Key words

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Serum Vascular Endothelial Growth Factor

and Interleukin−8 Levels in Epithelial Ovarian Tumors*

Stężenia czynnika wzrostu śródbłonka naczyniowego i interleukiny−8

w surowicy krwi pacjentek leczonych z powodu nabłonkowych guzów

jajnika

Department of Obstetrics and Gynecology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland

Adv Clin Exp Med 2009, 18, 2, 135–140 ISSN 1230−025X

© Copyright by Wroclaw Medical University

Abstract

Background.Epithelial ovarian cancer is the leading cause of cancer deaths from gynecological malignancies. Angiogenesis is considered essential for tumor growth and the development of metastases. VEGF and IL−8 are potent angiostimulatory molecules and their expression has been demonstrated in many solid tumors, including ovarian cancer.

Objectives. The aim of this study was to determine the levels of VEGF and IL−8 in the serum of patients with benign and malignant epithelial ovarian tumors.

Material and Methods.VEGF and IL−8 concentrations were measured by ELISA test (Quantikine R&D).

Results.The median VEGF and IL−8 levels were significantly higher in the sera of ovarian cancer patients than in those with benign tumors and in healthy controls.

Conclusions.Pre−treatment VEGF and IL−8 serum levels might be regarded as an additional tool in the differenti− ation of ovarian tumors (Adv Clin Exp Med 2009, 18, 2, 135–140).

Key words: VEGF, IL−8, angiogenesis, ovarian tumor.

Streszczenie

Wprowadzenie. Rak jajnika jest nowotworem o najwyższym wskaźniku umieralności spośród wszystkich nowo− tworów żeńskich narządów płciowych. Tworzenie nowych naczyń krwionośnych odgrywa istotną rolę we wzro− ście guzów nowotworowych i powstawaniu przerzutów. VEGF i IL−8 są silnymi czynnikami proangiogennymi, których ekspresję wykazano również w raku jajnika.

Cel pracy.Określenie stężeń VEGF i IL−8 w surowicy krwi pacjentek leczonych z powodu łagodnych i złośliwych nabłonkowych guzów jajnika.

Materiał i metody.Stężenia VEGF i IL−8 w surowicy krwi zostały oznaczone za pomocą zestawów ELISA (Qu− antikine R&D).

Wyniki. Średnie stężenia VEGF i IL−8 były istotnie większe w surowicy krwi pacjentek leczonych z powodu ra− ka jajnika w porównaniu do pacjentek z guzami łagodnymi jajnika oraz grupy kontrolnej.

Wnioski. Oznaczenie stężeń VEGF oraz IL−8 w surowicy krwi może być przydatne w różnicowaniu łagodnych i złośliwych guzów jajnika (Adv Clin Exp Med 2009, 18, 2, 135–140).

Słowa kluczowe:VEGF, IL−8, angiogeneza, guzy jajnika.

Ovarian cancer is the leading cause of death from gynecologic malignancies in western coun− tries. The primary reason for the high mortality rate is that most patients present with metastatic disease (FIGO stages III and IV) at the time of diagnosis. The possibility that detection of the dis− ease at an early stage might impact on survival has prompted the evaluation of different diagnostic methods.

Angiogenesis plays a very important role in both benign and malignant neoplastic processes [1]. Invasive cancer growth occurs as a result of the formation of blood vessels within a tumor that enable its growth, infiltration of tissues, and the genesis of metastases [2]. Indirect methods of detecting angiogenesis were created along with the development of knowledge about it and the recog− nition of various factors that either stimulate or inhibit vessel formation in neoplastic tissue. These methods are based on evaluating proangiogenic factor levels in body fluids or their expression in tumor tissue [2].

VEGF is a basic homodimeric glycoprotein with a mass of 45 kDa and belongs to the group of heparin−binding growth factors [3]. VEGF is a member of a family of six structurally related pro− teins (VEGF−A, VEGF−B, VEGF−C, VEGF−D, VEGF−E, and PlGF) that regulate the growth and differentiation of multiple components of the vas− cular system, especially blood and lymph vessels [4]. These factors bind to three different recep− tors: VEGFR−1, VEGFR−2, and VEGFR−3 [4, 5]. VEGFR−1 and VEGFR−2 play roles in angiogene− sis and vasculogenesis and VEGFR−3 participates in the formation of lymphatic vessels [6]. Many different factors control the expression of the gene encoding VEGF. Besides oxygen insufficiency, such factors as hypoglycemia, growth factors, proinflam− matory cytokines, some prostaglandins and hor− mones, and also mutated oncogenes and suppressor genes influence VEGF gene transcription [6].

