Vascular Endothelial Growth Factor, Wild-Type p53, and ... · over, no significant association of bcl-2 and c-erbB-2 onco-Received 6/8/98; revised 9/28/98; accepted 10/5/98. The costs

Vol. 4, 3017-3024, December 1998 Clinical Cancer Research 3017

Vascular Endothelial Growth Factor, Wild-Type p53, and

Angiogenesis in Early Operable Non-Small Cell

Lung Cancer’

Alexandra Giatromanolaki,

Michael I. Koukourakis, Stylianos Kakolyris,

Helen Turley, Ken O’Byrne,

Prudence A. E. Scott, Francesco Pezzella,

Vassilios Georgoulias, Adrian L. Harris, and

Kevin C. Gatter�Department of Radiotherapy and Oncology and Laboratory of CancerBiology, University Hospital of Iraklion, Iraklion 71110, Crete,Greece [A. G., M. I. K., S. K., V. G.]; Department of Cellular Science

and ICRF Medical Oncology Unit, Oxford Radcliffe Hospital,

Headington, Oxford OX3 7U, United Kingdom [H. T., P. A. E. S.,F. P., A. L. H., K. C. G.]; and Department of Oncology, LeicesterRoyal Infirmary, Leicester LE1 5WW, United Kingdom [K. 0.]

ABSTRACTVascular endothelial growth factor (VEGF) is a cyto-

kine that is involved in tumor angiogenesis. Wild-type p53

(wt-p53) protein has been shown in cell lines to suppress

angiogenesis through thrombospondin regulation. In this

study, we immunohistochemically examined the expression

of VEGF, nuclear and wild-type cytoplasmic p53, bcl-2,

epidermal growth factor receptor, and c-erbB-2 oncopro-

tein; vascular grade; proliferation index; and extent of ne-

crosis in non-small cell lung cancer (NSCLC). We analyzed

120 cases of early-stage NSCLCs (81 squamous cell carcino-

mas and 39 adenocarcinomas) treated with surgery alone

(median follow-up, 63 months; range, 45-74 months). VEGFexpression showed a positive association with high vascular

grade (microvessel score of >75 per x250 field; P 0.008),

although about half of the LVG cases also expressed VEGF.

None of the p53 antibodies examined correlated with angio-

genesis. However, wt-p53 expression was inversely associ-

ated with VEGF expression, suggesting that wt-pS3 is in-

volved in the suppression of the VEGF gene. Combined

analysis of VEGF, wt-p53, and microvessel counting showed

that, although wt-p53 loss associates with VEGF switch-on,p53 protein may not be involved in the regulation of the

angiogenic events downstream of VEGF expression. More-

over, no significant association of bcl-2 and c-erbB-2 onco-

Received 6/8/98; revised 9/28/98; accepted 10/5/98.

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.

C The study was financially supported by the Imperial Cancer Research

Fund and the Tumor and Angiogenesis Research Group.

2 To whom requests for reprints should be addressed, at Department ofCellular Science, Room 5501, Level 5, John Radcliffe Hospital, Head-

ington, Oxford OX3 9DU, United Kingdom. Fax: 44-1865-222916.

protein expression with VEGF expression was observed.

T/N stage, grade, Ki67 proliferation index, and extent of

necrosis were not correlated with VEGF expression. Sur-

vival analysis showed that VEGF correlated with poor sur-

vival (P = 0.04) and was significant in node-negative cases(P = 0.03). We conclude that VEGF is an important angio-

genic factor in NSCLC, its expression being dependent on

wt-p53 loss.

INTRODUCTIONThe prognostic role of the angiogenic activity in human

tumors is currently under investigation. New antibodies recog-

nizing endothelial cells have been introduced, and attempts have

been made to find a common language for microvessel assess-

ment of immunohistochemically stained tumor samples (1).

Several factors have also been recognized to be involved in

endothelial cell migration, proliferation, and tube-like structure

formation. Fibroblast growth factor, a heparin-binding protein,

was the first factor recognized to have angiogenic properties (2).

Scatter factor, TP,’ and VEGF are among the angiogenic mol-

ecules that have recently attracted attention as potential prog-

nostic markers in human tumors (3).

VEGF, also known as vascular permeability factor, is a

cytokine with a well-established angiogenic activity. Human

cells express four different VEGF molecular species of 121,

165, 189, and 206 amino acids, which are all encoded by a gene

located on chromosome 6p2l.3 (4, 5). VEGF was first recog-

nized as a factor that renders vessels hyperpermeable to mac-

romolecules ( 1 ), a step that precedes the process of angiogene-

sis. Subsequently, VEGF was shown to be a potent rnitogen for

endothelial cells (2), enhancing endothelial cell migration and

tube-like vessel structure formation (3). Its role in maintaining

the differentiated state of blood vessels has also been recently

shown (4). VEGF seems to exert its activity exclusively on

endothelial cells, although chemotactic activity for macrophages

has been also reported (6).

The role of VEGF in the pathogenesis and prognosis of

human cancers has recently been shown by several investigators

(7-10). In this study, we examined the patterns of VEGF ex-

pression in human lung cancer, using an antibody recognizing

the 121-, 165-, and 189-amino acid isoforms of VEGF. We also

examined possible correlation of VEGF reactivity with survival,

angiogenesis, and other histopathological parameters, such as

3 The abbreviations used are: TP, thymidine phosphorylaze; VEGF,vascular endothelial growth factor; EGFR, epidermal growth factorreceptor; NSCLC, non-small cell lung cancer; MoAb, monoclonal an-

tibody; APAAP, alkaline phosphatase/antialkaline phosphatase; wt-p53,

wild-type p53; TBS, Tris-buffered saline; MS, microvessel score; HVG,

high vascular grade; LVG, low vascular grade.

