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Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance
YGYNO-975462; No. of pages: 6; 4C: Gynecologic Oncology xxx (2014) xxx–xxx
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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance Pauline Wimberger a,b, Issam Chebouti a, Sabine Kasimir-Bauer a, Robert Lachmann b, Eberhard Kuhlisch c, Rainer Kimmig a, Ergün Süleyman d,1, Jan Dominik Kuhlmann a,b,⁎,1 a
Department of Gynecology and Obstetrics, University Hospital of Essen, Germany Department of Gynecology and Obstetrics, Technische Universität Dresden, Germany Department for Medical Informatics and Biometry, Technische Universität Dresden, Germany d Department of Anatomy and Cell Biology, University of Wuerzburg, Germany b c
H I G H L I G H T S • We systematically analyzed expression pattern and compartmental distribution of VEGF-receptor family in ovarian cancer. • Total VEGF-receptor expression correlated with tumor cell dissemination to the bone marrow and VEGF-R1 positivity indicated decreased progression-free survival. • We suggest VEGF-receptor status as a molecular biomarker for ovarian cancer, paralleling tumor cell spread and recurrence risk.
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Article history: Received 27 January 2014 Accepted 30 March 2014 Available online xxxx Keywords: Ovarian cancer Vascular endothelial growth factor receptor Angiogenesis Disseminated tumor cells Bevacizumab
a b s t r a c t Objectives. The identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patient's survival. Methods. A total of 73 patients with primary ovarian cancer were enrolled into this study. Primary tumor tissue was analyzed for the expression of VEGF-R1, VEGF-R2 and VEGF-R3 by immunohistochemistry. The presence of disseminated tumor cells (DTC) in the BM was analyzed by immunocytochemistry using the pancytokeratin antibody A45B/B3 and subsequent automatic detection based on staining and cytomorphology. Results. In primary ovarian cancer tissue, VEGF-receptor expression, detected with an overall frequency of 44%, was mostly located in the vascular wall and across the stroma; positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 were 34%, 18% and 26%, respectively. Total VEGF-receptor expression correlated with residual tumor after primary debulking surgery and the presence of DTC at primary diagnosis (p = 0.035, p = 0.023, respectively). Interestingly, VEGF-R1 positivity significantly correlated with decreased progression-free survival (p = 0.026). Conclusions. This is the first report, suggesting total VEGF-receptor status as a molecular biomarker for monitoring tumor cell spread to the BM and, particularly, revealing prognostic significance of VEGF-R1. © 2014 Elsevier Inc. All rights reserved.
Introduction Ovarian cancer is the leading cause of death among women with gynecologic malignancies [1]. Standard treatment of ovarian cancer
⁎ Corresponding author at: Department of Gynecology and Obstetrics, Technische Universität Dresden, Fetscherstraße 74, D-01307 Dresden, Germany. Fax: +49 351 458 5844. E-mail address: [email protected] (J.D. Kuhlmann). 1 Authors equally contributed to the underlying study.
constitutes primary radical surgery, aiming at macroscopically complete tumor resection, and subsequent platinum- and paclitaxel-based chemotherapy [2]. So far, residual tumor burden after primary surgery is believed to be one of the most relevant prognostic factors for ovarian malignancies [3,4]. Notably, advanced ovarian cancer is a comparatively chemo-sensitive tumor with overall clinical response rate of 70–80% [5]. However, despite this profound sensitivity to platinum-based chemotherapy and despite continuous attempts to implement maintenance therapies into the present treatment regime, more than half of all patients still experience recurrence, resulting in poor overall prognosis
