek, and fig on Tumor Endo-thelia and Tumor Epithelial Cells. As shown in Table 3,
positivity for KDR on tumor endothelium occurred significantly
more often (39.2%) in intestinal-type tumors than in diffuse-
type tumors (15.8%; P = 0.02). Seventeen (77.3%) of 22
intestinal-type tumors positive for KDR on endothelium were
from patients with stage 3 or 4 disease. There were no differ-
ences in positivity for the other VEGF and bFGF receptors
(fit-I, bek, and fig) on tumor endothelium in either intestinal-
type or diffuse-type gastric tumors.
KDR was not detected on tumor cells, and < I 0% had
minimal staining for fit-I. In contrast, the receptors for bFGF
(bek and fig) were detected on tumor cells in 83.5 and 77.8%,
respectively, of the tumors studied. Expression of bFGF on
tumor cells correlated with the presence of bek andfig on tumor
cells (P = 0.004 and P = 0.003, respectively).
Correlations between Vessel Count and Receptor Pos-
itivity. The mean vessel count in intestinal-type gastric tumors
in which the endothelium stained positive for KDR was signif-
icantly higher than in those in which the endothelium stained
negative (P = 0.02); however, there was no difference in vessel
count between diffuse-type tumors with KDR-positive- or KDR-
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�- . _ -,� . � � ‘ � : � �..
1682 Angiogenesis in Gastric Cancer
Fig. 2 Immunohistochemical staining of an intestinal-type gastric cancer (A-C) and a diffuse-type gastric cancer (D-F) using antibodies to factoryIn (A and D), VEGF (B and E), and bFGF (C and F). Vessel count was higher in the intestinal-type carcinoma (A) than in the diffuse-type gastriccarcinoma (D). The intestinal-type carcinoma (B and C) also showed stronger positive staining for VEGF and bFGF than did the diffuse-type
carcinoma (E and F). X200.
negative-staining tumor endothelium. Furthermore, there was no
difference in vessel count in either diffuse-type or intestinal-
type tumors in patients with fit-i-, bek-, or fig-positive or
-negative staining tumor endothelium.
DISCUSSION
Both the growth patterns and the biological behavior of
intestinal-type and diffuse-type gastric carcinomas are distinct.
Intestinal-type gastric cancers demonstrate exophytic growth
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p=O.03B
Fig. 3 Vessel count (A) and VEGF expres-
sion (B) as a function of stage of disease in
intestinal-type gastric cancer. Increasing stage
of disease correlated with an increase in vesselcount. Increasing stage of disease also corre-lated with an increase in VEGF expression,except that there was no significant increase in
VEGF expression between stages 3 and 4 dis-ease. Bars, SD.
A80
60#{149}C
00
.� 4�J.
(I)U)C)
>
Stage of disease
1 2 3 4
Stage of disease
Clinical Cancer Research 1683
C)
C
(5
(I)
LI.C,w
0
(11
CC)
C
Table 3 Tum or endothel ium positivity for r eceptors for VEGF and bFGF in gastric cancer patients
Type of gastric tumor
Receptors for VEGF Receptors for bFGF
KDR flt-l bek fig
Intestinal type
Diffuse type
(n
(a
51)
= 38)
20#{176}(39.2%)7
I6”(l5.8%)J
P=0.02
10” (19.6%)
7” 18.4%)
5” (9.87%)
5”(13.2%)
3” (5.9%)
2”(5.3%)
a Number of tumors where receptors are present on tumor endothelium.
and commonly metastasize to the liver, whereas diffuse-type
tumors demonstrate invasive growth and peritoneal metastasis
( 1 , 2). The Lauren classification of gastric cancer is based on the
pathobiology of cell cohesion (20, 22). In intestinal-type gastric
cancer, the neoplastic cells adhere to each other, forming struc-
tures that resemble intestinal mucosa. In contrast, cells of dif-
fuse-type gastric cancer lack these adhesive qualities and infil-
trate the gastric wall either as individual cells or in nests (22).
Those countries with a high rate of gastric cancer (such as
Japan) have a high percentage of patients who present with the
intestinal-type of disease (22). In our study of Japanese patients
with gastric cancer, nearly 60% presented with the intestinal
type. In other areas of the world, the distribution of this disease
is relatively equally divided between intestinal- and diffuse-type
gastric cancer. Despite the relatively distinct microscopic char-
acteristics and growth patterns of the two main types of gastric
cancer, the overall survival for both patient populations is sim-
ilar (35-45%; Ref. 22). Although some believe that the specific
types of gastric cancer may evolve from one type to another
(intestinal to diffuse), we do not believe this to be the case.
