2008;14:3030-3035.

Clin Cancer Res

Timothy J. Duncan, Ahmad Al-Attar, Phil Rolland, et al.

Cancer: A Model for Targeted Use of Novel Therapies?
Vascular Endothelial Growth Factor Expression in Ovarian

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Vascular Endothelial Growth Factor Expressionin Ovarian
Cancer: AModel forTargeted Use of Novel Therapies?
TimothyJ. Duncan,
1
Ahmad Al-Attar,
1
Phil Rolland,
1
Ian V. Scott,
4
Suha Deen,
2
David T.Y. Liu,
3
Ian Spendlove,
1
and Lindy G. Durrant
1
Abstract
Purpose: Angiogenesis has a vital role in tumor growth and metastasis, and vascular endothelial
growth factor (VEGF) represents a potent cytokine in this process. However, the influence of
VEGF in ovarian cancer remains controversial. Interest has focused on the use of antiangiogenic
drugs in ovarian cancer. This study aims to establish the pattern of expression and effect on
prognosis of VEGF in a large population of ovarian cancer patients and to potentially identify a
cohort in whomantiangiogenic therapy is appropriate.
Experimental Design: Usinga tissue microarray of 339 primary ovariancancers, the expression
of VEGF was assessed immunohistochemically. Coupled to a comprehensive database of
clinicopathologic variables, its effect on these factors and survival was studied.
Results: Tumors expressing high levels of VEGF had significantly poorer survival (P = 0.04).
Factors shown to predict prognosis independently of each other were age, International Federa-
tion of Gynecologists and Obstetricians stage, and the absence of macroscopic disease after
surgery. VEGF was independently predictive of prognosis on multivariate analysis (P = 0.02).
There was no correlation betweenVEGF and any clinicopathologic variable. High expression of
VEGF was seen in only 7% of the tumors, suggesting that the role of antiangiogenic drugs may
be limited to a small subset of patients.
Conclusion: High VEGF expression occurs in a small proportion of ovarian cancers, and this
independently predicts poor prognosis. The small percentage of tumors with high levels of
VEGF activity suggests that the role of bevacizumab may potentially be limited to a few patients;
these patients could be targeted by molecular profiling.
Ovarian cancer is the leading cause of death from gynecologic
malignancies. As symptoms are vague and nonspecific, these
tumors often present at an advanced stage. Developments in
chemotherapy and surgical techniques have had minimal effect
on patient survival over the last few decades (1).
Tumor stage and residual tumor mass, following primary
cytoreductive surgery, have been shown to most reliably predict
outcomes in patients with ovarian cancer (2) but offer no
information with regard to the potential sensitivity to
molecular targeted therapy. Investigation of novel prognostic
markers offers an insight into the mechanisms of tumor
development and suggests potential avenues for the develop-
ment of new therapeutic agents particularly through the use of
monoclonal antibody therapies.
Angiogenesis has been established as a vital component in
the mechanisms involved in tumor growth and metastasis
(3, 4). The angiogenic potential of tumors can be assessed by
microvessel density. Earlier studies illustrated the importance of
angiogenesis in tumor development, with microvessel density
directly correlating with a poor prognosis in ovarian (5) and
other tumors (6, 7).
Vascular endothelial growth factor (VEGF) is a multifunc-
tional cytokine that stimulates angiogenesis and increases
microvascular permeability through binding to specific recep-
tors expressed on vascular endothelial cells (8, 9). Although
VEGF is produced by several tumors and hypoxic tissues (10),
its receptors are expressed primarily by endothelial cells. It has
been shown to have a crucial role in neovascular formation in
tumors, providing nourishment for the highly metabolic tumor
cells as well as providing access to the host vasculature (11).
Studies have suggested a specific role for VEGF in various
phases of ovarian carcinogenesis, with effects on tumor
growth and neovascularization seen in animal models and
in humans (12, 13). Higher levels of VEGF are shown in
ovarian carcinomas when compared with normal ovaries
(14, 15).
Recent interest has focused on the use of antiangiogenic
drugs in an attempt to inhibit the protumor effects of VEGF
and other such cytokines. These studies have shown some
antitumor effects but have shown significant side effects.
Imaging, Diagnosis, Prognosis
AuthorsAffiliations:
1
Academic and Clinical Department of Oncology, University
of Nottingham;
2
Division of Histopathology and
3
Department of Obstetrics and
Gynaecology, University Hospitals Nottingham, Nottingham, United Kingdom and
4
Department of Obstetrics and Gynaecology, Derby City General Hospital, Derby,
United Kingdom
Received 8/1/07; revised1/27/08; accepted 2/7/08.
