Expression of the Vascular Endothelial Growth Factor (VEGF) Gene in Epithelial Ovarian Cancer: An Approach to Anti-VEGF Therapy

8/11/2019 Expression of the Vascular Endothelial Growth Factor (VEGF) Gene in Epithelial Ovarian Cancer: An Approach to Ant

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Abstract. Aim: A monoclonal antibody that targeted

vascular endothelial growth factor (VEGF) resulted in a

dramatic suppression of tumor growth in vivo, which led to

the development of bevacizumab, a humanized variant ofanti-VEGF antibody, as an anticancer agent. The aims of

this study were to clarify the significance of VEGF gene

expression in relation to clinicopathological parameters and

to identify potential candidates for anti-VEGF therapy with

bevacizumab. Patients and Methods: VEGF gene expression

was analyzed by real-time quantitative reverse transcription-

polymerase chain reaction in 178 surgical epithelial ovarian

cancer specimens. This gene expression was correlated with

clinicopathological parameters and patient survival. Results:

The median VEGF gene expression level and range relative

to GAPDH were 0.147 and 0.0162.44, respectively. Patients

were dichotomized into two groups with low and highexpression levels by using the median value as the cutoff.

VEGF gene expression did not affect prognosis of patients

overall (p=0.541). Although statistical significance was not

noted, we found the prognosis of patients with high VEGF

gene expression tended to be worse than that of those with

low VEGF gene expression by univariate Cox regression

analysis (p=0.085) in patients with stage IIIIV cancer.

Macroscopic residual disease (positive; p=0.012) was

significantly associated with poor prognosis in univariate

Cox regression analysis in patients with stage IIIIV cancer.

Moreover, presence of macroscopic residual disease was

positively associated with VEGF gene expression (p=0.030)

in patients with stage III-IV cancer. Conclusion: Patients

with epithelial ovarian cancer with tumors with positive

macroscopic residual disease and high VEGF gene

expression could be potential candidates for anti-VEGFtherapy with bevacizumab.

Vascular endothelial growth factor (VEGF) has been identified

as the most potent and specific mitogen for endothelial cells

and has essential role in activating the angiogenic switch (1,

2). VEGF is a key regulator of physiological angiogenesis

during embryogenesis, skeletal growth, and reproductive

function (3). VEGF also seems to be important in pathological

angiogenesis, such as that associated with tumor growth (4).

Overexpression of VEGF has been associated with tumor

progression and poor prognosis in tumors of the colorectum

(5), stomach (6), pancreas (7), breast (8), prostate (9), and lung(10). Shen et al. (11) showed that VEGF expression assessed

by immunohistochemistry was an independent prognostic

indicator of overall survival time in patients with epithelial

ovarian cancer. Raspollini et al. (12) presented similar results to

those of Shen et al. (11) in advanced disease (FIGO stage III).

Recently, by using a tissue microarray of 339 primary ovarian

tumors, Duncan et al. (13) immunohistochemically assessed

VEGF expression and noted that VEGF was an independent

predictor of prognosis on multivariate analysis, although there

was no correlation between VEGF and any clinicopathological

variables. By performing real-time quantitative reverse

transcription-polymerase chain reaction (RT-PCR) in cases of

epithelial ovarian cancer, we demonstrated that tumors mightacquire an aggressive tumor phenotype in the presence of

VEGF expression (14). These results prompted us to consider

the potential for anti-VEGF therapy in malignant tumors,

including epithelial ovarian cancer.

Bevacizumab is a humanized monoclonal antibody

directed against VEGF. It binds to and neutralizes all human

VEGF isoforms and bioactive proteolytic fragments (15).

The usefulness of bevacizumab has been witnessed in

colorectal (2, 16), breast (17), and lung (1, 18) carcinomas.

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Correspondence to: Kohkichi Hata, MD, Ph.D., Department of

Tumor Biology, Kagawa Prefectural University of Health Sciences,

Takamastu 761-0123, Japan. Tel +81 878701578, Fax: +81

878701204, e-mail: [email protected]

Key Words: Vascular endothelial growth factor, VEGF, gene

expression, anti-VEGF therapy, bevacizumab, epithelial ovarian

cancer.