Poorly differentiated intestinal-type carcinoma has solid (i.e.,
nongland-forming) areas, but this is not equivalent to evolve-
ment to the diffuse-type cancer. Occasionally, tumors may be
mixed with components of both intestinal and diffuse types. Our
study excluded the mixed type of gastric cancer from analysis
(-5% of gastric cancers).
We hypothesized that angiogenesis and its regulating fac-
tors play a role in determining the growth characteristics and
patterns of metastasis in diffuse-type and intestinal-type gastric
cancers. We found that vessel count, VEGF expression, and
bFGF expression were significantly higher in intestinal-type
gastric tumors than in diffuse-type tumors. Significant correla-
tions among stage of disease, vessel count, and VEGF expres-
sion were observed in intestinal-type tumors. Moreover, tumors
associated with liver metastasis (all were intestinal-type tumors)
showed higher vessel counts and VEGF expression than tumors
associated with peritoneal dissemination. These results suggest
that the processes of growth and metastasis in intestinal-type
tumors are more angiogenesis dependent than they are in dif-
fuse-type tumors. Furthermore, the correlation of VEGF expres-
sion and vessel count implies that VEGF may induce the an-
giogenic response in intestinal-type gastric cancer. These data
strongly suggest that VEGF may be the more important of the
two angiogenic factors studied in inducing neovascularization in
intestinal-type gastric tumors.
Expression of bFGF also was higher in intestinal-type
gastric tumors than in diffuse-type gastric tumors, but bFGF
expression did not correlate with vessel count or with stage of
disease. bFGF is a ubiquitous, potent growth factor that can act
as a tumor cell mitogen as well as an angiogenic factor. This
study demonstrated the presence of bFGF receptors on gastric
tumor cells, which suggests that bFGF acts as a tumor cell
mitogen for gastric carcinoma cells in an autocrine and/or para-
crine fashion. This theory is supported further by the fact that
tumor cells with high bFGF expression were more likely to stain
positive for bFGF receptors than tumor cells with lower bFGF
expression. Thus, it is possible that bFGF acts as a tumor cell
mitogen and VEGF acts as an angiogenic factor in intestinal-
type gastric cancer.
To confirm the hypothesis that VEGF plays a role in
angiogenesis in intestinal-type gastric cancer, it must be dem-
onstrated that the receptors for this factor are present on tumor
endothelium. The significance of expression of VEGF and its
receptors has been reported in other human carcinomas, such as
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1684 Angiogenesis in Gastric Cancer
colon cancer (15), breast cancer (17), and brain tumors (1 8). In
a small series of patients, Brown et a!. (15) demonstrated the
presence of VEGF and its receptors in gastric cancer specimens.
To date, no study has examined the importance of angiogenic
factors, neovascularization, and the clinical course of patients
with specific types of gastric cancer.
In our study, a greater percentage of intestinal-type gastric
tumors than diffuse-type gastric tumors expressed KDR on
tumor endothelium (39.2% versus 15.8%; P = 0.02). Most
KDR-positive tumors were from patients with advanced stages
of disease. Moreover, intestinal-type tumors in which the endo-
thelium stained positive for KDR were associated with higher
vessel counts than were KDR-negative tumors. There were no
differences in endothelial positivity for the other receptors (fit-i,
bek, and fig) between intestinal-type and diffuse-type tumors.
These results suggest that VEGF and KDR may be the important
receptor-ligand system in the process of angiogenesis in intes-
tinal-type gastric cancer.
If VEGF is indeed responsible for tumor angiogenesis in
intestinal-type gastric cancer, strategies using antibodies or an-
tisense RNA to VEGF may inhibit tumor angiogenesis. To
address this hypothesis, we plan to down-regulate VEGF ex-
pression in tumor cells using an orthotopic implantation model
of gastric cancer.
The different growth characteristics and metastatic patterns
of intestinal-type and diffuse-type gastric cancers may be due, in
part, to the increased vascularity of intestinal-type tumors. This
may be caused by tumor secretion of VEGF and binding to its
receptor on tumor endothelium. The correlation of vessel count,
VEGF expression, and stage of disease in intestinal-type gastric
cancer suggests that angiogenesis may be a potential prognostic
marker in this type of gastric cancer. In addition, VEGF may
provide a potential target for therapy in intestinal-type gastric
cancer.
ACKNOWLEDGMENTS
We thank Ricky Sanchez and Velma Harris for technical assistanceand Melissa Burkett and Cindy Lhamon for editorial assistance.
REFERENCES
1. Nishi, M., and Tamura, T. Clinical study of stomach cancer withhepatic metastasis. Jpn. J. Cancer Clin., 8: 422-433 (in Japanese), 1962.
2. Duarte, I., and Llanos, 0. Patterns of metastases in intestinal anddiffuse types of carcinoma of the stomach. Hum. Pathol., 12: 237-242,
1981.