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Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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[5,6]. Innovative therapy approaches are therefore needed to improve the patient's outcome. In this regard, the discovery of predictive molecular biomarkers is of high clinical interest, in order to individualize prognosis and therapy decision for each patient. The formation of new vessels from pre-existing vasculature, referred to as angiogenesis, is usually regulated through a dynamic balance of pro- and anti-angiogenic factors, in order to maintain physiological homeostasis. However, angiogenesis is also critically involved in ascites development and peritoneal dissemination of ovarian cancer [7]. In this context, it has been shown that vascular endothelial growth factor (VEGF) receptor signaling is one of the major regulators of angiogenesis [8,9] and that VEGF-ligands (comprising VEGFA, VEGFB, VEGFC, VEGFD and VEGFE) are capable of interacting with three different tyrosine kinase VEGF-receptors (VEGF-R1, VEGF-R2, and VEGF-R3). This interaction, in turn, triggers downstream signaling pathways and promotes angiogenesis, by regulating e.g. migration, proliferation and survival of endothelial cells [10]. Given that VEGF-overexpression has been observed in tumor tissue and in the circulation [11,12], the addition of the monoclonal antibody bevacizumab, directed against VEGF, combined with platinum- and taxane-based chemotherapy, followed by bevacizumab monotherapy, significantly prolonged PFS in first-line therapy [13,14]. In addition, in patients with response to adjuvant platinum- and taxane-based chemotherapy, maintenance monotherapy with pazopanib, a tyrosine kinase inhibitor against VEGF-receptor, PDGF-receptor and c-kit, showed significantly prolonged PFS, too [15]. Moreover, in platinum-sensitive and platinum-resistant relapse, the addition of bevacizumab to chemotherapy also significantly improved PFS [16,17]. However, despite recent efforts to reveal components of the VEGFaxis as novel biomarkers for response to anti-angiogenic treatment and despite the well described prognostic relevance of intratumoral and circulating VEGF [18,19], only little is known about expression pattern and clinical relevance of VEGF-receptors in ovarian cancer. Therefore, the present study aimed at systematically analyzing VEGFR1, VEGF-R2 and VEGF-R3 protein expressions and its compartmental distribution in primary ovarian cancer tissue and at assessing clinical relevance of the VEGF-receptor family with regard to established clinicopathological parameters, the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and survival (PFS, OS). Patients and methods Characterization of study patients The present study was conducted at the Department of Gynecology and Obstetrics at the University Hospital of Essen, Germany. In total, 73 patients with primary epithelial ovarian cancer were studied from January 2006 until November 2010. Survival data of these patients were obtained from the local municipal registry. The median followup time was 2.6 years, ranging from 0.08 to 6.58 years. Informed written consent was obtained from all patients, and the study was approved by the local Essen Research Ethics Committee (05/2856). Clinical data of the patients are summarized in Table 1. The whole study population received primary radical surgery aiming at macroscopically complete tumor resection. Individual patients, relapsing within the first six months after the end of chemotherapy, were defined to be clinically platinum resistant. Immunohistochemical VEGF-receptor staining VEGF-receptor analysis was performed in formalin fixed paraffin embedded (FFPE) primary ovarian cancer tissue, obtained during debulking surgery. Immediately after resection, specimens were incubated in buffered paraformaldehyde (4%, 24 h) and subsequently embedded in paraffin. For immunohistochemical investigation, 4 consecutive sections of 5 μm thickness were processed from each FFPE-block,
Table 1 Patient characteristics at primary diagnosis of ovarian cancer. Total 73 Age Tumor stage FIGO I–II FIGO III FIGO IV Lymph node metastasis N0 N1 Nx Tumor grading I–II III Histologic subtype Serous histology Mucinous histology Any other histology Residual tumor Macroscopic complete resection Any residual tumor Platinum resistance Platinum sensitive Platinum resistant DTC in the bone marrow DTCa-negative DTC-positive No status available Survival PFSb No relapse Relapse OSc Alive Dead
Mean: 61 years (range 21–89 years) 11 (15%) 49 (67%) 13 (18%) 23 (32%) 28 (38%) 22 (30%) 37 (51%) 36 (49%) 60 (82%) 3 (4%) 10 (14%)
38 (52%) 35 (48%) 49 (67%) 13 (18%) 43 (59%) 25 (34%) 5 (7%) 28 months (range 2–77 months) 20 (27%) 48 (66%) 36 months (range 1–79 months) 29 (40%) 44 (60%)
The present table recapitulates the patient's characteristics at primary diagnosis of ovarian cancer and comprises clinicopathological variables, platinum-sensitivity status, information on the presence of disseminated tumor cells in the bone marrow and survival data. a DTC, disseminated tumor cells in the bone marrow. b PFS, progression-free survival. c OS, overall survival.