Research. on June 15, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

3018 VEGF, p53, and Angiogenesis in Lung Cancer

bcl-2 staining was performed on frozen sections with the

TIN stage, differentiation, Ki67 mitotic index, necrosis, and

EGFR, c-erbB-2, bcl-2, and p53 expression in NSCLC.

MATERIALS AND METHODS

We examined 120 tumor samples from patients with early

operable NSCLC (8 1 squamous cell carcinomas and 39 adeno-

carcinomas). Histological diagnosis, grading, and N staging

were performed on H&E-stained sections. Patients dying within

60 days after operation were excluded to avoid bias from perio-

perative death. There were 94 male patients and 26 female

patients, ages 45-74 years (median age, 63 years). The fol-

low-up at the time of analysis was 3-7 years (median, 45

months).

Immunohistochemistry for VEGF. VEGF expression

was assessed with the VGI MoAb recognizing the 121-, 165-,

and 1 89-amino acid isofonms of VEGF. The VG1 antibody was

raised using recombinant VEGF 1 89-amino acid protein, and the

specificity of the antibody was confirmed using COS cells

transfected with cDNA coding for VEGF 121-, 165-, and 189-

amino acid proteins and by Western blotting studies (1 1). Stain-

ing was performed with the horseradish peroxidase technique.

Sections were dewaxed and incubated in 0.5% H2O2 in metha-

nol for 30 mm. After microwaving and washing in PBS, sections

were incubated with the primary antibody for 60 mm. After

washing in PBS for 5 mm, sections were incubated with goat

antimouse immunoglobulins (1 :200) for 30 mm (DAKO, United

Kingdorn), washed again with PBS for 5 mm, and incubated

with rabbit antigoat immunoglobulins (1:100) for 30 mm. The

peroxidase reaction was developed using diaminobenzidine

(Sigma Fast tablets) as chromogen, and sections were counter-

stained with hematoxylin. Normal rabbit IgG was substituted for

primary antibody as the negative control (same concentration as

the test antibody). The percentage of VEGF-positive cancer

cells (0-100%) was assessed by three observers. Taking into

account the extent of positive staining, we divided our cases into

three categories: low reactivity (0-29% positive cells), interme-

diate reactivity (30-69% positive cells), and high reactivity

(70-100% positive cells). This grouping was used for survival

analysis.

Angiogenesis Assessment. The JC7O MoAb (DAKO),

recognizing CD3 I (platelet/endothelial cell adhesion molecule;

Ref. I 2), was used for microvessel staining on 5-p.m paraffin-

embedded sections using the APAAP procedure. Sections were

dewaxed, rehydrated, and predigested with protease type XXIV

for 20 mm at 37#{176}C.JC7O ( 1 :50) was applied at room tempera-

tune for 30 mm and washed in TBS. Rabbit antirnouse antibody

1 :50 (v/v) was applied for 30 mm, followed by application of

mouse APAAP complex 1 : 1 (v/v) for 30 mm. After washing in

TBS, the last two steps were repeated for 10 mm each. The color

was developed by a 20-mm incubation with New Fuchsin so-

lution.

Microvessel counting was used for angiogenesis assess-

ment. For eye appraisal, sections were scanned at low power

( X40 and X 100) and afterward at X250 field to group cases into

three vascular grade categories (low, medium, and high). The

areas of the highest vascularization were chosen at low power

( X 100) and microvessel counting followed on three chosen

X250 fields of the highest density. The MS was the sum of the

vessel counts obtained in these three fields. Microvessels adja-

cent to normal lung were excluded from the appraisal. Vessels

with a clearly defined lumen or well-defined linear vessel shape

but not single endothelial cells were taken into account for

microvessel counting. HVG was defined as a MS of >74,

whereas LVG was defined as a MS of <75. This cutoff point

was based on a previous study, in which a MS of >75 defined

a group of cases with the highest death rate, compared with

other cutoff points (13)

Other Immunohistochemical Assessment. Proliferative

index was assessed with the MoAb Ki67 (DAKO A/S. Glostrup,

Denmark). Frozen material was taken from two separate areas of

the tumor, and the Ki67 assessment was based on the average

value. Three groups were considered, based on the percentage of

stained nuclei: 0-10%, low proliferative index (Pil); 10-40%,

medium proliferative index (Pi2); and >40%, high proliferative

index (Pi3; Ref. 14).

EGFR was identified by a murine MoAb (EGFR1) raised

against an epidermoid carcinoma cell line (15). Cryostat sec-

tions were processed by means of an indirect immunoperoxidase

technique. The positive control was human placenta, and for the

negative control, the primary antibody was omitted. Two groups

were considered: negative or very weak staining was considered

negative, and moderate or positive staining was considered

positive.

c-erbB-2 oncoprotemn expression was assessed with the

MoAb NCL-CB 1 1 (Novocastra Laboratories, Newcastle upon

Tyne, United Kingdom), which recognizes the internal domain

of the c-erbB-2 protein amino acid sequence (16). Staining was

performed with an indirect immunoperoxidase technique. Sec-

tions were dewaxed and incubated in 6% H2O7 in methanol for

30 mm. After being washed in TBS, sections were incubated

with the primary antibody at a dilution of 1 :40 for 60 mm.

Sections were washed in TBS for 5 mm and covered with

peroxidase-conjugated goat antimouse immunoglobulin

(DAKO) diluted at I :50 for 45 mm. The peroxidase reaction was

developed using diaminobenzidine as chromogen, and sections

were counterstained with hematoxylin. For a positive control,

we used a breast carcinoma with 15-fold amplification of the

c-erbB-2 gene, and for the negative control, the primary anti-

body was omitted. Two groups were identified for cytoplasmic

staining; the positive reactivity group (strong staining intensity

in >70% of cells) and the negative/weak reactivity group (all

other cases).