IL−8 is a small polypeptide with a mass of 8 kDa that strongly binds to heparin [7]. There are two receptors, A and B, that have the same affinity for IL−8 [8]. IL−8 receptor expression was discovered on vascular endothelial cells [8]. The mechanisms of the proangiogenic activity of IL−8 have still not been completely clarified. The direct activity of IL−8 consists of induction of migration and pro− liferation of vascular endothelial cells [9]. The indirect action of IL−8 results in increased secre− tion of other proangiogenic factors, for example VEGF [10].

The aim of this study was to evaluate VEGF and IL−8 levels in blood serum and determine the applicability of these proangiogenic factors in diagnosing ovarian tumors.

Material and Methods

Sixty−six female patients hospitalized at the Department of Obstetrics and Gynecologcy of the Collegium Medicum in Bydgoszcz took part in this study. All the patients had provided their medical history and had undergone clinical and ultrasound examination of the pelvic organs before they were qualified for the study. There were 30 female patients with ovarian cancer (21 serous, 3 mucinous, 2 endometrioid, 3 clear cell, and 1 un−differentiated) in the group. The average age of this group was 55.80 ± 10.43 years and postmenopausal patients accounted for 60% of the group (n = 18). Twenty−five patients with benign ovarian tumors (11 serous cyst adenoma, 6 muci− nous, and 8 endometrial cyst) formed a compara− tive group. The average age of this group was 49.08 ± 14.31 years and postmenopausal patients accounted for 40% (n = 10). All the patients in this group had a tumor located in one of the ovaries. The control group was composed by 11 healthy women with an average age of 52.45 ± 11.28 years, postmenopausal women making up 55% (n = 6). Results of postoperational histologi− cal tests were the basis for verification of the tumor types. The research project was accepted by the Bioethics Commission of Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun (KB/522/2004). All patients gave their informed consent to participate in this study.

10−ml blood samples were taken from a cu− bital vein of the patients with ovarian tumors qual− ified for surgical treatment. The blood was taken in the first phase of the cycle in the case of the pre− menopausal patients. The blood serum was kept frozen at −70°C until measurements were per-formed. Proangiogenic factor levels were deter-mined by ELISA using Quantikine ELISA kits (R&D Systems Inc., Minneapolis, MN, USA). The kits were designed to measure isoform VEGF-A165 and IL-8 levels in cell culture,

super-natant, serum, and plasma. The method’s sensitiv-ity for VEGF-A165 was 9 pg/ml, with reference

values in the serum of 62-707 pg/ml. The method’s sensitivity for IL-8 was 1.5–7.5 pg/ml, with refer-ence values in the serum below the value of the lowest calibrator (31.2 pg/ml). Concentration was measured by a Labsystems Multiskan RC device.

Statistical analysis was done using Statistica for Windows by StatSoft®_{. A normal distribution}

thods were evaluated on the basis of histological results, having the sensitivity, specificity, and accuracy of both tests.

Results

Serum VEGF and IL−8 levels were statistical− ly significantly higher in the patients with ovarian cancer than in those with benign tumors and in the control group (Table 1). The concentrations of

clinical stage of disease and the grade of histolog− ical differentiation of ovarian cancer (Table 2). There were no statistically significant differences in proangiogenic factor concentrations in the blood serum for clinical stages I/II and III/IV ovar− ian cancer. Similarly, no statistically significant differences in proangiogenic factor concentrations were observed between separate grades of histo− logical differentiation of ovarian cancer.

Confidence intervals for the means were cal− culated with the aim to evaluate the diagnostic

Table 1.Serum VEGF and IL−8 levels in patients with ovarian cancer, benign tumors and in healthy controls

Tabela 1.Stężenia VEGF i IL−8 w surowicy krwi pacjentek z rakiem jajnika, guzami łagodnymi jajnika oraz w grupie kontrolnej

I II III

Ovarian cancer Benign tumors Healthy controls p (Rak jajnika) (Guz łagodny (Grupa kontrolna)

n = 30 jajnika) n = 11

n = 25

VEGF Me 459.15 131.80 158.00 0.0020I–II

[pg/ml] Q1 283.00 104.70 74.80 0.0006I–III

Q3 658.10 351.00 213.30 NSII–III

IL−8 Me 24.00 10.30 8.20 0.0001I–II

[pg/ml] Q1 16.30 6.70 6.70 0.0001I–III

Q3 37.40 14.30 10.30 NSII–III

Me – mediana. Q1 – pierwszy kwartyl. Q3 – trzeci kwartyl.

NS – nieistotne statystycznie. Me – median.

Q1 – first quartile. Q3 – third quartile. NS – not significant.

