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.
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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.
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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
Research. on June 15, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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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.
Research. on June 15, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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Clinical Cancer Research 3021
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.
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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-
Research. on June 15, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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vascular grade and wt-p53 (PAb248) expression categories. #{149},HVG/wt-p53(-): �. HVG/wt-p53(+): EL LVG/wt-p53(-: L LVG/wt-p53(+).
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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
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Clinical Cancer Research 3023
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.
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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
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