Nuclear p53 expression was assessed on 8-jim cryostat

sections with the APAAP technique, using the CM-I 1 and DO-7

antibodies (dilution, 1:30; DAKO). These antibodies are con-

sidered markers of mutant p53 activity, although wt-p53 expres-

sion may be also detected (17, 18). Cytoplasmic-perinuclear p53

was assessed in cryostat sections using the PAb248 MoAb. This

antibody recognizes the wild-type cytoplasmic p53 protein (19).

Two cutoff points were used for p53 positivity [10% (17) and

20%]. Although the 10% is the more frequently used cutoff

point, in a recent paper, the 20% cutoff was used to assess

VEGF correlation with angiogenesis in NSCLC (20). Moreover,

p53 staining was considered as a continues variable using a

score from 1 to 5, according to the percentage of p53-positive

cells.



Clinical Cancer Research 3019

APAAP method, using a MoAb that is specific for bcl-2 (clone

100 raised to a synthetic peptide; dilution, 1:20). A strong

diffuse expression was used to define positive cases (21).

Necrosis Assessment. The percentage of optical fields

(X250) with necrosis was recorded by three observers sepa-

rately. Necrotic areas in >50% of the examined fields (mean

value of the score given by the observers) was scored as exten-

sive, and in <50%, the necrotic areas were scored as limited.

Statistical Analysis. Statistical analysis and graphic

presentation were performed using Stata Version 3. 1 (Stata

Corporation, College Station, TX) and the GraphPad Prism

Version 2.01 package (GraphPad Software, Inc., San Diego,

CA). Unpaired two-tailed t test was used for testing relation-

ships between categorical tumor variables as appropriate. Linear

regression analysis was used to assess correlation between con-

tinuous variables. Survival curves were plotted using the

Kaplan-Meier method, and the log-rank test was used to deter-

mine statistical differences between life tables. A Cox propor-

tional hazard model was used to assess the effects of patient and

tumor variables on overall survival. A P of <0.05 was consid-

ered significant.

RESULTS

Normal Lung and Tumor VG1 Immunostaining. Al-

veolar epitheliurn was always negative, whereas bronchiolar and

differentiated columnar cells showed persistently positive reac-

tivity (Fig. 1A). Bronchial basal cells were negative. The normal

lung endothelium and fibroblast were not stained with the VGI

MoAb. Alveolar macrophages were positive (Fig. 1B). The

pattern of VG 1 staining was granular cytoplasmic. Immunore-

activity was heterogeneous, with no differences between the

central and marginal tumor areas. Intensity of tumor staining,

when present, was equal or stronger than normal epithelial

reactivity. Both squamous cell and adenocarcinomas showed a

varying degree of VEGF expression (Fig. 1, C and D).

Tumor stromal fibroblasts and macrophages were only

occasionally positive (Fig. 1E). Some tumor vessels also

showed a positive reactivity (Fig. lF). VG1-positive vessels

were identified in 53% (63 of 1 20) of the NSCLC cases.

VGI-reactive lymphocytic infiltration was also observed in

<10% (12 of 120) of NSCLC cases (Fig. 1, G and H).

Serum areas, observed in 77 of 120 NSCLC cases, were

consistently stained for VEGF (Fig. 11). In 82 of 120 NSCLC

cases, well-defined necrotic areas were observed. Intense VEGF

tumor cell expression around areas of necrosis was observed in

30% (24 of 82) of these cases (Fig. lJ).

Expression and Histopathological Parameters of

VEGF. Interobserver variability assessed with linear regres-

sion analysis was minimal (P < 0.0001, r > 0.93). The per-

centage of positive cells (mean ± SD) was 52 ± 33% (95%

confidence interval, 41-64%; median, 70%). Table 1 shows the

correlation of VEGF expression and MS with histopathological

(histology, T/N stage, grade, Ki67 mitotic index, and extent of

necrosis) and patient (age and sex) parameters. The VEGF

expression was used as a continuous variable according to the

percentage of positive cells. No statistically significant correla-

tion with the remaining parameters was found. A trend of VEGF

to be more frequently expressed in cases with a lower mitotic

index was observed (low/medium Ki67 versus high Ki67 index,

P = 0.10).

Correlation of VEGF with Angiogenesis. A significant

association of the percentage of positive VEGF cells with the

vascular grade was observed. HVG cases (MS of >75) had a

mean value of VEGF positive cells of 62 ± 3 1 % versus 45 ±

32% of cell positivity observed in LVG cases (MS <75), which

was significant (P = 0.008: unpaired two-tailed a’ test). The

association was significant for squamous cell carcinomas (72 ±

24% versus 41 ± 32%; P 0.0002) but not for the adenocar-

cinomas (47 ± 35% versus 54 ± 30%; P = 0.56). Linear

regression analysis between microvessel counting, and percent-

age of VEGF-positive cells showed a marginal statistically

significant association (P 0.06, r 0.20). In Fig. 2, 36 of 82

(44%) of the LVG cases had tumors with >70% positive VEGF

cells (inset A), showing that VEGF expression alone is not

sufficient for the onset of neovascularization. Twenty-one of 32

(66%) HVG tumors had >70% VEGF-positive cells (P = 0.05:

Fisher’s exact test).

Correlation of VEGF with p53 Staining. Linear regres-

sion analysis between p53 antibodies (score of 1-5) and per-

centage of VEGF-positive cells showed a significant inverse

correlation of VEGF with PAb248 cytoplasrnic expression (P =

0.003, r = 0.26). Taking 10% and 20% of positive cells as

cutoff points for p53 positivity, unpaired t test analysis showed

a significant association of high VEGF expression with negative

PAb248 expression (P = 0.01 and 0.009, respectively: Table 2).

Although there was a trend for the CM-l I antibody to directly

correlate with VEGF expression, the difference was not signif-

icant (P = 0.10 and P 0.06 for 10% and 20% cutoff points,

respectively).