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
with18 U.S.C. Section1734 solely to indicate this fact.
Requests for reprints: Lindy G. Durrant, Institute of Infections and Immunity,
University of Nottingham, Nottingham City Hospital NHS Trust, Hucknall Road,
Nottingham NG5 1PB, United Kingdom. Phone: 44-115-82-31862; Fax: 44-115-
82-31849/44-121-353-1482; E-mail: lindy.durrant@nottingham.ac.uk.
F2008 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-07-1888
www.aacrjournals.org Clin Cancer Res 2008;14(10) May15, 2008 3030
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The future role of such therapies is yet to be established
(1618).
Previous research has produced inconsistent evidence with
regard to the importance of VEGF in ovarian cancer and its
relation to prognosis. These studies often suffered from low
patient numbers and the use of subset analysis (1921). This
study was designed to determine VEGF status in patients with
ovarian carcinoma and investigate its relation to prognosis. This
was done through the assessment of over 350 consecutive
patients using tissue microarray technology. Second, elucida-
tion of the prognostic role of VEGF in patients with ovarian
cancer might enable more individualized adjuvant treatments
using developing novel therapies to inhibit tumor angiogenesis;
these therapies are likely to be most effective in tumors
expressing high levels of VEGF.
Materials and Methods
Patients. A total of 358 patients with ovarian cancer were entered in
this study, and these consisted of patients undergoing a laparotomy for
primary ovarian cancer. Information on cancer size, stage, presence or
absence of residual disease after surgery, histologic type and grade, age
at diagnosis, and type of adjuvant treatment were collected for all
patients (Table 1). From this original population, histologic material
was available for analysis in 339 cases. The paraffin-embedded tissue
blocks from these patients dated back from January 1, 1984 until
December 31, 1997. Disease-specific survival was calculated from the
operation date until November 31, 2005 when any remaining survivors
were censored. The database was audited to ensure validity; there were
no major discrepancies with >97% of data available.
From each of the 339 patients with histologic material available, two
tumor samples were used for analysis. In total, 320 carcinomas were
analyzed for VEGF following the omission of the cores, which were
uninterpretable due to tissue loss during processing (6% core loss).
During the study period, patients with high-grade stage I and stage
II to IV disease received chemotherapy. The specific chemotherapy
varied but reflected the best current practice; most recently, this
treatment was platinum based. Sixty-two patients participated in the
International Collaborative Group for Ovarian Neoplasia trials I to IV
during which the allocated chemotherapy was randomized.
Although the study spans a 14-y period, there was no significant
change in the survival of patients treated in the earlier or latter part of
the study. This is in line with the unaltered survival of ovarian cancer
patients over the last 30 y (22).
Tissue microarray construction. All tumors received following
resection in the operating theater were incised, fixed immediately in
10% neutral buffered formalin overnight, and then embedded in
paraffin wax, ensuring optimal tissue fixation and preservation for
histologic examination.
Tissue microarrays were constructed as described previously (23). For
each tumor, 5-Am section slides stained with H&E were first used to
locate representative areas of viable tumor tissue. Needle core biopsies
(0.6 mm) from the corresponding areas on the paraffin-embedded
tumor blocks were then placed at prespecified coordinates in recipient
paraffin array blocks using a manual tissue arrayer (Beecher Instru-
ments). Array blocks were constructed with between 76 and 133 cores
in each, and five copies of the array were assembled using different
points within the representative tumor area. Fresh 5-Am sections were
obtained from each tissue microarray block and placed on coated glass
slides to allow the immunohistochemical procedures to be done,
preserving maximum tissue antigenicity.