ANTICANCER RESEARCH 31: 731-738 (2011)

Expression of the Vascular Endothelial GrowthFactor (VEGF) Gene in Epithelial Ovarian Cancer:

An Approach to Anti-VEGF Therapy

KOHKICHI HATA1, YOH WATANABE2, HIDEKATSU NAKAI2, TOSHIYUKI HATA3 and HIROSHI HOSHIAI2

1Department of Tumor Biology, Kagawa Prefectural University of Health Sciences, Takamastu 761-0123, Japan;2Department of Obstetrics and Gynecology, Kinki University School of Medicine, Osaka-Sayama 589-8511, Japan;

3Department of Perinatology and Gynecology, Kagawa University School of Medicine, Kagawa 761-0793, Japan

0250-7005/2011 $2.00+.40


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Burger et al. (19) and Cannistra et al. (20). reported that

bevacizumab was effective in recurrent epithelial ovarian

cancer as a single agent. Moreover, bevacizumab has been

combined with a variety of cytotoxic agents, and its

feasibility has been demonstrated (21-24). However, these

studies were performed regardless of clinicopathological

factors and without assessing the status of VEGF expression.

In this study, we sought to determine mRNA expression

of VEGF using real-time quantitative RT-PCR in cases of

epithelial ovarian cancer. VEGF gene expression was

correlated with established clinicopathological prognostic

parameters and their impact on patient survival was

evaluated. The objectives of the current study were to clarify

the significance of VEGF gene expression in relation to

clinicopathological parameters and to identify potential

candidates for anti-VEGF therapy with bevacizumab.

Patients and Methods

Patients. Patients with epithelial ovarian cancer treated between

March 2000 and August 2009 at Kinki University Hospital, Osaka-

Sayama, Japan, were included in this study. Eligible patients had a

histological diagnosis of primary epithelial ovarian cancer and were

suitable for adequate surgical staging. Patients were excluded from

this study when surgically resected specimens were not available or

if they had undergone any kind of preoperative therapy, had cancer

other than ovarian cancer, or had severe complications. All research

was conducted after the patients provided informed consent to have

their tissues banked for future unspecified studies. The present study

conformed to the ethical standards of the World Medical

Associations Declaration of Helsinki.

The median age of the 178 eligible patients was 55 years (range,

19-84 years), and 57 of them were premenopausal. Patients werestaged according to the 1987 criteria recommended by FIGO (25).

There were 72 stage I patients, 14 stage II patients, 81 stage III

patients, and 11 stage IV patients. The staging system defined by

FIGO, as described elsewhere (19, 20), assumes that an adequate

staging operation has been performed. Tumors were classified

histologically according to World Health Organization (WHO) criteria

(26) as serous (n=86), mucinous (n=49), endometrioid (n=23), clear

cell (n=17), transitional cell (n=1), and undifferentiated (n=2). The

tumors were classified histologically as well-differentiated (n=91),

moderately differentiated (n=41), or poorly differentiated (n=28) (27).

The number of poorly differentiated tumors is smaller than that of

well-differentiated tumors. This seems to be unusual compared to

European series; however, the population in this study was a typical

Japanese population with ovarian cancer (14, 28, 29).

Surveillance for recurrent disease usually consisted of physical

examination, Papanicolaou smear and serology with tumor marker

examination (e.g., CA 125, CA 19-9, carcinoembryonic antigen,

sialyl Tn) every month for the first year, every 2 months for the

second and third year, and every 3 months for the fourth and fifth

years. Patients were examined semiannually after 5 years. A chest

radiograph and computed tomography (CT) scan or ultrasonography

were obtained every 6 months for 5 years after surgery and every

year thereafter; magnetic resonance imaging (MRI) was performed

as needed. Recurrent disease was confirmed pathologically,

radiographically, or serologically. Follow-up information was

obtained from medical records, by letter or telephone contact with

patients, and from the referring physicians. Survival data were

available for all patients (median follow-up, 42 months; range, 2-

114 months). Of these, 172 patients received platinum and/or

paclitaxel-based chemotherapy. Five patients with stage Ia tumors

(1 serous cystadenocarcinoma, 3 mucinous cystadenocarcinomas,

and 1 endometrioid adenocarcinoma) and 1 with a stage IV serous

cystadenocarcinoma required no further treatment after surgery.

Eighty patients in the current study had participated in the

previous study (29).