Correlation of p53 with Angiogenesis. Linear regres-

sion analysis between CM-l 1, DO-7, and PAb248 p53 antibod-

ies showed no significant association of any of the three anti-

bodies with MS (P = 0.19, 0.10, and 0. 16, respectively). Taking

cutoff points of 10% and 20% p53 cell positivity, we observed

no significant association of p53 expression with angiogenesis.

Linear regression analysis between PAb248 and the expression

of CM-l 1 and DO-7 showed no inverse association (P = 0.62

and 0.34, respectively).

VEGF, PAb248, and Angiogenesis Analysis. To exam-

me whether wild-type cytoplasmic p53 (PAb248 MoAb) is

involved in the regulation of VEGF angiogenic activity, we

performed further analysis, taking all three factors into account

(Fig. 3). The percentage of VEGF-positive cells was signifi-

cantly higher (73.3 ± 25%) in cases with HVG and loss of

wt-p53 expression than it was in cases with HVG but rnainte-

nance of wt-p53 (48.4 ± 36%; P = 0.03). Cases with HVG and

maintenance of wt-p53 expression had a percentage of VEGF

positive cells similar to that observed in the LVG cases (47 ±

3 1%: P > 0.58). This shows that high angiogenesis in wt-p53-

positive cases is not dependent on VEGF activity.

Correlation with Other Parameters. There was no as-

sociation of VEGF expression with c-erbB-2 and EGFR expres-

sion. Analysis of c-erbB-2 together with VEGF showed a higher

MS for c-erbB-2-negative cases with VEGF expression as com-

pared to c-erbB-2-positiveNEGF-positive cases (MS 67 +-50

versus 46+-43), but the difference was not significant (P =

0.09). Although a lower percentage of VEGF-positive cells was



�. .

�-‘�-, . - m #{149}-,

3020 VEGF, pS3, and Angiogenesis in Lung Cancer

Fig. 1 A, differentiated columnar epithelium

expressing VEGF. B, alveolar macrophageswith positive reactivity. C and D. a squamous

cell carcinoma (C) and an adenocarcinoma

(D) that are positive for VEGF. E and F,

positive tumor stromal macrophages (E) and

vessels (fl. G and H. VEGF-expressingtumor-infiltrating lymphocytes. 1. serum

area stained for VEGF. J, cancer cells aroundnecrotic areas expressing VEGF.



100’

80

60

40

20

Ut

UtUUt

>I


Table I Correlation of V EGF expression and MS with histo logical and pa tient parameters in 120 NSCLC cases

Parameter (no. of patients)

% VEGF� cells MS

Mean ± SD P Mean ± SD P

Histology

Squamous cell (81) 50.3 ± 33 0.81 57.0 ± 43 0.16

Adenocarcinoma (39) 51.9 ± 32 69.6 ± 51

T stageTC (52) 48.3 ± 35 0.47 60.7 ± 50 0.87

T, (68) 52.9 ± 30 62.1 ± 43

N stage

N0 (83) 48.9 ± 32 0.35 47.9 ± 36 <0.0001

NC (37) 55.2 ± 33 86.9 � 50

GradeI/Il (51) 54.1 ± 28 0.37 58.1 ± 42 0.50

III (69) 48.4 ± 35 63.8 ± 48

Ki67”

L/M (97) 53.2 � 32 0.10 63.3 ± 45 0.35

H (23) 40.2 ± 36 52.9 ± 47

Necrosis

Limited (39) 53.8 ± 35 0.53 67.0 ± 47 0.61

Extensive (81) 49.6 ± 31 63.9 ± 41

Age

<65 yr (43) 50.9 ± 31 0.97 63.6 ± 46 0.52

>64 yr (37) 50.7 ± 34 58.1 ± 44

SexFemale (26) 53.9 ± 36 0.62 73.7 ± 62 0.15

Male (94) 50.1 ± 31 58.7 ± 40

aL, low; M, medium: H, high.

A

� #{149} � #{149}#{149}

�__#{149}#{149}_ #{149}#{149},� #{149} #{149}L J

#{149}#{149} #{149} p=O.06r=O.20

#{149}#{149}#{149}_ #{149}

0 50 100 150 200

Microvessel counting

Fig. 2 Linear regression analysis of microvessel counting and percent-age of VEGF-expressing cells. Inset A shows that a good proportion oflow VG cases had a high percentage of VEGF-positive cells (36 of 82:44%).

observed in bcl-2-positive cases (39% versus 5 1%), the differ-

ence was not significant (P 0. 1 1).

Overall Survival Analysis. NSCLC cases that were

evaluable for survival analysis (1 14 of 120) were divided into

three categories: low reactivity (37 of 1 14 cases; 0-29% posi-

tive cells), intermediate reactivity (42 of 1 14 cases; 30-69%

positive cells), and high reactivity (35 of 1 14 cases; 70-100%

positive cells). In univariate analysis, vascular grade (P =

0.0006), N stage (P = 0.001), bcl-2 (P = 0.008), and T-stage

(P = 0.02) were significant prognostic parameters. Cases with

high VEGF reactivity had a poorer prognosis than did cases with

low VEGF reactivity, although the significance was marginal

(P = 0.04; Fig. 4a). This was also significant in squamous cell

type (P = 0.04) but not in adenocarcinomas (P = 0.58). Anal-

ysis within the N0-staged cases showed that VEGF expression

defined a node-negative group of patients with statistically

worse prognosis (P 0.03: Fig. 4b).

Survival analysis for p53 expression showed no association

of any of the three p53 antibody staining patterns with progno-

sis. Expression of p53 was not associated with prognosis in

either LVG or HVG groups of patients. Double stratification for

VEGF and p53 expression did not reveal a subgroup of patients

with statistically significant worse prognosis. Stratifying the

LVG and HVG cases according to VEGF expression, we ob-

served no significant difference in survival. In multivariate

analysis (taking into account all of the parameters that were

significant in the univariate), none of the examined parameters

had an independent prognostic meaning. This was probably

because of the strong association between vascular grade and

nodal involvement, as well as between the VEGF expression

and vascular grade.

