significance ofvessel count andvascular endothelial growth ... · cruz biotechnology) ata1:200...

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

Research. on September 10, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

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.



,, 1� ,

.�? �

A B

Clinical Cancer Research 1681

.� -*

+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-



:‘ #{149}� ‘#{176}‘� -‘:‘,‘�. � &,,� � - �.

.� . .-‘, S�... � � ‘ � 4%�

� , ‘ �.#{176}..,�,; ;:. #{149}‘�. :�.,-�

�- . _ -,� . � � ‘ � : � �..


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



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




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.

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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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