Immunohistochemical staining. A prediluted rabbit anti-human
VEGF antibody was used (SP28, Abcam). Optimization of the staining
was done on ovarian cancer whole-section mounts using a range of
incubation times. Two-hour incubation was chosen to stain the arrays
as it showed the best results with minimal background staining. Two
copies of the ovarian tissue microarrays were cut from the paraffin
blocks (4 Am thickness), transferred to extra-adhesive glass slide, and
stained in a single run using a routine streptavidin-biotin peroxidase
technique. Briefly, slides were dewaxed in xylene (2 10 min) and
rehydrated in three grades of ethanol (99%, 90%, and then 70%) for
1 min each. Endogenous peroxidase activity was blocked by immersing
the slides in a 0.03% solution of H
2
O
2
in methanol for 25 min. Heat-
induced epitope retrieval was done using an 800-W rotary microwave
oven. Slides were put in a plastic vessel containing 10 mmol/L sodium
citrate buffer (pH 6.0) and treated for 10 min on high power and then
for 10 min on low power. Slides were then taken out and cooled down
under running tap water for 20 min and washed with TBS (Dako) for
10 min more. To reduce nonspecific adsorption of antibodies to tissue,
the slides were incubated with normal swine serum (Dako) diluted
1:20 in TBS for 10 min. The test sections were then left to incubate
with the anti-VEGF antibody for 1 h at room temperature. Negative
control sections were incubated with normal swine serum under the
same conditions. Following a thorough wash with TBS, 100 AL of the
biotinylated anti-mouse antibody, diluted 1:100 (Dako), were applied
for 30 min, followed by another TBS wash and then 100 AL of the
streptavidin-biotin/horseradish peroxidase complex solution (prepared
30 min in advance). This was left to incubate with the sections for
60 min. The color was developed using 3,3-diaminobenzidine (Dako)
and enhanced with 0.5% CuSO
4
solution, and the sections were
then counterstained with Mayers hematoxylin solution (Dako) for
1 min.
Scoring of cores. The intensity of the staining was estimated on a
four-tiered scale, encoded as 0 (absent), 1 (weak), 2 (moderate), and 3
(strong). The pathologist and researcher who reviewed the immunos-
taining of the tissue samples were blinded to the clinicopathologic data
of the patients.
Table 1. Patient characteristics
Frequency (n) %
SEER age categories (n = 357)
<30 y at diagnosis 2 1
30-60 y at diagnosis 143 40
>60 y at diagnosis 212 59
FIGO stage (n = 348)
I 95 27
II 38 11
III 175 50
IV 40 12
Macroscopic residual disease (n = 344)
Absent 143 42
Present 201 58
Histologic type (n = 358)
Serous cystadenocarcinoma 178 49
Mucinous cystadenocarcinoma 35 10
Endometrioid 42 12
Clear cell 25 7
Undifferentiated 54 15
Others 24 7
Tumor grade (n = 337)
1 39 11
2 73 22
3 225 67
Adjuvant therapy (n = 350)
No 101 29
Yes 249 71
Abbreviations: SEER, Surveillance, Epidemiology, and End
Results; FIGO, International Federation of Gynecologists and
Obstetricians.
VEGFExpression in Ovarian Cancer
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Statistical analysis. The correlation between VEGF expression levels
and other prognostic variables was statistically analyzed by means of
Pearsons m
2
test.
Survival rates were examined using Kaplan-Meier plots for analysis of
censored data. The statistical significance of differences between the
survival rates of groups with different VEGF expression was assessed
using the log-rank test. The independent prognostic significance of
variables was assessed in multivariate analysis by means of a
multivariant Cox regression model and the -2 log likelihood test
(omnibus test). P values V0.05 were assumed statistically significant. All
statistical analysis was done using the computer statistical program
Statistical Package for the Social Sciences 15.0 (SPSS).
Results
Clinicopathologic characteristics. In the current cohort of
ovarian cancer patients, the mean age at diagnosis was 61 years
Fig. 1. Photomicrographs of ovariantissuemicroarraycoresimmunohistochemicallystainedforVEGF.Thelevel of expressionrangedfromnone(A) toweak(B) tomoderate(C)
to strong (D). Magnifications: 100 (A-D) and 400 (inset).
Table 2. Survival times, 95% confidence intervals (A) and survival rates at 12 and 24 mo (B)
A. Median survival time in relation to VEGF expression
VEGF expression Median
Estimate (mo) 95% CI
Lower bound Upper bound
Low 24.1 18.9 29.4
High 13.8 1.5 26.0
Overall 23.5 18.7 28.3
B. 12- and 24-mo survival rates
VEGF expression 12-mo survival 24-mo survival
% alive 95% CI % alive 95% CI
Lower bound Upper bound Lower bound Upper bound
Low 66.4 63.6 69.2 50.0 47.1 52.9
High 56.0 45.5 66.5 38.1 27.7 48.5
Imaging, Diagnosis, Prognosis
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(range, 24-90). Using the current Surveillance, Epidemiology,
and End Results Program age categorization system, 59% of the
patients were in group 3 (>60 years at diagnosis), 40% were 30
to 60 years at diagnosis, and only 2 of 357 were <30 years.