deparaffinised with xylene and subsequently rehydrated with descending alcohol concentrations. After rehydration, sections were incubated with primary antibodies for 12 h at RT, detecting VEGF-R1, VEGF-R2 or VEGF-R3 (IgG rabbit polyclonal, Santa Cruz, Dallas, Texas, 1:50, 1:50, 1:100, respectively). Subsequent staining was performed by the streptavidin–biotin–peroxidase method [20,21]. Briefly, sections were incubated with a secondary antibody (anti-rabbit polyclonal, 1:200, 60 min, RT), followed by incubation with alkaline-phosphatasecomplex (PAP, 1:200, 30 min, RT) and avidin–biotin-complex (ABC, 1:250 30 min, RT). Specific immunostaining was visualized by a modified nickel-enhanced glucose oxidase method [20,22]. In order to analyze the underlying tissue structure, all sections were counterstained with calcium red and separate consecutive Hematoxylin & Eosin stained sections were prepared for each patient. Detection of disseminated tumor cells in the bone marrow Between 5 and 10 ml blood of BM per site was aspirated from the anterior iliac crests (local anesthesia with mepivacain) and processed within 24 h. Tumor cell detection was performed according to recommendations by the German Consensus group of Senology [23]. Briefly, mononuclear cells (MNC) were isolated from heparinized BM (5000 U/ml BM) by Ficoll-Hypaque density gradient centrifugation (density 1.077 g/mol; Pharmacia, Freiburg, Germany) at 400 g for 30 min. In total, 8 × 106 MNC per patient were analyzed for the presence of cytokeratin (CK)-positive cells using the murine monoclonal antibody A45-B/B3, directed against a common epitope of CKpolypeptides, including the CK-heterodimers 8/18 and 8/19 (Micromet,
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Munich, Germany). Microscopic evaluation of the slides was carried out using the ARIOL-system (Applied Imaging), according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) evaluation criteria and the DTC-consensus [23,24]. Statistical analysis Association between VEGF-receptor expression and the patient's clinicopathological parameters, including the presence of DTC in the BM, was analyzed by the Fisher's exact test and additionally, in terms of an explorative analysis, by the proportional hazard model (Cox regression). Prognostic significance of VEGF-receptor immunoreactivity was described by Kaplan–Meier curves and the Log-rank (Mantel–Cox) test. A p-value less than 0.05 was considered statistically significant; all indicated p-values are two-tailed. Statistical analysis was performed using GraphPad Prism version 6.01 for Windows (GraphPad Software) and IBM SPSS Statistics version 21 for Windows. Results Positivity and immunostaining pattern of VEGF-receptor in primary ovarian cancer tissue Protein expression of VEGF-R1, VEGF-R2 and VEGF-R3 was evaluated in primary ovarian cancer tissue (n = 73) by immunohistochemistry. In 32/73 patients (44%), positivity for at least one of the three receptors was detected. Positivity of VEGF-R1 was observed in 25/73 patients (34%), whereas 13/73 patients (18%) were VEGF-R2 positive and 19/73 patients (26%) were positive for VEGF-R3. Positivity rates for the receptor combinations VEGF-R1 + 2, VEGF-R1 + 3 and VEGFR2 + 3 were 15%, 19% and 12%, respectively (Fig. 1). Subsequently, VEGF-receptor expression was further evaluated with regard to its immunostaining pattern in ovarian cancer tissue. On average, we were able to detect and assign two different immunostained compartments per analyzed tissue section. Altogether, in our entire patient cohort, we observed VEGF-receptor positivity in the vasculature (comprising endothelial cells or smooth muscle cells of the vascular wall), in lymph endothelial cells, across the stroma (comprising fibroblasts, inflammatory cells, fibro-muscular cells) and in malignant ovarian cancer cells (Fig. 2). VEGF-receptor immunoreactivity in the vasculature and in lymph endothelial cells appeared as sharply defined, moderate to locally intense staining (Fig. 2a–c), whereas VEGF-receptor
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positivity in stromal compartments emerged comparatively weaker, more diffuse and covered large tissue areas (Fig. 2d). Moreover, in only two patients, we detected VEGF-receptor immunoreactive patches of irregularly shaped and intensively stained tumor cells (Fig. 2e). VEGFR1, VEGF-R2 and VEGF-R3 expressions were predominantly observed in the vasculature and across the stroma, whereas immunoreactive lymph endothelial cells were exclusively detected in VEGF-R3 stained tissue sections (5%) and staining in malignant ovarian cancer cells was restricted to positivity for VEGF-R1 (4%) or VEGF-R3 (5%, Fig. 3). Correlation of VEGF-receptor expression with the patient's clinicopathological parameters In our patient cohort, residual tumor burden after surgery was an independent predictive factor for PFS (HR = 2.09, 95% CI = 1.08– 4.02, p = 0.028) as well as for overall survival (OS, HR = 2.63, 95% CI 1.36–5.09, p = 0.004). Moreover, tumor stage revealed to be an independent predictor for PFS (HR = 3.85, 95% CI 1.11–13.31, p = 0.033), whereas the presence of distant metastasis (FIGO IV) and tumor grading were independent predictors for OS (HR = 2.24, 95% CI = 1.08–4.64, p = 0.029 and HR = 2.22, 95% CI = 1.19–4.16, p = 0.012, respectively, data not shown). To interrogate clinical relevance of VEGF-receptor expression in primary ovarian cancer, immunohistochemical data on VEGF-receptor analysis were subsequently correlated with the patient's clinicopathological features (Table 2). For a subset of 68 patients before surgery and 34 patients after chemotherapy, we were able to analyze BM aspirations for the presence of DTC. Likewise, these data on tumor cell dissemination to the BM were correlated with VEGF-receptor immunoreactivity in the corresponding primary tumor. VEGF-R1, VEGF-R2 and VEGF-R3 receptor positivity, as well as positivity for the receptor combinations 1 + 2, 1 + 3 and 2 + 3, significantly associated with the histological subtype (p = 0.043, p = 0.002, p = 0.019, p = 0.001, p = 0.007, p = 0.001, respectively). Moreover, interestingly, total VEGFreceptor expression correlated with residual tumor after surgery as well as with the presence of DTC in the BM at primary diagnosis (p = 0.035, p = 0.023, respectively). Contrarily, no associations between VEGF-receptor immunostaining and tumor stage, nodal status, distant metastasis, tumor grading or platinum-resistance were observed. Prognostic significance of VEGF-receptor expression in ovarian cancer patients In the following, Kaplan–Meier analyses were performed, in order to describe prognostic significance of VEGF-R1, VEGF-R2 or VEGF-R3 expression as well as of combined receptor positivity (VEGF-R1 + 2, 1 + 3 and 2 + 3) for ovarian cancer. Interestingly, we revealed that VEGFR1 immunoreactivity in the primary tumor, at the time of primary diagnosis, significantly correlated with a decreased PFS (p = 0.026, Fig. 4a). Moreover, we observed a trend that VEGF-R1 positivity also indicated decreased OS, but, however, the difference in the survival curves did not reach statistical significance (p = 0.059, Fig. 4b). Discussion
Fig. 1. VEGF-receptor expression in primary ovarian cancer tissue. The bar chart depicts positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 in primary ovarian cancer tissue. Percentages were calculated for total VEGF-receptor expression (represented by observed positivity for at least one of the three receptors), for each receptor alone and for denoted receptor combinations, represented by positivity of two defined receptors, respectively.
In the present study, we systematically investigated expression, compartmental distribution and clinical relevance of the VEGFreceptor family in ovarian cancer and observed that total VEGFreceptor immunoreactivity associates with tumor cell spread to the BM. Moreover, as our key finding, we demonstrate prognostic capacity of VEGF-receptor expression and show that VEGF-R1 positivity indicates increased risk of recurrence. By allocating VEGF-receptor expression to the different histological compartments in the given tissue sections, we primarily observed VEGF-receptor immunoreactivity in the vasculature and across the stroma, whereas only a small proportion of patients showed VEGF-receptor
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Fig. 2. VEGF-receptor staining in different histological compartments. The figure shows representative examples for VEGF-receptor immunoreactivity, observed in different histological compartments of primary ovarian cancer tissue. Regions of interest are indicated by red arrowheads. VEGF-receptor positivity was observed a) in endothelial cells b) in the vascular wall c) in lymph endothelial cells d) across stromal cells and e) within patches of rounded and irregularly shaped malignant ovarian cancer cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
positive malignant tumor cells. Profound VEGF-receptor expression in the vasculature appears reasonable, because in a cancerous condition, angiogenic VEGF, e.g. derived from tumor cells, primarily acts on pre-existing vasculature and initiates vessel sprouting. Moreover, this finding is principally accordant with a recent investigation, reporting that in human solid cancers (including ovarian cancer), VEGF-R2 and VEGF-R3 are primarily localized in the tumor vasculature [25]. However, in this study, immunoreactivity was exclusively observed in the vascular endothelium, whereas in our investigation, VEGF-receptors were found not only in endothelial cells but also in mural cells, such as pericytes or smooth muscle cells of tumor associated blood vessels. This may be consistent with a previous observation that VEGF, produced by tumor cells, interacts with VEGF-R1 of vascular smooth muscle cells and increases their proliferative and migratory capacity, thereby recruiting also smooth muscle cells for tumor vessel formation [26].