DISCUSSION

VEGF is a factor involved in vascular permeability, endo-

thelial cell migration, proliferation, and vessel maturation (1-3,

22). In this study, we investigated the expression of VEGF in

lung cancer using a MoAb recognizing the 121-, 165-, and

189-amino acid VEGF isoforms (1 1). VEGF was expressed

(expression in >50% ofcells) in 58% ofboth squamous cell and

adenocarcinomas. In a previous study, Mattern et a!. (23) also

found VEGF expression in 54 of 91 (59%) of squamous cell

lung cancer cases. Although he found a positive correlation of

VEGF expression with proliferating cell nuclear antigen label-



p=0.03

p=O.02

p=0.001

100-�

UtUUt 75

.?

.�

Ut00. 50

U-0w> 25

0

p=NS

1�Thm�VG and wt-p53 status

a

bFig. 3 Percentage of VEGF positive cells within the four combined

vascular grade and wt-p53 (PAb248) expression categories. #{149},HVG/wt-p53(-): �. HVG/wt-p53(+): EL LVG/wt-p53(-: L LVG/wt-p53(+).

100�

Ut.� 80

Cl) 60

Ut

� 40#{149}0

�20

0�

100

Ut.? 80

U) 60

Ut

� 400

� 20

500 1000 1500 2000 2500

Days

0 500 1000 1500 2000 2500

3022 VEGF, p53. and Angiogenesis in Lung Cancer

Table 2

For p53. 20% cell positivity w

Correlation of VEGF expression and MS w

as considered the cutoff point.

ith p53 expression in 120 NSCLC cases

Parameter (no. of_patients)

% VEGF� cells MS

± SD P Mean ± SD P

p53 (CM-lI)

Negative (61)

Positive (59)

p53 (D0-7)

Negative (85)

Positive (35)p53 (PAb248)

Negative (48)

Positive (72)

43.9 ± 34

55.2 � 31

47.5 ± 33

54.2 ± 32

59.1 ± 30

43.0 ± 34

0.06

0.31

0.009

54.1 ± 42

63.3 ± 48

55.3 ± 42

65.2 ± 51

66.5 ± 4555.8 ± 43

0.27

0.26

0.19

ing index, in our study, a trend of VEGF-positive cases to

associate with a low Ki67 proliferation index was observed. In

a study by Zhang et al. (24). transfection of the VEGF12I-

encoding gene in breast showed that the growth rate of V I 2 cells

in vitro was indistinguishable from that of MCF-7 wild-type

cells, although s.c. implantation of transfected cells formed

faster growing tumors in vivo because of intense neovascular-

ization. Plate et al. (25) also found no increased cellular prolif-

eration in glioma cell lines expressing VEGF as compared to

negative cell lines. Still. the EGFR immunoreactivity was not

increased in VEGF-expressing cell lines, which was also ob-

served in our study.

Peripheral blood, tumor-infiltrating lymphocytes, and

macrophages have been shown to express VEGF and a possible

role of VEGF of inflammatory origin in tumor neoangiogenesis

is postulated (26, 27). However. in our study, lymphocytes and

macrophages infiltrating the tumor as well as fibroblasts only

occasionally stained for VEGF. This may show that the stroma-

lly derived VEGF is probably of limited angiogenic importance

in NSCLC. In a previous study, we showed that stromal fibro-

blast TP overexpression substantially contributed to the appear-

ance of the angiogenic phenotype (28). Of interest is that tumor-

infiltrating macrophages that only occasionally expressed

VEGF strongly expressed the angiogenic factor TP (28). Be-

Days

Fig. 4 Kaplan-Meier overall survival curves for VEGF expression

within all analyzed cases (a) and within N0 stage group (b). a: , low

VEGF (A: 37 patients): - - - -, weak VEGF (B: 42 patients); - - -. high

VEGF (C: 35 patients). A versus B, P = 0.48: A versus C, P 0.04: Bversus C, P = 0.13. b: . low VEGF (A: 26 patients): - - - -, medium

VEGF (B: 27 patients): - - -. high VEGF (C: 27 patients). A versus B,

P = 0.10: A versus C. P = 0.03; B versus C. P = 0.37.

cause VEGF is shown to exert chemotactic activity on macro-

phages (29) a complex interplay among angiogenic and chemo-

tactic events may exist, which requires further investigation.

Several recent clinicopathological studies have shown a

varying degree of VEGF expression and correlation with angio-

genesis in different tumors. Guidi et al. (30) showed that VEGF




mRNA expression was higher in invasive cervical cancer as com-

pared to low-grade intraepithelial lesions. Toi et al. (7) reported that

VEGF was an important angiogenic factor in breast cancer. In two

previous studies, VEGF expression was significantly associated

with the degree of vascularization in NSCLC (20, 23). This is in

accordance with our study, in which a significant association of the

percentage of positive VEGF cells with vascular grade was ob-

served. However, linear regression analysis considering VEGF and

microvessel counting as continuous variables gave a marginal

significance. This was because a group of patients with LVG had

high VEGF expression. Taking this observation into account, we

made the assumption that VEGF expression per se is not sufficient

to switch on angiogenesis in NSCLC. Cooperation with other

angiogenic factors or loss of angiogenesis suppressing genes may

be of importance. In support of this hypothesis is a study by Toi et

a!. (31), in which TP was frequently coexpressed with VEGF in

breast cancer, and a very high MS was observed with TP and

VEGF coexpression.