Serous cystadenocarcinoma was the commonest histologic type
(49%) followed by undifferentiated (15%), endometrioid
(12%), mucinous cystadenocarcinoma (10%), clear cell (7%),
and other types (7%). All patients were treated surgically, of
which 42% had their masses optimally debulked with no
macroscopic disease left. Clinicopathologic staging showed that
the majority of patients (50%) were stage III followed by 27%
in stage I, 11% in stage IV, and 11% in stage III. When
histologic grading was applicable, almost two thirds of the
tumors were poorly differentiated (grade 3). Twenty-two
percent were moderately differentiated, and only 11 were
deemed well differentiated. All patients characteristics are
summarized in Table 1.
VEGF analysis. VEGF analysis was done on 320 primary
ovarian tumors, with the remaining 19 tumor cores not being
interpretable due to tissue loss during the immunohistochem-
ical processing. When staining was positive, it was primarily of
cytoplasmic location, and its pattern was uniform among the
cancer cells within each core. Only 22 tumors (6.9%) were
strongly positive, whereas the remaining tumors (298 of 320)
exhibited either weak (135 of 320, 42.2%) or moderate
(126 of 320, 39.4%) staining. Thirty-seven tumor cores
(11.6%) failed to show any noteworthy staining. Photomicro-
graphs of each category are shown in Fig. 1A to D. The scoring
system was designed to identify potentially sensitive tumors to
anti-VEGF therapies, and as such, cases were categorized as
either high expressers (represented by the strongly stained
group) or low expressers (composed of the negative, weak,
and moderate groups).
VEGF correlations. Using m
2
test, VEGF expression did not
correlate with the patients age, tumor grade, stage, or histologic
type, nor was it associated with the presence or absence of
residual disease or with the administration of adjuvant
chemotherapy.
Survival analysis revealed that patients who had tumors with
low levels of VEGF had a median survival time of 24.1 months,
whereas that of high VEGF expression was 13.7 months
(Table 2A). The 12- and 24-month survival rates for high VEGF
expression were 56.0% and 38.1%, respectively, compared with
66.4% and 50.0% for low expression (Table 2B). This
difference in survival was shown to be statistically significant
with the log-rank test (test statistic = 4.2; P = 0.04), and a
Kaplan-Meier survival plot is shown in Fig. 2.
Using the Cochrane-Armitage test, VEGF did not correlate
with any of the established clinicopathologic prognostic
variables (e.g., age, stage, and grade). On its own, high VEGF
expression correlated with poor prognosis, yielding an unad-
justed hazard ratio (HR) of 1.59 [95% confidence interval
(95% CI), 1.02-2.49; P = 0.042]. However, to ensure the
findings were truly independent of other clinicopathologic
variables, the Cox model was expanded to incorporate stage,
residual disease, and the receipt of adjuvant therapy. The
adjusted HR for high VEGF expression was 1.78 (95% CI, 1.08-
2.94; P = 0.023; Table 3). The independent effect of VEGF was
reinforced by doing the Cox model excluding VEGF as a
variable; the HRs for stage, residual disease, and adjuvant
therapy were unaltered. Using the -2 log likelihood test
(omnibus test), the effect of VEGF on our multivariate model
was shown to be significant (P = 0.036). This suggests that the
model as a whole is significant and the covariate effect of VEGF
is different from zero.
Discussion
Despite the assumed importance of VEGF in the progression
of cancer, relatively few studies of the expression of this protein
in ovarian cancer have been linked to comprehensive clinical
data, particularly data on survival.
The distribution of histologic subtypes is consistent with
established literature, with serous carcinoma being the most
prevalent followed by endometrioid and mucinous (1). The
Fig. 2. Kaplan-Meier graph and log-rank two-tailed (Mantel-Cox) test for
VEGF = 4.2 (P = 0.04).
Table 3. Cox multivariant regression model
HR 95% CI for HR P
Lower Upper
FIGO stage
I 1 <0.001
II 2.25 1.28 3.97
III 4.52 2.74 7.47
IV 4.99 2.78 8.96
Macroscopic residual disease
Absent 1 <0.001
Present 2.38 1.69 3.36
Adjuvant therapy
No 1 <0.001
Yes 0.48 0.33 0.69
VEGF
Low 1 0.023
High 1.78 1.08 2.94
VEGFExpression in Ovarian Cancer
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typical distribution of ovarian cancer in relation to stage and
differentiation is seen with the majority presenting with poorly
differentiated, advanced (stage III and IV) disease (24). The
clinicopathologic features correlate with established character-
istics of ovarian cancer, suggesting that the study contains a
representative cohort of patients, and hence, the study findings
will be of relevance to a general population.
The expression of VEGF in our patients exhibited a similar
pattern to that of Goodheart et al. (19), with a strong
expression seen in 6.9% of the tumors. Lesser expression
ranged from moderate (39.4%) to weak (42.2%) to no
expression in 11.6% of the patients. Other groups reported
varying levels of VEGF using different antibodies to stain the
tissue, different scoring systems, and different cutoff points.
