To study pre-treatment serum VEGF of patients with invasive cervical cancer and its possible role as prognostic indicator.
Methods
VEGF was measured using ELISA in the largest patient group (n = 167) to date.
Reults
Serum VEGF was significantly higher in advanced tumor stage (P = 0.01), large tumor size (tumors larger than 2 cm) (P = 0.03), and the presence of vascular space invasion (P = 0.05). Serum VEGF was associated with disease free and overall survival [DFS: Hazard Ratio (HR) = 2.61; 95% CI 1.32–5.17; P = 0.006; for OS: HR = 2.09; 95% CI 1.54–2.84; P < 0.001, respectively]. In multivariate Cox regression serum VEGF retained its prognostic value for DFS (HR = 2.10, P = 0.03) and OS (HR = 1.92, P = 0.04).
Conclusions
Serum VEGF levels correlate with more advanced and more aggressive disease in cervical cancer and may be a useful prognostic factor in patients with cervical cancer.
Introduction
At present, results of primary surgery on one hand and radiotherapy on the other as the primary treatment modality in low-stage cervical carcinoma are comparable. Many patients with cervical cancer are relatively young. In those cases most gynecologic oncologists prefer surgery as this preserves ovarian function and affects sexual function to a lesser extent than primary radiotherapy. Furthermore, surgery provides information on the presence of positive lymph nodes, which in that case leads to a worse prognosis. The presence of positive lymph nodes will create the necessity of adjuvant radiotherapy. Radiotherapy given in an adjuvant setting leads to a high incidence of severe complications. A prognostic factor that can accurately predict aggressive disease may identify patients with clinical stage IB or IIA in whom radiotherapy should be considered as primary therapy.
Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is essential for both tumor growth and tumor spread (Folkman 1990, 1995). It seems likely that a tumor capable of inducing an extensive angiogenic response will show aggressive behavior and worse prognosis. This implies that quantifying the extent of angiogenesis could serve as an indicator of tumor behavior and prognosis. Among the various angiogenic factors vascular endothelial growth factor (VEGF) has a pivotal role in tumor angiogenesis and is known to participate in neovascularisation by promoting the differentiation of endothelial cells and increasing the permeability of capillaries (Ferrara 1995; Ferrara and Vis-Smyth 1997). Furthermore, tumor associated stroma has also been shown to produce VEGF (Fukumura et al. 1998).
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The relationship between tumor or serum VEGF and tumor behavior in cervical cancer has been the focus of a number of studies, with conflicting data as result. Cheng et al. showed that intra-tumoral protein levels of VEGF correlated well with local tumor progression and tumor metastasis (Cheng et al. 2000). Furthermore, they found that patients with high protein levels of VEGF had poorer disease-free and overall survival rates. Tissue expression of VEGF was found to be significantly increased in patients with cervical intra-epithelial neoplasia compared to patients with healthy cervices and to be highest in patients with squamous cervical carcinoma (Dobbs et al. 1997). Some studies showed that tumor expression of VEGF was a highly significant and independent prognostic factor in cervical cancer patients treated with primary radiotherapy (Loncaster et al. 2000). Others, however, did not find tissue VEGF expression to be of any prognostic value (Lee et al. 2002; Tjalma et al. 2000). Serum VEGF levels were significantly higher in both patients with cervical intraepithelial neoplasia and cervical cancer compared to healthy controls (Lebrecht et al. 2002; Moon et al. 2000; Bachtiary et al. 2002; Mitsuhashi et al. 2005; Yang et al. 2006; Gadducci et al. 2007). Interestingly, serum VEGF levels often respond to successful treatment (Moon et al. 2000; Mitsuhashi et al. 2005; Yang et al. 2006) and can have a prognostic value (Bachtiary et al. 2002), although this was not confirmed in a larger study (Lebrecht et al. 2002). These results indicate the tumor as source of elevated circulating VEGF levels. Even so, it offers the possibility of an easily obtainable biomarker with possible diagnostic and prognostic potential.