Tissue specimens and RNA preparation. Fresh surgical specimens

were obtained from all patients. A dissecting microscope was used

to prevent cross-contamination of non-cancerous tissue with

cancerous tissue. Tissue samples were stored at 80C for

subsequent quantification of mRNA expression. Total RNA was

isolated from frozen tissues using a commercially available

extraction method (Isogen; Nippon Gene Inc., Tokyo, Japan).

Real-time quantitative RT-PCR procedures. Complementary DNA

(cDNA) was prepared by random priming from 1,000 ng of total RNA

using a First-Strand cDNA Synthesis kit (Pharmacia-LKB, Uppsala,

Sweden). Real-time quantitative PCR was performed using the TaqMan

system (Applied Biosystems). Gene expression levels of VEGFand

internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

were measured using TaqMan probes labeled with 6-carboxyfluorescein

(FAM) or VIC, respectively. Primers of the VEGFgene were designed

from sequences within exon 2 to exon 3, which are common to all

isoforms of VEGFmRNA (10). The sequences of the primer and the

TaqMan probe (forward primer, reverse primer, TaqMan probe) were 5-

GCACCCATGGCAGAAGG-3, 5-CTCGATTGGATGGCAGTAGCT-

3, and 5-TTCATGGATG TCTATCAGC-3, respectively. Pre-

Developed TaqMan Assay Reagent, GAPDHprimer/probe set were also

purchased from Applied Biosystems. Real-time PCR amplification and

product detection were performed using an ABI PRISM 7300 Sequence

Detection System (Applied Biosystems) as recommended by themanufacturer. The quantity of cDNA for the VEGFgene was normalized

to the quantity of GAPDHcDNA in each sample. Relative expression

was determined using the Ct (threshold cycle) method according to

the manufacturers protocol (User Bulletin #2). Each assay included a

standard curve sample in duplicate, a no-template control, and a cDNA

sample from the tumor specimen in triplicate. All samples with a

coefficient of variance higher than 10% were retested.

Statistical analysis. Mann-Whitney U-test and Kruskal-Wallis one-way

analysis of variance by ranks were used as appropriate for the evaluation

of differences between end-points. The Cox proportional hazards model

was used in survival analysis. Maximum likelihood parameter estimates

and likelihood ratio statistics (LRS) in the Cox proportional hazards

models were obtained with the use of the statistical package EPICURE(30). Kaplan-Meier curves were compared by the univariate Cox

regression analysis. Allp-values presented were two-sided. P-values of

less than 0.05 were considered significant.

Results

VEGF gene expression and clinicopathological features. The

median relative VEGFgene expression level and range were

0.147 and 0.016-2.44, respectively Patients were divided into

groups of low or high VEGFgene expression by using the


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median value as the cutoff. Values of VEGFgene expression

in epithelial ovarian cancer were classified according to

patient age at diagnosis, stage of disease, presence or

absence of macroscopic residual disease after initial surgery,

and histological subtype and grade in all patients (Table I)

and in patients with stage III-IV cancer (Table II). VEGF

gene expression significantly differed among histological

grade (p=0.013) in all patients (Table I). Presence ofmacroscopic residual disease was positively associated with

VEGFgene expression (p=0.030) in patients with stage III-

IV cancer (Table II, Figure 1).

VEGF gene expression and prognosis. Tables III and IV

present the results of univariate and multivariate Cox

regression analysis in all patients, respectively. In univariate

Cox regression analysis, FIGO stage (stage IIIIV;

p


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than that of those with low VEGFgene expression level by

univariate Cox regression analysis in patients with stage III-

IV cancer (p=0.085) (Table V and Figure 3). Only

macroscopic residual disease (positive; p=0.012) was

significantly associated with poor prognosis in univariate

Cox regression analysis in patients with stage IIIIV cancer

(Table V).

Discussion

Studies on the importance of VEGF in epithelial ovarian

cancer and its relationship to prognosis have shown

inconsistent results (11-14, 31, 32). We reported that the

expression of VEGF assessed by immunohistochemistry

(32) and real-time quantitative RT-PCR (14) did not

significantly affect prognosis. However, the significance of

VEGF expression in carcinogenesis was confirmed (32),

and it was reported that the tumors with up-regulation of

angiopoietin-2 gene expression compared to angiopoietin-

1 gene expression might acquire an aggressive tumor

phenotype in the presence of VEGF expression (14).