The wt-p53 oncogene has been shown in vitro to inhibit

angiogenesis through regulation of thrombospondin- 1 , an inhib-

itor of angiogenesis (32). Moreover, in vitro data show that

bcl-2 may inhibit wt-p53 functions (33), which may result in

increased neovascularization. However, in previous studies, we

reported that bcl-2 and c-erbB-2 genes are expressed in poorly

vascularized lung tumors (34, 35). Several clinicopathological

studies also showed an inverse correlation of bcl-2 with mutant

p53 expression in lung, breast, and gastric cancer (36-38) or

even a positive correlation of mutant p53 with neovasculariza-

tion (20). In this study, no association of bcl-2 and c-erbB-2

with VEGF expression was observed, showing that these two

genes are unlikely to be involved in the regulation of VEGF-

mediated angiogenesis. Fontanini et a!. (20) reported a positive

association of mutant p53 (CM-l 1 Ab) and VEGF expression

with angiogenesis in NSCLC (20). In this study we observed

that, although there was a trend, mutant and wt-p53 did not

associate with angiogenesis. However, a statistically significant

inverse association of the wt-p53 expression with VEGF expres-

sion was observed. This shows that maintenance of wt-p53

activity may suppress the expression of VEGF. Although wt-

p53 loss permitted VEGF expression, p53 was not clearly in-

volved in the regulation of angiogenic events downstream of

VEGF expression, suggesting once again the existence of an

unknown factor cooperating with the VEGF.

Hypoxia and glucose deficiency are well known to induce

VEGF expression in vitro (39, 40). Both normal and cancer cells

produce VEGF under hypoxic stress and endothelial cell VEGF

receptors are up-regulated (4 1). The induction of VEGF around

necrotic tumor areas has been shown (42). However, in our

study, the extent of necrosis was not related to the expression of

VEGF throughout the tumor, although focal overexpression was

observed. This may show that hypoxia and nutrient deprivation

may not be sufficient for VEGF induction. It may be that, in

vivo, complementary factors such as hypoxia inducible factor

expression (43) or even inflammatory cell cytokine expression

(44) are also of importance.

VEGF expression in breast, colon, gastric, and bladder cancer

associates with poorer outcome and/or early relapse (7-10). A

lower survival of VEGF positive squamous cell lung cancers has

been also reported (45), whereas flt-l receptor status was not of

prognostic significance. In our study, survival analysis showed that

VEGF expression in NSCLC defined a poorer prognosis, espe-

cially in node negative patients. A similar observation was reported

in a previous study of ours, in which TP expression defined poor

prognosis in patients without lymph node involvement (46).

We conclude that VEGF associates with angiogenesis in

NSCLC, although its activity may depend on other angiogenic

or angio-suppressing proteins. wt-p53 protein seems to suppress

VEGF expression, but it is unclear whether it is involved in the

VEGF downstream events. Because both TP and VEGF expres-

sion are shown to associate with a favorable response to chem-

otherapy (47, 48) and to confer poor prognosis, even in the

absence of nodal metastasis, adjuvant chemotherapy and/or ra-

diotherapy in early operable NSCLC should be recommended in

TP- and VEGF-positive tumors.

REFERENCES

1. Vincenti, V., Cassano. C., Rocchi. M., and Persico. G. Assignment of

the vascular endothelial growth factor gene to the human chromosome

flp2l.3. Circulation, 93: 1493-1495, 1996.

2. Tischer. E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D.,

Fidders, J. C.. and Abraham, J. A. The human gene for vascular

endothelial growth factor. Multiple protein forms are encoded through

alternative exon splicing. J. Biol. Chem., 266: 1 1947-1 1954, 1991.

3. Ferrara, N.. and Davis-Smyth, T. The biology of vascular endothelial

growth factor. Endocr. Rev.. 18: 4-25, 1997.

4. Vermeulen, P. B., Gasparini, G., Fox, S. B.. Toi, M., Martin, L..McCulloch, P.. Pezzella, F.. Viale, G., Weidner, N., Harris, A. L., andDirix, L. Y. Quantification of angiogenesis in solid tumors: an interna-

tional consensus on the methodology and criteria of evaluation. Eur. J.

Cancer, 14: 2474-2484, 1996.

5. Folkman, J., and Klagsbrun. M. Angiogenic factors. Science (Wash-

ington DC). 235: 442-447, 1987.

6. Clauss, M., Gerlach, M., Gerlach, H., Brett, J., Wang, F., Familletti,P. C., Pan, Y. C., Olander, J. V., Connolly, D. T., and Stern, D. Vascularpermeability factor: a tumor-derived polypeptide that induces endothe-

hal cell and monocyte procoagulant activity. and promotes monocyte

migration. J. Exp. Med., 172: 1535-1545, 1990.

7. Toi, M., Inada, K.. Suzuki, H.. and Tominaga, T. Tumor angiogen-esis in breast cancer: its importance as a prognostic indicator and the

association with vascular endothelial growth factor expression. Breast

Cancer Res. Treat.. 36: 193-204, 1995.

8. 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.

9. Maeda, K., Chung, Y. S.. Ogawa. Y., Takatsuka, S.. Kang. S. M..Ogawa, M., Sawada, T., and Sowa, M. Prognostic value of vascularendothelial growth factor expression in gastric carcinoma. Cancer(Phila.), 77: 858-863, 1996.

10. O’Brien, T., Cranston. D., Fuggle, S., Bicknell, R., and Harris, A. L.

Different angiogenic pathways characterize superficial and invasive

bladder cancer. Cancer Res., 55: 510-513, 1995.

1 1 . Thang, L., Scott, P., Turley, H., Leek, R.. Lewis. C. E., Gatter, K. C..

Harris, A. L.. Mackenzie, I. Z., Rees, M. P., and Bicknell, R. Validation of

anti-vascular endothelial growth factor (anti-VEGF) antibodies for immu-

nohistochemical localization of VEGF in tissue sections: expression of

VEGF in the human endometrium. J. Pathol., 185: 402-408, 1998.

12. Giatromanolaki, A.. Koukourakis, M., O’Byrne, K.. Fox, S., White-house. R., Talbot. D.. Harris, A. L., and Gatter. K. C. Angiogenesis is a

significant prognostic marker in operable non small cell lung cancer.