Currently, it is still difficult to predict tumor behavior in patients with cervical cancer and efforts are being made to find prognostic factors that could contribute to customizing patient care. The purposes of the present study were to determine if serum concentration of VEGF is a possible tumor marker for invasive cervical carcinoma, and to determine its role as a prognostic factor in patients with cervical carcinoma, by correlating serum VEGF levels with established prognostic factors and disease outcome in the largest group of patients with cervical carcinoma so far.
Materials and methods
Patients
The Institutional Review Board of the Radboud University Nijmegen Medical Centre approved the study. Patients diagnosed with cervical cancer, scheduled to have an examination under anesthesia in combination with radiographic and/or endoscopic techniques to determine tumor-stage had their blood samples taken whilst being admitted for this procedure. Blood samples were obtained by vena puncture and centrifuged at 3,000×g for 10 min; serum was aliquoted and stored at −20°C until further analysis. From August 1983 until May 2000, serum samples of 167 patients were collected for testing. The case-notes of these patients were carefully reviewed. We also determined the serum VEGF levels in a group of 20 healthy female controls, comprising blood donating volunteers. Women with FIGO stage IB1 and IIA underwent a radical hysterectomy. Adjuvant radiotherapy was given in case of positive lymph nodes or positive resection areas. In women with FIGO stage IB2, chemoradiotherapy was the treatment of choice. Women with advanced stage disease (IIB, III and IVA) were treated with chemoradiotherapy or radiotherapy with hyperthermia. In the only woman with FIGO stage IVB, treatment was only palliative. The median age of patients was 42 years (range 20–90 years). The median duration of follow-up was 2.8 years, ranging from 0.4 to 15.9 years. Most patients (79%) were diagnosed with stage IB and IIA disease and squamous cell carcinoma was the predominant histological type. Seventy percent of the patients were pre-menopausal. Among the patients 46% were smokers. The control group of 20 female healthy blood-donating volunteers had a median age of 37 years (range 19–64; P = 0.67). Eight of the volunteers were smokers (P = 0.71).
VEGF measurement
VEGF levels were determined in sera with a quantitative enzyme-linked immunosorbent assay (ELISA). The details of the assay, including those regarding specificity and performance, have been described previously (Span et al. 2000). This assay is based on the combination of 4 polyclonal antibodies (Ab) raised in four different animal species including duck, chicken, rabbit and goat, which are employed in a sandwich assay format. The assay measures VEGF165 and VEGF121, the main isoforms of VEGF. There is no cross-reactivity with VEGF B, VEGF C, VEGF D, platelet derived growth factor AB (PDGF AB), insulin growth factor type 1 (IGF-1), human growth hormone (hGH), placental growth factor (PlGF), nerve growth factor (NGF), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor (TGF). The analytical sensitivity for VEGF is 5 pg/ml. For VEGF, the within-run CV and between-run CV are found to be 8.7 and 13.4%, respectively.
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Statistical analysis
Serum VEGF concentrations are presented as median values and expressed in μg/l (interquartile range). Differences in serum VEGF levels between patients and controls, and between clinical subgroups were tested using the Mann–Whitney U test and the Kruskal–Wallis test, where appropriate. Furthermore, the correlation between serum VEGF levels and disease free (DFS) and overall survival (OS) were tested using Cox-univariate regression analyses. Multivariate Cox regression was performed, with only tumor stage and VEGF entered as variables in the analysis due to limited number of events. A two-sided P value below 0.05 was considered statistically significant. The software packages SAS (version 6.12, SAS Institute Inc., Cary, NC) and SPSS (version 12.0.1, SPSS Inc. Chicago, IL) were used for the statistical analyses.
Results
Descriptive analyses
Table 1 summarizes descriptive data of our patient group (n = 167). The median VEGF levels differed significantly for patients with cervical carcinoma compared to the group of healthy controls: 0.54 (interquartile range 0.28–1.26 μg/l) versus 0.46 μg/l (interquartile range 0.29–0.60 μg/l) (P = 0.01; Table 2).