Table 2.Comparison of serum VEGF and IL−8 levels with ovarian cancer stage and grade

Tabela 2.Porównanie stężeń VEGF i IL−8 w surowicy krwi w zależności od stopnia zaawansowania klinicznego oraz zróżnicowania histopatologicznego raka jajnika

VEGF IL−8

n Me p Me p

(Q1/Q3) (Q1/Q3)

[pg/ml] [pg/ml]

Stage (Stopień)

I/II 8 407.10 NS 21.45 NS

(110.30/584.75) (15.80/31.25)

III/IV 22 459.15 NS 24.50 NS

(307.50/671.60) (16.80/55.30)

Grade

(Zróżnicowanie)

G1 6 251.50 NS 20.90 NS

(110.30/484.00) (16.30/36.40)

G2 10 577.55 NS 25.05 NS

(340.60/671.60) (13.30/57.40)

G3 14 416.90 NS 24.50 NS

(283.00/684.10) (17.90/36.40)

Me – mediana. Q1 – pierwszy kwartyl. Q3 – trzeci kwartyl.

NS – nieistotne statystycznie. Me – median.

accuracy of the serum VEGF and IL−8 levels. Correlations with the histological diagnosis of ovarian cancer were determined on the assumption that the lower limit of the confidence interval is a threshold value. Then the sensitivity, specificity, and accuracy of VEGF and IL−8 level evaluation in the differential diagnostics of ovarian tumors were determined (Table 3). VEGF values above 366.65 pg/ml correlated with a histological diag− nosis of ovarian cancer with 60% sensitivity, 86.11% specificity, and 74.24% accuracy. Values of IL−8 above 23.66 pg/ml correlated with a histo− logical diagnosis of ovarian cancer with 46.67% sensitivity, 97.22% specificity, and 74.24% accu− racy. These data indicate that determining serum VEGF and IL−8 levels might be applicable in the differential diagnostics of ovarian tumors.

Discussion

Malignant ovarian neoplasms continue to be one of the main issues in gynecological oncology. This is due to the great difficulties in specifying high−risk groups, the lack of symptoms at an early stage of the disease, and the lack of a sufficiently specific and sensitive diagnostic method that could be applicable in cross−sectional studies. It is now believed that clinical examination complemented by image diagnostics should be backed up by a de− termination of tumor marker levels in serum [11].

Many studies have shown that VEGF−depen− dent angiogenesis plays a very important role in ovarian tumors. According to various authors, serum VEGF concentrations in patients with ovar− ian cancer are significantly higher than in patients with benign or borderline malignancy tumors [12]. These observations confirm the present analysis of serum VEGF−A levels in patients with ovarian cancer, benign ovarian tumors, and healthy women. There were statistically significantly higher serum VEGF levels in the patients with ovarian cancer than in the patients with benign ovarian tumors (459.15 vs. 131.80 pg/ml, p = 0.002) and the control group (459.15 vs. 158.00 pg/ml, p = 0.0006). These results confirm those presented in studies by Cooper and Demirkiran,

who discovered significantly higher blood serum VEGF levels in patients with ovarian cancer than in patients with benign ovarian tumors and healthy women [12, 13]. Moreover, Cooper and Hefler concluded that a serum VEGF level above 380 pg/ml was an independent factor linked with worse prognosis [12, 14]. In the present study, lev− els of VEGF above 366.65 pg/ml correlated with a histological diagnosis of ovarian cancer with 60% sensitivity, 86.11% specificity and 74.24% accura− cy. In the available literature, authors assumed var− ious borderline values for serum VEGF concentra− tions. Demirkiran et al. [13] evaluated VEGF lev− els in blood serum and in tumor fluid from 43 patients with malignant ovarian tumors and 45 patients with benign ovarian tumors. The assumption that the borderline value for VEGF is 410 pg/ml allowed detecting malignant ovarian tumors with 95% sensitivity and 78% specificity [13]. Obermair et al. [15] analyzed serum VEGF levels in 44 patients with ovarian cancer, 81 pa− tients with benign ovarian tumors, and 113 healthy women. The assumption of a borderline value for VEGF at 363.7 pg/ml allowed the authors to diag− nose ovarian cancer with just 54% sensitivity and 77% specificity [15].