J. Pathol.. 179: 80-88. 1996.

13. Giatromanolaki. A., Koukourakis. M., Theodossiou, D.. Barbatis,C., Harris, A. L., and Gatter, K. C. Comparative evaluation of angio-

genesis assessment with anti-Factor VIII and anti-CD3 1 immuno-



3024 VEGF, pS3, and Angiogenesis in Lung Cancer

staining in non small cell lung cancer. Clin. Cancer Res., 3: 2493-2500,

1997.

14. Tungekar, M. F.. Gatter. K. C., Dunnill, M. S., and Mason, D. Y.Ki-67 immunostaining and survival in operable lung cancer. Histopa-

thology, /9: 545-550, 1991.

15. Veale, D., Aschroft, T., Gibson. G. J., and Harris, A. L. Epidermal

growth factor receptors in non small cell lung cancer. Br. J. Cancer, 55:

513-516. 1987.

16. Giatromanolaki. A., Gorgoulis, V., Cheny, R., Koukourakis, M.,Whitehouse, R.. Kittas, C., Veslemes, M., Gatter, K. C., and lordano-

glou, I. C-erbB-2 oncoprotein expression in operable non-small cell lungcancer. Anticancer Res., /6: 987-994, 1996.

17. McLaren, R., Kuzu, I., Dunnill, M., Harris, A. L., Lane, D., and

Garter, K. C. The relationship of p53 immunostaining to survival in

carcinoma of the lung. Br. J. Cancer, 66: 735-738, 1992.

18. Pezzella, F., Turley, H., Kuzu, I., Gatter, K. C., and Mason, D. Y.

Reversible p53 expression in lung cancer. Lancet. 340: 922, 1992.

19. Pezzella, F., Micklem, K., Turley, H., Jones, M., Kocialkowski, S.,Delia, D., Aiello, A., Bicknell, R.. Smith, K., Harris. A. L., Gatter. K. C..

and Mason, D. Y. Antibody for detecting p53 protein by immunohisto-

chemistry in normal tissues. J. Clin. Pathol., 47: 592-596, 1994.

20. Fontanini, G., Vignati, S., Lucchi, M., Mussi, A., Calcinai, A..

Boldrini, L., Chine, S., Silvestri, V., Angeletti, C. A., Basolo, F., andBevilacqua, G. Neoangiogenesis and p53 protein in lung cancer: their

prognostic role and their relation with vascular endothelial growth factor

(VEGF) expression. Br. J. Cancer. 75: 1295-1301, 1997.

21 . Pezzella, F., Turley, H., Kuzu, I., Tungekar. M. F., Dunnill, M. S.,Pierce, C. B., Harris, A., Gatter, K., and Mason, D. Y. bcl-2 protein innon small cell lung carcinoma. N. Engl. J. Med., 329: 690-694, 1993.

22. Leung, D. W., Cachianes, G., Kuang. W. J., Goeddel, D. V., and

Ferrara, N. Vascular endothelial growth factor is a secreted angiogenicmitogen. Science (Washington DC), 246: 1306-1309, 1989.

23. Mattern, J., Koomagi, R.. and VoIm, M. Association of vascularendothelial growth factor expression with intratumoral microvessel den-sity and tumor cell proliferation in human epidermoid lung carcinoma.

Br. J. Cancer, 73: 93 1-934, 1996.

24. Zhang, H. T., Craft, P., Scott, P. A., Ziche, M., Weich, H. A.,Hams, A. L.. and Bicknell, R. Enhancement of tumor growth and

vascular density by transfection of vascular endothelial cell growthfactor into MCF-7 human breast carcinoma cells. J. Nail. Cancer Inst.

(Bethesda), 87: 213-219, 1995.

25. Plate. K. H.. Breier, G., Weich, H. A., Mennel, H. D.. and Risau, W.

Vascular endothelial growth factor and glioma angiogenesis: co-ordinate

induction of VEGF receptors, distribution of VEGF protein and possible in

vivo regulatory mechanisms. Int. J. Cancer, 59: 520-529, 1994.

26. Freeman, M. R., Schneck, F. X., Gagnon, M. L., Corless. C., Soker,S., Niknejad, K., Peoples, G. E., and Klagsbrun, M. Peripheral blood Tlymphocytes and lymphocytes infiltrating human cancers express vas-

cular endothelial growth factor: a potential role for T cells in angiogen-esis. Cancer Res., 55. 414#{216}4145 1995.

27. Berse, B., Brown, L. F., Van de Water, L., Dvorak, H. F., and

Senger, D. R. Vascular permeability factor (vascular endothelial growthfactor) gene is expressed differentially in normal tissues, macrophagesand tumors. Mol. Biol. Cell, 3: 21 1-220, 1992.

28. Koukourakis, M., Giatromanolaki, A., Kakolyris, S., O’Byrne, K.,

Apostolikas. T., Skarlatos, J., Gatter, K. C., Harris, and A. L. Differentpatterns of stromal and cancer cell thymidine phosphorylaze reactivityin non small cell lung cancer. Impact on neoangiogenesis and survival.Br. J. Cancer, in press, 1998.

29. Clauss, M., Cerlach. M., Cerlach, H., Brett, J., Wang, F., Familletti,P. C., Pan, Y. C., Olander, J. V., Connolly. D. T., and Stern, D. vascularpermeability factor: a tumor derived polypeptide that induces endothe-

hal cell and monocyte procoagulant activity, and promotes monocyte

migration. J. Exp. Med., 172: 1535-1545, 1990.

30. Guidi, A. J., Abu-Jawdeh, G., Brygida, B.. Jackman, R. W., Tognazzi,K., Dvorak, H. F., and Brown, L. F. Vascular permeability factor (vascularendothelial growth factor) expression and angiogenesis in cervical neopla-sia. J. Nat!. Cancer Inst. (Bethesda), 87: 1237-1245, 1995.