Table 1
Clinical characteristics of patients with cervical cancer (n = 167)
Characteristics
Number (%) or (range)
Age (years) (range)
42 (20–90)
Follow up (years) (range)
2.8 (0.4–15.9)
Stage
IB
100 (60%)
IIA
32 (19%)
IIB
19 (11%)
III
11 (7%)
IV
5 (3%)
Histology
Squamous
114 (68%)
Adeno
37 (22%)
Adenosquamous
14 (8%)
Unknown
2 (1%)
Grade
I
3 (2%)
II
81 (48%)
III
61 (37%)
Unknown
22 (13%)
Lymph nodes
Positive
23 (14%)
Negative
102 (61%)
Unknown
42 (25%)
Recurrence
Yes
40 (24%)
No
116 (69%)
Unknown
11 (7%)
Table 2
Serum VEGF levels in controls and different clinicopathological categories of patients
N
Median VEGF(μg/l)
Interquartile range
P
Group
Controls
20
0.46
0.31
0.01
Patients
167
0.54
0.98
Stage
IB
100
0.44
0.61
0.01
IIA
32
0.63
0.72
IIB
19
0.52
0.72
III/IV
16
1.04
1.28
Histology
Squamous
114
0.61
0.83
0.62
Adeno
37
0.52
0.45
Adenosquamous
14
0.55
0.53
Grade
I/II
84
0.50
0.68
0.22
III
61
0.61
0.89
Lymph nodes
Negative
102
0.51
0.61
0.67
Positive
23
0.45
1.07
VSI
No
21
0.39
0.46
0.05
Yes
81
0.50
0.73
Depth of invasion
<0.7 mm
29
0.50
0.72
0.77
≥0.7 mm
56
0.48
0.57
Tumor size
≤2 cm
31
0.38
0.73
0.03
>2 cm
76
0.64
1.00
VSI vascular space invasion; n number, P is for non parametric Mann–Whitney U or Kruskal–Wallis test, where appropriate
Associations of serum VEGF with clinicopathological characteristics
We correlated the serum VEGF levels to established prognostic factors in cervical carcinoma, i.e. tumor stage, histological subtype, grade, lymph node involvement, vascular space invasion, depth of invasion and tumor size (Table 2) (Kristensen et al. 1999; Nguyen and Averette 1999). Serum VEGF levels were significantly higher in patients with higher tumor stages (1.04 μg/l for III and IV) as compared to early stage tumors (0.44 μg/l for IB) (P = 0.01). When vascular space invasion was involved higher serum VEGF levels were found as compared to cases with no vascular space invasion (0.50 vs. 0.39 μg/l, respectively; P = 0.05). Patients with tumors larger than 2 cm also showed markedly raised serum VEGF levels as compared to patients with small tumor size (0.64 vs. 0.38 μg/l, respectively; P = 0.03). VEGF levels did not correlate with histology, grade, lymph node involvement and depth of invasion. As other factors such as age, menopausal status, smoking, exogenous hormone use and parity might also influence serum VEGF levels, we included these in our analyses. None of these factors, however, showed statistically significant differences in serum VEGF levels.
Survival analyses
Forty women (24%) showed recurrence of disease. Cox regression analysis showed that tumor stage, positive lymph nodes, tumor size, and serum VEGF levels were all correlated with DFS and OS in univariate analyses (Table 3). For VEGF, this prognostic value was significant when entered as a continuous factor (for DFS: Hazard Ratio (HR) = 2.61; 95% CI 1.32–5.17; P = 0.006; for OS: HR = 2.09; 95% CI 1.54–2.84; P < 0.001), precluding the definition of a subjective cut-off value, and when the patients were dichotomized using the median value of serum VEGF (for DFS: HR = 2.00; 95% CI 1.15–3.50; P = 0.01; for OS: HR = 2.32; 95% CI 1.25–4.30; P = 0.006) (shown in Fig. 1). Twenty-four women died within 1 year after diagnosis. These patients had significantly higher serum VEGF levels at the time of surgery than women who survived the first year [1.10 μg/l (interquartile range 0.38–5.36 μg/l) versus 0.61 μg/l (interquartile range 0.21–0.1.86 μg/l)] (P = 0.02).