Fig. 3. Compartmental distribution of VEGF-receptor immunostaining. The bar chart depicts compartmental distribution of VEGF-receptor immunoreactivity in primary ovarian cancer tissue. In patients with positive VEGF-receptor staining, immunoreactivity could be allocated to the vasculature (comprising endothelial cells or smooth muscle cells of the vascular wall), to the stroma, to lymph endothelial cells or to malignant ovarian cancer cells.
The coverage of endothelial cells by smooth muscle cells from outside, in turn, might result in stabilization and persistence of tumor vessels, leading to improved tumor perfusion and to resistance against antiangiogenic drugs [27,28]. Moreover, in only two patients, we found ovarian cancer cells, being intensively immunoreactive for VEGF-R1 and VEGF-R3, respectively. Interestingly, VEGF-receptor positivity seems to be of variable occurrence in ovarian cancer, as another study reported on complete VEGF-R2 absence in epithelial cells of normal ovaries but observed VEGF-R2 positive malignant ovarian tumor cells in 75% of analyzed invasive cancer specimen [29]. Moreover, we frequently detected VEGF-receptor immunoreactive stromal cells across the analyzed tumor tissues. These observations may be explained by the fact that the VEGF-receptor axis, besides its profound angiogenic activity, may likewise be involved in pro-tumorigenic signaling, taking place in the tumor cell itself in terms of autocrine loops, or in terms of a cross-talk with tumorassociated stromal cells [29–31]. Since anti-angiogenic treatment has been implemented into first line treatment of ovarian cancer [13,14], a variety of studies intended to discover novel biomarkers for stratifying response to bevacizumab, by assessing e.g. micro-vessel density, components of the VEGF/VEGFreceptor signaling network, circulating endothelial cells, or genetic polymorphisms [9]. However, despite these efforts, there is no validated biomarker available at present, which could predict a patient's response to anti-angiogenic treatment [32]. Moreover, a variety of other studies investigated components of the VEGF/VEGF-receptor signaling pathway and primarily focused on clinical relevance of VEGF for ovarian cancer, independently from the context of anti-angiogenic treatment. In this regard, it was observed that VEGF is highly abundant in tumor tissue as well as in the circulation and is of prognostic significance [11,12,18, 19]. Complementarily to these previous findings, our results suggest clinical relevance of VEGF-receptor expression in primary ovarian cancer: We demonstrated that total VEGF-receptor positivity is associated with residual tumor load after primary surgery, which is principally accordant to another study, showing that VEGF-R3 overexpression in ovarian cancer paralleled debulking status [33]. Given that residual tumor burden is one of the most relevant prognostic factors for ovarian
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Table 2 Clinical relevance of VEGF-receptor expression in ovarian cancer.
Tumor stage Nodal status Distant metastasis Grading Histology Residual tumor Platinum resistance DTCa before surgery DTC after chemotherapy
VEGF-R1
VEGF-R2
p p p p p p p p p
p p p p p p p p p
= 0.613 N 0.999 = 0.118 = 0.744 = 0.043 = 0.054 = 0.309 = 0.296 N 0.999
N 0.999 N 0.999 N 0.999 N 0.999 = 0.002 = 0.763 N 0.999 = 0.754 = 0.682
VEGF-R3
Total VEGF-R
VEGF-R 1 + 2
p p p p p p p p p
p p p p p p p p p
p p p p p p p p p
= 0.876 N 0.999 = 0.087 = 0.887 = 0.019 = 0.060 = 0.445 = 0.401 N 0.999
= 0.496 N 0.999 = 0.064 = 0.613 = 0.096 = 0.035 = 0.526 = 0.023 = 0.502
N 0.999 N 0.999 N 0.999 = 0.822 = 0.001 = 0.748 N 0.999 N 0.999 = 0.682
VEGF-R 1 + 3 p p p p p p p p p
N 0.999 = 0.715 = 0.258 = 0.715 = 0.007 = 0.074 = 0.203 N 0.999 = 0.693
VEGF-R 2 + 3 p p p p p p p p p
= 0.749 = 0.647 N 0.999 = 0.468 = 0.001 = 0.729 = 0.615 = 0.715 N 0.999
The present table shows a correlation of VEGF-receptor expression and the patient’s clinicopathological variables, including platinum-sensitivity status and the presence of disseminated tumor cells in the bone marrow. p-Values were calculated by the Fisher's exact test, a p-value b0.05 was considered statistically significant. a DTC, disseminated tumor cells in the bone marrow.