The above reference data and the results of the present study may provide evidence that VEGF may be secreted by neoplastic cells of a tumor directly to the peritoneal cavity. In the present study, VEGF levels were analyzed in relation to tumor histological type, clinical stage, and histo− logical grade of ovarian cancer. Taking into con− sideration the reference data, it can be concluded that serum VEGF levels often correlate with the histological grade of ovarian cancer. High VEGF concentrations in blood serum were usually found in poorly differentiated ovarian cancer [16–18]. This may suggest that serum VEGF level reflects the intensity of cell proliferation and differentia− tion rather than the size of the tumor. On the basis of reference literature values it can be concluded that the relationship between the level of VEGF and FIGO clinical stage of ovarian cancer is unam− biguous. Some authors, however, do not confirm such a relationship, which may suggest that VEGF−dependent angiogenesis remains constant

Table 3.Serum VEGF and IL−8 concentrations in the diagnosis of ovarian cancer

Tabela 3.Stężenia VEGF i IL−8 w diagnostyce raka jajnika

Cutoff Sensitivity Specificity Accuracy Value (Czułość) (Swoistość) (Dokładność)

[pg/ml] [%] [%] [%]

VEGF 366.65 60.00 86.11 74.24

VEGF plays a major role in ovarian cancer pro− gression not only due to its proangiogenic proper− ties, but also due to its ability to increase vessel permeability, leading to the presence of peritoneal fluid, which promotes the intraperitoneal dissemi− nation of ovarian cancer. Chechlinska et al. [20] claims that measuring VEGF in peritoneal fluid might be useful in the differential diagnosis of ovarian tumors, but not in examining residual or recurrent disease. Moreover, Hu et al. [21] con− cluded that neutralization of VEGF activity, lead− ing to inhibition of angiogenesis and the accumu− lation of cancer exudation in the peritoneal cavity, may have clinical application in ovarian cancer therapy.

IL−8 influences tumor cell proliferation and participates in angiogenesis [22]. The proangio− genic activity of IL−8 is based on inducing the migration and proliferation of vascular endothelial cells and fibroblasts and on increasing proangio− genic factor secretion, such as that of VEGF [23, 24]. In the present study, IL−8 level in blood serum was evaluated in patients with ovarian cancer and those with benign ovarian tumors and healthy women. There were significantly higher serum IL−8 concentrations in the patients with ovarian cancer than in those with benign ovarian tumors (24.0 vs. 10.3 pg/ml, p < 0.0001) and the control group (24.0 vs. 8.2 pg/ml, p < 0.0001). Similar results were obtained by Nowak et al. [11], who discov− ered that serum IL−8 levels in patients with ovari− an cancer were significantly higher than in patients with benign ovarian tumors. High levels of IL−8 were observed in 90% of patients with ovarian cancer, while the levels of IL−8 in patients with benign ovarian tumors and in a control group did not differ significantly [11]. Fasciani and Daraï obtained different results when analyzing serum IL-8 levels in patients with ovarian tumors. In their

nificantly in patients with ovarian cancer and those with benign ovarian tumors [25, 26]. In the present study it was found that IL-8 levels above 23.66 pg/ml correlated with a histological diagnosis of ovarian cancer with 46.67% sensitivity, 97.22% specificity, and 74.24% accuracy. This indicates that determining serum IL-8 level might be applic-able in the differential diagnostics of ovarian tumors. Taking into consideration the low sensitiv-ity (46.67%) and simultaneous high specificsensitiv-ity (97.22%) of this test, its application would be pos-sible with more sensitive diagnostic methods. In the present study there were no statistically signif-icant differences in IL-8 level in relation to histo-logical type of ovarian tumor and the clinical stage and histological differentiation of ovarian cancer. Fasciani et al. did not find any significant differ-ences in IL-8 blood serum levels in relation to the histological type of ovarian tumors [26]. Kaminska et al. observed significantly higher IL-8 blood serum levels in patients with FIGO stages III and IV ovarian cancer [27]. The lack of corre-lation between IL-8 levels and the main prognos-tic factors in ovarian cancer may suggest that IL-8 is secreted steadily, independently of the clinical stage of the disease. Analysis of reference data and the results of the present study suggest that IL-8 is a very important factor in neoplastic angiogenesis.

The authors concluded that pretreatment VEGF and IL−8 serum levels might be regarded as an additional tool in the differentiation of ovarian tumors. Evaluation of VEGF and IL−8 levels in blood serum and peritoneal fluid may also be help− ful in diagnostics and in monitoring malignant ovarian tumor therapy. Neutralization of proangio− genic factor activity, leading to an inhibition of angiogenesis and the accumulation of cancer exu− dation in the peritoneal cavity, may have clinical application in ovarian cancer therapy in the future.

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Address for correspondence:

Paweł Sadłecki

Department of Obstetrics and Gynecology Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Torun Ujejskiego 75

85−168 Bydgoszcz Poland

Tel.: +48 52 36 55 799 E−mail: [email protected]

Conflict of interest: None declared