Table 3
Univariate Cox regression analysis of disease free and overall survival
DFS
OS
HR
95% CI
P
HR
95% CI
P
Stage
IIA vs. IB
1.21
0.56–2.58
<0.001
0.94
0.38–2.33
<0.001
IIB vs. IB
2.26
1.01–5.02
3.23
1.36–7.67
III/IV vs. IB
6.50
3.20–13.2
8.05
3.80–17.1
Histology
Adeno vs. squamous
1.71
0.94–3.10
0.29
1.59
0.72–3.55
0.39
Adenosquamous vs. squamous
1.56
0.65–3.74
1.83
0.62–5.40
Grade
III vs. I/II
1.58
0.89–2.79
0.11
1.84
0.95–3.55
0.07
Lymph nodes
Positive vs. negative
1.84
1.12–3.02
0.02
8.08
1.62–40.3
0.005
VSI
Yes vs. no
1.02
0.95–1.09
0.59
2.80
0.66–11.9
0.11
Depth of invasion
≥0.7 vs. <0.7 mm
0.86
0.32–2.31
0.77
0.50
0.17–1.50
0.23
Tumor size
>2 vs. ≤2 cm
4.83
1.47–15.8
0.009
5.88
1.38–24.9
0.002
Serum VEGF
Continuous (log transformed)
2.61
1.32–5.17
0.006
2.09
1.54–2.84
<0.001
Serum VEGF
>Median vs. ≤median
2.00
1.15–3.50
0.010
2.32
1.25–4.30
0.006
HR Hazard ratio; vs versus; VSI vascular space invasion; 95% CI 95% confidence interval
Fig. 1
Kaplan–Meier analysis of serum VEGF concentration. Dashed line indicates < than the median, continuous line indicates ≥ than the median. The median serum VEGF concentrations in this study population is 0.54 μg/l
×
Multivariate analysis
To assess whether the prognostic value of serum VEGF was attributable to its strong association with tumor stage, multivariate Cox regression analysis was performed with tumor stage and VEGF as variables in the analysis. After correction for tumor stage, serum VEGF retained its prognostic value for both DFS (HR = 2.10, P = 0.031) and OS (HR = 1.92, P = 0.047; Table 4).
Table 4
Multivariate Cox regression analysis of disease free and overall survival
DFS
OS
HR
95% CI
P
HR
95% CI
P
Stage
IIA vs. IB
1.21
0.52–2.80
0.099
0.80
0.32–2.02
<0.001
IIB vs. IB
2.83
1.23–6.51
2.99
1.26–7.12
III/IV vs. IB
1.80
0.52–6.26
6.60
3.05–14.3
Serum VEGF
>Median vs. ≤median
2.10
1.07–4.10
0.03
1.92
1.01–3.64
0.04
HR Hazard ratio, vs versus, 95% CI 95% confidence interval
To further characterize this prognostic value of serum VEGF, Kaplan–Meier curves were made after stratification for tumor stage (Fig. 2). Interestingly, serum VEGF discriminated between good and poor prognosis mainly in the clinically relevant tumor stage IIA/B patients. However, due to the low number of samples per group these analyses were not significant.
Fig. 2
Kaplan–Meier curves for DFS (left) and OS (right) of patients dichotomized by the median level of serum VEGF after stratification for tumor stage. Dashed line indicates < than the median, continuous line indicates ≥ than the median. The median serum VEGF concentrations in this study population is 0.54 μg/l. VEGF discriminates between good and poor prognosis mainly in tumor stage IIA/B patients
×
Discussion
The results of recent studies have suggested that neovascularisation is one of the most important processes in tumor growth and metastasis. VEGF induces the formation of new capillaries from the pre-existing vasculature. Our results show that serum VEGF levels correlate well with more advanced disease, as levels were significantly raised in patients with higher stages of the disease, in patients with tumors with vascular space invasion and larger tumors. Furthermore, serum VEGF was an independent factor influencing disease-free and overall survival in our study. In a multivariate Cox regression analysis with tumor stage and VEGF as variables in the analysis, serum VEGF retained its prognostic value for both DFS and OS. To further characterize this prognostic value of serum VEGF, Kaplan–Meier curves were made after stratification for tumor stage (Fig. 2). Although the number of events is too small for a meaningful statistical analysis of the Kaplan–Meier curves for serum VEGF in every single tumor stage, these analyses show that the prognostic value of VEGF is particularly discernable in the clinically relevant cohort of patients with stage IIA/B disease.