cancer [3,4], our finding is of clinical interest and might imply that ovarian tumors with elevated VEGF-receptor expression and profound angiogenic capacity, may provide complex growth patterns, leading to insufficient debulking. For ovarian cancer, tumor cell spread to blood and BM has already been demonstrated to be a negative prognostic factor [34–36]. However, to the best of our knowledge, this is the first study, showing a relationship between VEGF-receptor expression in the primary tumor and the presence of DTC in the BM. Considering that VEGF-signaling contributes to intra-peritoneal dissemination and ascites formation [7], this finding appears reasonable and thus VEGFreceptor status may constitute a potential molecular biomarker to estimate tumor cell spread at the time of primary diagnosis. Finally, we reported that VEGF-R1 positivity indicates increased risk of recurrence. So far, prognostic significance of the VEGF-receptor family has rarely and incompletely been studied for ovarian cancer, with partly controversial results [33,37]. However, prognostic significance of VEGF-R1, our key finding, has never been described for ovarian cancer, yet. From a functional perspective, it was previously shown that VEGF-R1, in contrast to VEGF-R2 and VEGF-R3, is not required as a signaling receptor for endothelial cells and instead captures VEGF in order to spatially control VEGF-R2 signaling in terms of a negative regulator of angiogenesis [38]. Contrarily, other reports showed that VEGF-R1 signaling can modulate growth of Ras/MAPK-pathway driven
mouse tumor models or can promote migration and invasion of cancer cell lines [39,40]. Given these observations and also considering the role of VEGF-R1 in pro-angiogenic support of smooth muscle cell proliferation [26], we may hypothesize a pro-tumorigenic role of VEGF-R1 expression, being associated with tumor aggressiveness and high risk of recurrence. However, at the first glance, it might appear counterintuitive that only VEGF-R1 positivity is prognostically relevant, whereas VEGFR-2 and VEGFR-3 are prognostically non-informative, albeit obviously expressed across the analyzed cancer tissues. However, given the complex network of VEGF-receptors in terms of pro-tumorigenic and angiogenic signaling as well as its interaction with other signaling pathways, it may be possible that expression of VEGF-R2 or VEGF-R3 is collinear to tumor progression, but not necessarily associated with prognostic capacity. Conclusively, to the best of our knowledge, this is the first report suggesting that VEGF-receptor expression status in primary ovarian cancer tissue can serve as molecular biomarker for monitoring tumor cell spread to the BM and recurrence risk. Nevertheless, the present study is of explorative character, due to the limited total number of patients and, moreover, collinearity of VEGF-receptor expression with established risk factors of ovarian cancer, at least to a certain extent, cannot completely be ruled out at present. However, only very little is known about VEGF-receptor expression and its clinical relevance for
Fig. 4. Prognostic relevance of VEGF-receptor expression in ovarian cancer. Kaplan–Meier plots were drawn in order to estimate prognostic significance of VEGF-receptor expression in primary ovarian cancer tissue with regard to a) PFS and b) OS. Top curves refer to patients, being negative for VEGF-receptor expression, bottom curves refer to patient with VEGFreceptor positive primary tumor sections.
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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ovarian cancer so far. Therefore, we suppose the present study to be a relevant cornerstone, raising hypotheses for further investigation, such as the role of VEGF-receptor expression as a potential companion diagnostic for neoadjuvant treatment or clinical significance of soluble VEGF-R1 in the patient's blood. Conflict of interest statement The authors declare that no conflict of interest exists.
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Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance Pauline Wimberger a,b, Issam Chebouti a, Sabine Kasimir-Bauer a, Robert Lachmann b, Eberhard Kuhlisch c, Rainer Kimmig a, Ergün Süleyman d,1, Jan Dominik Kuhlmann a,b,⁎,1 a
Department of Gynecology and Obstetrics, University Hospital of Essen, Germany Department of Gynecology and Obstetrics, Technische Universität Dresden, Germany Department for Medical Informatics and Biometry, Technische Universität Dresden, Germany d Department of Anatomy and Cell Biology, University of Wuerzburg, Germany b c
H I G H L I G H T S • We systematically analyzed expression pattern and compartmental distribution of VEGF-receptor family in ovarian cancer. • Total VEGF-receptor expression correlated with tumor cell dissemination to the bone marrow and VEGF-R1 positivity indicated decreased progression-free survival. • We suggest VEGF-receptor status as a molecular biomarker for ovarian cancer, paralleling tumor cell spread and recurrence risk.