Kabbinavar et al. (2, 16) have shown that a monoclonal

antibody to VEGF (bevacizumab) added to first-line 5-

fluorouracil/leucovorin chemotherapy in patients withmetastatic colorectal cancer provides statistically

significant and clinically meaningful improvements in

response rate, progression-free survival, and overall

survival. Kuramochi et al. (33) measured the VEGFgene

expression levels in primary and liver metastasis tissue of

colorectal cancer by real-time quantitative RT-PCR. They

found that the VEGFgene expression levels of the patients

who had two or more liver metastatic tumors were

significantly higher than those of the patient who had

solitary liver metastatic tumor, in both the primary cancer

and liver metastases, and concluded that the risk of

multiple metastatic tumors might be predicted by

measuring VEGF gene expression in primary colorectalcancer (33). Higher VEGFexpression in primary cancer

tissue would indicate multiple metastases; in such cases,

bevacizumab therapy would be selected to avoid

unnecessary surgery (33). On the basis of these findings,

we surmised that the measurement of VEGF gene

expression levels in tumor tissue correlated with

established clinicopathological prognostic parameters and

could be used to determine the optimal use of bevacizumab

in patients with epithelial ovarian cancer.


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Table III.Results of univariate Cox regression analysis in all patients.

Variable HR 95% CI P-value

Age at the time of

diagnosis 1.021 0.998-1.045 0.070

FIGO stage

I-II (n=86) referentIII-IV (n=92) 13.496 4.818-37.809


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Bevacizumab can reduce tumor vessels in patients; thus,

it kills a fraction of cancer cells. Bevacizumab can also

normalize tumor vasculature and microenvironment (34, 35).

These effects lead to apoptosis of tumor endothelial cells and

decrease interstitial fluid pressure within the tumor, which

then allows for greater amounts of oxygen and

chemotherapeutic drugs to reach specific target sites (36).

Considering the mechanisms of action of bevacizumab, it is

speculated that anti-VEGF therapy by bevacizumab might be

useful when viable tumor tissue exists. In this study,

although statistical significance was not noted, the prognosis

of the patients with high VEGFgene expression tended to beworse than that of those with low VEGFgene expression

according to univariate Cox regression analysis by Kaplan-

Meier curves in patients with stage III-IV cancer (p=0.085).

Macroscopic residual disease (positive; p=0.012) was

significantly associated with a poor prognosis in univariate

Cox regression analysis in patients with stage III-IV cancer.

Moreover, the presence of macroscopic residual disease was

positively associated with VEGFgene expression (p=0.030)

in patients with stage III-IV cancer. Epithelial ovarian cancer

patients with tumors with positive macroscopic residual

disease and high VEGF expression could be potential

candidates for anti-VEGF therapy with bevacizumab after

initial surgery. However, precise cutoff values for high VEGFgene expression levels have not yet been determined. It

might be reasonable to consider that when a tumor is

completely removed during the initial surgery, bevacizumab

therapy is not recommended. Moreover, further evaluation of

VEGF expression levels in prospective clinical trials

evaluating the benefit of bevaczumab in the upfront treatment

of advanced ovarian cancer may be helpful in further

defining the optimal candidates for the addition of anti-

VEGF-based therapies.

Multiple isoforms of VEGF result from differential

splicing of pre-mRNA from 8 exons, resulting in at least 6

mRNA and peptide species that can be identified by exon

composition and amino acid length of the final proteins

(37). Uthoff et al. (38) reported that the pattern of specific

VEGF isoform expression might influence the response to

therapy without substantial changes in total VEGFmRNA

expression in human colorectal cancer. Bevacizumab

treatment of human colorectal cancer may also depend on

the balance of VEGF isoform expressions (39). Taking these

findings into account, the assessment of differential

expression of VEGF isoforms might lead to more preciseselection of candidates for bevacizumab therapy in epithelial

ovarian cancer.

Conflict of Interest Statement

None declared.

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Figure 2. Comparison of survival between groups with high and low

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Received November 26, 2010

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Accepted January 25, 2011

Hata et al: VEGFin Epithelial Ovarian Cancer

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Documents

Expression of the Vascular Endothelial Growth Factor (VEGF) Gene in Epithelial Ovarian Cancer: An Approach to Anti-VEGF Therapy