Our results indicate that patients with tumors that express
high levels of VEGF have worse survival rates compared with
those with medium, low, or no VEGF. A median survival
advantage of f10 months is seen among the latter group when
compared with the former (P = 0.04; Fig. 2). This result is
further substantiated when a multivariant Cox regression
model was constructed in which established prognostic factors
were accounted for. VEGF maintained statistical significance
with regard to patient survival (P = 0.023; Table 3). The HR
indicates that patients with high VEGF have f75% higher risk
of dying than their low VEGF counterparts.
The role of angiogenesis in ovarian carcinoma development
remains unclear; there are contradictory studies with regard to
the influence of microvessel density in ovarian cancer
prognosis (5, 25, 26). Immunohistochemical assessment of
VEGF within a tumor offers further information about the
potential for angiogenic activity and its effects on tumor
behavior and subsequent prognosis for the patient. The
literature has been equally controversial with regard to VEGF
expression within ovarian tumors, some authors showing no
independent relationship with prognosis (27, 28), whereas
other studies have shown a significant independent prognos-
tic influence. Patients with early-stage disease (International
Federation of Gynecologists and Obstetricians stages I and II)
showed poorer prognosis with increased VEGF expression
within the tumor (29). Shen et al. (15) showed elevated
expression of VEGF (using mRNA) to be predictive of a poor
prognosis; interestingly, there was no correlation with micro-
vessel density, which contradicts previous work (27). Ras-
pollini et al. (21) illustrated that VEGF and microvessel
density were both independent predictors of survival in
advanced disease (International Federation of Gynecologists
and Obstetricians stage III) and also correlated with the
likelihood of response to chemotherapy; similar findings
were also seen when including early-stage disease (20).
Because we have such a large number of cases, we can
clearly show that the prognostic effects of VEGF are seen at
all disease stages and that these effects are independent of
confounding variables such as stage, grade, and residual
disease.
Our study illustrated no clear associations between VEGF
and any of the clinicopathologic variables, including stage and
grade of tumor; this agrees with some studies, most of
which had a typical distribution of disease according to stage
(5, 20, 27, 30, 31). Some studies have suggested that stage and
grade are associated with VEGF, although these studies tended
to have an unusually high proportion of early-stage disease
(up to 58%; refs. 15, 32, 33).
Zhang et al. (34), in a study of infiltrating T cells in ovarian
cancer, showed that the absence of intratumoral T cells was
associated with higher levels of VEGF. This group of patients
had early recurrence rates and short survival. It is therefore
thought that VEGF further affects the behavior of ovarian
tumors by reducing the number of T cells in the tumor milieu,
suppressing the defenses of the immune system against ovarian
cancer.
There is early evidence that VEGF-mediated angiogenesis may
be used as a novel pathway in the treatment of ovarian cancer
as has been witnessed in colon (35), breast (36), and lung (37)
carcinomas. Monk et al. (38) have shown some clinical benefit
from using a monoclonal antibody against VEGF (bevacizu-
mab) in recurrent ovarian cancer; this has also been used
successfully in the palliative treatment of ascites in refractory
disease (39). Another strategy devised to suppress angiogenesis
in ovarian cancer is the interception of VEGF with receptor
decoys, such as VEGF-Trap, which has shown encouraging
results in early-phase trials (16, 40).
Our subgroup of high VEGF-expressing tumors accounts for
<10% of patients, and hence, we are identifying a small group
who seem to have a much worse prognosis. The small
proportion of tumors expressing high levels of VEGF may
explain why smaller studies failed to find significant prognostic
associations (27, 28). The clinical value of identifying these
patients and adapting treatment will therefore have a limited
effect on overall population survival; however, we may have
identified a specific group of patients who are highly sensitive
to antiangiogenic drugs. VEGF status is independent of stage in
chemotherapy-naive patients, suggesting that there may be a
role for bevacizumab as first-line treatment in addition to
standard chemotherapy in selected patients. This would
represent a significant new role for such agents, as most
studies have looked at use in recurrent platinum-resistant
disease (17, 18).
We conclude that expression of VEGF is an independent
prognostic indicator in a large series of patients with all stages
of ovarian cancer. High VEGF expression only occurs in a small
proportion of ovarian cancers but may denote a specific group
in which antiangiogenic therapy is more effective.
Imaging, Diagnosis, Prognosis
www.aacrjournals.org Clin Cancer Res 2008;14(10) May15, 2008 3034
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