a r t i c l e
i n f o
Article history: Received 27 January 2014 Accepted 30 March 2014 Available online xxxx Keywords: Ovarian cancer Vascular endothelial growth factor receptor Angiogenesis Disseminated tumor cells Bevacizumab
a b s t r a c t Objectives. The identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patient's survival. Methods. A total of 73 patients with primary ovarian cancer were enrolled into this study. Primary tumor tissue was analyzed for the expression of VEGF-R1, VEGF-R2 and VEGF-R3 by immunohistochemistry. The presence of disseminated tumor cells (DTC) in the BM was analyzed by immunocytochemistry using the pancytokeratin antibody A45B/B3 and subsequent automatic detection based on staining and cytomorphology. Results. In primary ovarian cancer tissue, VEGF-receptor expression, detected with an overall frequency of 44%, was mostly located in the vascular wall and across the stroma; positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 were 34%, 18% and 26%, respectively. Total VEGF-receptor expression correlated with residual tumor after primary debulking surgery and the presence of DTC at primary diagnosis (p = 0.035, p = 0.023, respectively). Interestingly, VEGF-R1 positivity significantly correlated with decreased progression-free survival (p = 0.026). Conclusions. This is the first report, suggesting total VEGF-receptor status as a molecular biomarker for monitoring tumor cell spread to the BM and, particularly, revealing prognostic significance of VEGF-R1. © 2014 Elsevier Inc. All rights reserved.
Introduction Ovarian cancer is the leading cause of death among women with gynecologic malignancies [1]. Standard treatment of ovarian cancer
⁎ Corresponding author at: Department of Gynecology and Obstetrics, Technische Universität Dresden, Fetscherstraße 74, D-01307 Dresden, Germany. Fax: +49 351 458 5844. E-mail address: [email protected] (J.D. Kuhlmann). 1 Authors equally contributed to the underlying study.
constitutes primary radical surgery, aiming at macroscopically complete tumor resection, and subsequent platinum- and paclitaxel-based chemotherapy [2]. So far, residual tumor burden after primary surgery is believed to be one of the most relevant prognostic factors for ovarian malignancies [3,4]. Notably, advanced ovarian cancer is a comparatively chemo-sensitive tumor with overall clinical response rate of 70–80% [5]. However, despite this profound sensitivity to platinum-based chemotherapy and despite continuous attempts to implement maintenance therapies into the present treatment regime, more than half of all patients still experience recurrence, resulting in poor overall prognosis
http://dx.doi.org/10.1016/j.ygyno.2014.03.574 0090-8258/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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[5,6]. Innovative therapy approaches are therefore needed to improve the patient's outcome. In this regard, the discovery of predictive molecular biomarkers is of high clinical interest, in order to individualize prognosis and therapy decision for each patient. The formation of new vessels from pre-existing vasculature, referred to as angiogenesis, is usually regulated through a dynamic balance of pro- and anti-angiogenic factors, in order to maintain physiological homeostasis. However, angiogenesis is also critically involved in ascites development and peritoneal dissemination of ovarian cancer [7]. In this context, it has been shown that vascular endothelial growth factor (VEGF) receptor signaling is one of the major regulators of angiogenesis [8,9] and that VEGF-ligands (comprising VEGFA, VEGFB, VEGFC, VEGFD and VEGFE) are capable of interacting with three different tyrosine kinase VEGF-receptors (VEGF-R1, VEGF-R2, and VEGF-R3). This interaction, in turn, triggers downstream signaling pathways and promotes angiogenesis, by regulating e.g. migration, proliferation and survival of endothelial cells [10]. Given that VEGF-overexpression has been observed in tumor tissue and in the circulation [11,12], the addition of the monoclonal antibody bevacizumab, directed against VEGF, combined with platinum- and taxane-based chemotherapy, followed by bevacizumab monotherapy, significantly prolonged PFS in first-line therapy [13,14]. In addition, in patients with response to adjuvant platinum- and taxane-based chemotherapy, maintenance monotherapy with pazopanib, a tyrosine kinase inhibitor against VEGF-receptor, PDGF-receptor and c-kit, showed significantly prolonged PFS, too [15]. Moreover, in platinum-sensitive and platinum-resistant relapse, the addition of