So far, the relationship between serum VEGF and tumor behavior in cervical cancer has been the focus of a number of studies, but the results remained equivocal (Lebrecht et al. 2002; Moon et al. 2000; Bachtiary et al. 2002; Mitsuhashi et al. 2005; Yang et al. 2006; Gadducci et al. 2007) (Table 5), possibly to the overall low number of patients (n = 23–78). To our knowledge, pre-treatment serum VEGF levels of patients with cervical carcinoma have as yet not been reported in such a large patients group (n = 167). Almost all studies have found that serum VEGF levels are higher in patients when compared to controls (Lebrecht et al. 2002; Moon et al. 2000; Mitsuhashi et al. 2005; this study; Table 5). Also, a correlation with stage is often found (Lebrecht et al. 2002; Mitsuhashi et al. 2005; this study). Furthermore, the correlation with tumor size we describe here has been reported earlier (Mitsuhashi et al. 2005). However, the relationship between VEGF and recurrence we report here was not found by Lebrecht et al. (2002), whereas the study of Bachtiary et al. (2002), which also found a relation between serum VEGF and prognosis, only entered 23 patients. This number should have been considered too low for meaningful statistical analyses, especially multivariate regression analysis, due to low power and chance of spurious results. The relation between serum VEGF and prognosis we describe here suggests that angiogenesis may be a major mechanism in the pathogenesis of recurrence in cervical cancer. Previous literature showed that increased angiogenic activity correlated well with prognosis and overall survival in cervical cancer (Cheng et al. 2000; Kaku et al. 1998; Obermair et al. 1998; Wiggins et al. 1995). Another noticeable result is that in our study serum VEGF levels were not significantly different between patients with squamous cell carcinoma and those with adeno- or adenosquamous carcinoma, whereas others found adenocarcinoma to be the more angiogenic (Fujimoto et al. 1999) and aggressive subtype (Samlal et al. 1997).
RT radiotherapy; T/N tumor vs. normal; DFS disease free survival; OS overall survival; PFS progression free survival; ND not detectable/not disclosed
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As the range of serum VEGF levels in both patients and controls is very wide, varying from 0.03 to 5.36 μg/l, the practical use of VEGF as a tumor marker in individual patients with cervical carcinoma is limited. An explanation for this wide range in VEGF levels might be that angiogenesis in oncology is a dynamic process in which there is a continuous shift of balance between tumor growth, hypoxia, and angiogenesis, involving several inhibitors and promoters of angiogenesis, in which VEGF plays an important role, but is not the only determining factor. From this point of view the serum VEGF level is probably only a snapshot impression of this process and very susceptible to changes. Moreover, serum VEGF levels may also vary intra-individually due to factors such as diet and day- or night-time. Future research should focus on further analyzing the influence of angiogenesis on tumor behavior in cervical cancer. Furthermore, more research is needed to find more about the angiogenic switch that endothelial cells make in cancer tissue, as healthy endothelium is mainly dormant and not susceptible to high serum levels of VEGF (Ferrara 1995).
We conclude that serum VEGF levels are of prognostic value in cervical carcinoma. These findings may lead to future application of therapeutic trials with anti-angiogenic factors. Moreover, its clinical use as a prognostic indicator needs further evaluation. The FIGO classification of cervical cancer, based on clinical staging, is not an accurate way of defining a patient’s extend of disease, leading to misclassification in 15–25% of cases, mostly due to positive lymph nodes, or lymph or blood vessel invasion, possibly resulting in suboptimal care (Lagasse et al. 1980). Patients with positive lymph nodes or lymph or blood vessel invasion need adjuvant radiotherapy leading to a high incidence of complications. Determination of pre-treatment serum VEGF levels may help to improve the selection of the most appropriate therapy regime for an individual patient with low-stage cervical cancer. Perhaps even better might be the combination of VEGF with other markers for lymphogenic of vascular spread of disease in low-stage cervical cancer, such as squamous cell carcinoma antigen (SCC) and CA-125 serum levels (Massuger et al. 1997).
Acknowledgments
The authors highly appreciate the technical support of Mrs. A.Geurts-Moespot in analyzing VEGF concentrations.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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Open AccessThis is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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