3. Folkman, J. How is blood vessel growth regulated in normal andneoplastic tissue? Cancer Res., 46: 467-473, 1986.
4. Liotta, L., Kleinerman, J., and Saldel, G. Quantitative relation-ships of intravascular tumor cells, tumor vessels, and pulmonarymetastases following tumor implantation. Cancer Res., 34: 997-
1004, 1974.
5. Weidner, N., Semple, J. P., Welch, W. R., and Folkman, J. Tumorangiogenesis and metastasis: correlation in invasive breast carcinoma.N. Engl. J. Med., 324: 1-8, 1991.
6. Macchiarmni, P., Fontanini, G., Hardin, M. J., Squartini, F., andAngeletti, C. A. Relation of neovascularization to metastasis of non-small-cell lung cancer. Lancet, 340: 145-146, 1992.
7. Weidner, N., Carroll, P. R., Flax, J., Blumenfeld, W., and Folkman,J. Tumor angiogenesis correlates with metastasis in invasive prostate
carcinoma. Am. J. Pathol., 143: 401-409, 1993.
8. Graham, C. H., Rivers, J., Kerbel, R. S., Stankiewicz, K. S., and
White, W. L. Extent of vascularization as a prognostic indicator in thin
(<0.76 mm) malignant melanomas. Am. J. Pathol., 145: 510-514,
1994.
9. Smith-McCune, K. K., and Weidner, N. Demonstration and charac-terization of the angiogenic properties of cervical dysplasia. Cancer
Res., 54: 800-804, 1994.
10. Takahashi, Y., Kitadai, Y., Bucana, C. D., Cleary, K. R., and Ellis,L. M. Expression of vascular endothelial growth factor and its receptor,
KDR, correlates with vascularity, metastasis, and proliferation of humancolon cancer. Cancer Res., 55: 3964-3968, 1995.
1 1. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., andFerrara, N. Vascular endothelial growth factor is a secreted angiogenicmitogen. Science (Washington DC), 246: 1306-1309, 1989.
12. Ferrara, N., Houck, K., Jakeman, L., and Leung, D. W. Molecularand biological properties of the vascular endothelial growth factorfamily of proteins. Endocr. Rev., 13: 18-32, 1992.
13. Folkman, J., and Klagsbrun, M. Angiogenic factors. Science
(Washington DC), 235: 442-447, 1987.
14. New, B. A., and Yeoman, L. Identification of basic fibroblastgrowth factor sensitivity and receptor and ligand expression in humancolon tumor cell lines. J. Cell Physiol., 150: 320-326, 1992.
15. Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Manseau,
E. J., Senger, D. R., and Dvorak, H. F. Expression of vascular perme-ability factor (vascular endothelial growth factor) and its receptors in
adenocarcinomas of the gastrointestinal tract. Cancer Res., 53: 4727-
4735, 1993.
16. Millauer, B., Wizigmann-Voos, S., Achnurch, H., Martinez, R.,Moller, N. P. H., Risau, W., and UlIrich, A. High affinity VEGF binding
and developmental expression suggest KDR as a major regulator of
vasculogenesis and angiogenesis. Cell, 72: 835-846, 1993.
17. Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Guidi, A. J.,
Dvorak, H. F., Senger, D. R., Connolly, J. L., and Schnitt, S. J.Expression of vascular permeability factor (vascular endothelial growthfactor) and its receptors in breast cancer. Hum. Pathol., 26: 86-91,1995.
18. Samoto, K., Ikezaki, K., Ono, M., Shono, T., Kohno, K., Kuwano,
M., and Fukui, M. Expression of vascular endothelial growth factor andits possible relation with neovascularization in human brain tumors.
Cancer Res., 55: 1 189-1 193, 1995.
19. Kitadai, Y., Ellis, L. M., Takahashi, Y., Bucana, C. D., Anzai, H.,Tahara, E., and Fidler, I. J. Multiparametric in situ messenger RNA
hybridization analysis to detect metastasis-related genes in surgical
specimens of human colon carcinomas. Clin. Cancer Res., 1: 1095-1102, 1995.
20. Lauren, P. The two histological main types of gastric carcinoma:diffuse and the so-called intestinal type carcinoma. Acta Pathol. Micro-
biol. Scand., 64: 31-49, 1965.
2 1 . Japanese Research Society for Gastric Cancer. The general rules forthe gastric cancer study in surgery and pathology. I. Clinical classifica-
tion. Jpn. J. Surg., ii: 127-139, 1981.
22. Brenes, F., and Correa, P. Pathology of gastric cancer. Surg. Oncol.Clin. North Am., 2: 347-370, 1993.
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1996;2:1679-1684. Clin Cancer Res Y Takahashi, K R Cleary, M Mai, et al. factor and its receptor (KDR) in intestinal-type gastric cancer.Significance of vessel count and vascular endothelial growth
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