31. Toi, M., Inada, K., Hoshina, S., Suzuki, H., Kondo, S., and

Tominaga, T. Vascular endothelial growth factor and platelet-derivedendothelial cell growth factor are frequently coexpressed in highlyvascularized human breast cancer. Clin. Cancer Res., 1: 961-964, 1995.

32. Dameron, K. N., Volpert, 0. V., Tainsky. M. A., and Bouck, N.Control of angiogenesis in fibroblasts by p53 regulation of throm-bospondin-l. Science (Washington DC), 265: 1582-1584, 1994.

33. Ryan, J. J., Prochownik, E., Gottlieb, C. A., Apel, I. J., Memo, R.,Nunez, G., and Clarke, M. F. c-myc and bcl-2 modulate p53 function by

altering p53 subcellular trafficking during the cell cycle. Proc. Nat!.

Acad. Sci. USA, 91: 5878-5882, 1994.

34. Giatromanolaki, A., Koukourakis, M., O’Byrne, K., Kaklamanis,L., Dicoglou, C., Trichia, E., Whitehouse, R., Harris, A. L., and Gatter,K. C. Non small cell lung cancer: c-erbB-2 correlates with low angio-genesis and poor prognosis. Anticancer Res.. 16: 3819-3825, 1996.

35. Koukourakis, M., Giatromanolaki, A., O’Byrne, K.. Whitehouse,R., Talbot, D. C., Garter, K. C., and Harris, A. L. Potential role of bcl-2as a suppressor of tumor angiogenesis in non small cell lung cancer. Int.

J. Cancer, 74: 565-570, 1997.

36. Nakamura, S., Akazawa, K., Kinukawa, N., Yao, T., and Tsuneyo-shi, M. Inverse correlation between expression of bcl-2 and p53 proteinsin primary gastric lymphoma. Hum. Pathol., 27: 225-233, 1996.

37. Fontanini. G., Vignati, S., Bigini, D., Mussi, A., Lucchi, M., Angeletti,C. A., Basolo, F., and Bevilacqua, G. Bcl-2 protein: a prognostic factor

inversely correlated to p53. Br. J. Cancer. 71: 1003-1007, 1995.

38. Charpin, C., Garcia, S., Bouvier, C., Devictor, B., Andrac, L.,

Lavaut, M. N., and Allasia, C. Automated and quantitative immunohis-

tochemical assays of bcl-2 protein in breast carcinoma. Br. J. Cancer,

76: 340-346, 1997.

39. Shweiki, D., Neeman, M.. Itin, A., and Keshet, E. Induction ofvascular endothelial growth factor expression by hypoxia and by glu-cose deficiency in multicell spheroids: implications for tumor angiogen-

esis. Proc. Natl. Acad. Sci. USA, 92: 768-772, 1995.

40. Kuroki, M., Voest, E. E., Amano, S., Beerepoot, L. V., Takashima,S., Tolentino. M., Kim, R. Y., Rohan, R. M., Colby, K. A., Yeo, K. T.,and Adamis, A. P. Reactive oxygen intermediates increase vascularendothelial growth factor expression in vitro and in vivo. J. Clin. Invest.,98: 1667-1675, 1996.

41. Brogi, E., Schatteman. G., Wu, T., Kim, E. A., Varticovski, L.,Keyt, B., and Isner, J. M. Hypoxia-induced paracnne regulation ofvascular endothelial growth factor receptor expression. J. Clin. Invest.,

97: 469-476, 1996.

42. Shweiki, D., Itin, A., Soffer, D., and Kashet, E. Vascular endothelial

growth factor induced by hypoxia may mediate hypoxia-initiated angio-genesis. Nature (Lond.). 359: 843-845, 1992.

43. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W.,

Koos, R. D., and Semenza, G. L. Activation of vascular endothelialgrowth factor gene transcription by hypoxia-inducible factor I . Mol.Cell. Biol., 16: 4604-4613, 1996.

44. Cohen, T., Nahari. D., Cerem, L. W., Neufeld, G., and Levi, B. Z.Interleukin-6 induces the expression of vascular endothelial growthfactor. J. Biol. Chem., 271: 736-741, 1996.

45. Volm, M., Koomagi, R., and Mattern, J. Prognostic value of vas-cular endothelial growth factor expression and its receptor fit- 1 in

squamous cell lung cancer. Int. J. Cancer. 74: 64-68, 1997.

46. Koukourakis, M. I., Giatromanolaki, A., O’Byrne, K., Comley, M.,Whitehouse, R., Talbot. D. C., Gatter, K. C., and Harris, A. L. Platelet-

derived endothelial cell growth factor expression correlates with tumor

angiogenesis and prognosis in non-small cell lung cancer. Br. J. Cancer, 4:

477-481, 1997.

47. VoIm, M., Koomagi, R.. and Mattern, J. Interrelationships between

microvessel density, expression of VEGF and resistance to doxorubicin

of non-small lung cell carcinoma. Anticancer Res., 16: 213-217, 1996.

48. Patterson, A., Zhang, H., Moghaddam, A., Bicknell, R., Talbot, D.,Stratford, I., and Harris, A. L. Increased sensitivity to the pro-drug5’-deoxy-5-fluorounidine and modulation of 5’-fluoro-2’-deoxyuridine

sensitivity in MCF-7 cell transfected with thymidine phosphorylaze.

Br. J. Cancer, 72: 669-675, 1995.



1998;4:3017-3024. Clin Cancer Res A Giatromanolaki, M I Koukourakis, S Kakolyris, et al. angiogenesis in early operable non-small cell lung cancer.Vascular endothelial growth factor, wild-type p53, and

Updated version

http://clincancerres.aacrjournals.org/content/4/12/3017

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/4/12/3017To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Vascular Endothelial Growth Factor, Wild-Type p53, and ... · over, no significant association of bcl-2 and c-erbB-2 onco-Received 6/8/98; revised 9/28/98; accepted 10/5/98. The costs