bevacizumab to chemotherapy also significantly improved PFS [16,17]. However, despite recent efforts to reveal components of the VEGFaxis as novel biomarkers for response to anti-angiogenic treatment and despite the well described prognostic relevance of intratumoral and circulating VEGF [18,19], only little is known about expression pattern and clinical relevance of VEGF-receptors in ovarian cancer. Therefore, the present study aimed at systematically analyzing VEGFR1, VEGF-R2 and VEGF-R3 protein expressions and its compartmental distribution in primary ovarian cancer tissue and at assessing clinical relevance of the VEGF-receptor family with regard to established clinicopathological parameters, the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and survival (PFS, OS). Patients and methods Characterization of study patients The present study was conducted at the Department of Gynecology and Obstetrics at the University Hospital of Essen, Germany. In total, 73 patients with primary epithelial ovarian cancer were studied from January 2006 until November 2010. Survival data of these patients were obtained from the local municipal registry. The median followup time was 2.6 years, ranging from 0.08 to 6.58 years. Informed written consent was obtained from all patients, and the study was approved by the local Essen Research Ethics Committee (05/2856). Clinical data of the patients are summarized in Table 1. The whole study population received primary radical surgery aiming at macroscopically complete tumor resection. Individual patients, relapsing within the first six months after the end of chemotherapy, were defined to be clinically platinum resistant. Immunohistochemical VEGF-receptor staining VEGF-receptor analysis was performed in formalin fixed paraffin embedded (FFPE) primary ovarian cancer tissue, obtained during debulking surgery. Immediately after resection, specimens were incubated in buffered paraformaldehyde (4%, 24 h) and subsequently embedded in paraffin. For immunohistochemical investigation, 4 consecutive sections of 5 μm thickness were processed from each FFPE-block,
Table 1 Patient characteristics at primary diagnosis of ovarian cancer. Total 73 Age Tumor stage FIGO I–II FIGO III FIGO IV Lymph node metastasis N0 N1 Nx Tumor grading I–II III Histologic subtype Serous histology Mucinous histology Any other histology Residual tumor Macroscopic complete resection Any residual tumor Platinum resistance Platinum sensitive Platinum resistant DTC in the bone marrow DTCa-negative DTC-positive No status available Survival PFSb No relapse Relapse OSc Alive Dead
Mean: 61 years (range 21–89 years) 11 (15%) 49 (67%) 13 (18%) 23 (32%) 28 (38%) 22 (30%) 37 (51%) 36 (49%) 60 (82%) 3 (4%) 10 (14%)
38 (52%) 35 (48%) 49 (67%) 13 (18%) 43 (59%) 25 (34%) 5 (7%) 28 months (range 2–77 months) 20 (27%) 48 (66%) 36 months (range 1–79 months) 29 (40%) 44 (60%)
The present table recapitulates the patient's characteristics at primary diagnosis of ovarian cancer and comprises clinicopathological variables, platinum-sensitivity status, information on the presence of disseminated tumor cells in the bone marrow and survival data. a DTC, disseminated tumor cells in the bone marrow. b PFS, progression-free survival. c OS, overall survival.
deparaffinised with xylene and subsequently rehydrated with descending alcohol concentrations. After rehydration, sections were incubated with primary antibodies for 12 h at RT, detecting VEGF-R1, VEGF-R2 or VEGF-R3 (IgG rabbit polyclonal, Santa Cruz, Dallas, Texas, 1:50, 1:50, 1:100, respectively). Subsequent staining was performed by the streptavidin–biotin–peroxidase method [20,21]. Briefly, sections were incubated with a secondary antibody (anti-rabbit polyclonal, 1:200, 60 min, RT), followed by incubation with alkaline-phosphatasecomplex (PAP, 1:200, 30 min, RT) and avidin–biotin-complex (ABC, 1:250 30 min, RT). Specific immunostaining was visualized by a modified nickel-enhanced glucose oxidase method [20,22]. In order to analyze the underlying tissue structure, all sections were counterstained with calcium red and separate consecutive Hematoxylin & Eosin stained sections were prepared for each patient. Detection of disseminated tumor cells in the bone marrow Between 5 and 10 ml blood of BM per site was aspirated from the anterior iliac crests (local anesthesia with mepivacain) and processed within 24 h. Tumor cell detection was performed according to recommendations by the German Consensus group of Senology [23]. Briefly, mononuclear cells (MNC) were isolated from heparinized BM (5000 U/ml BM) by Ficoll-Hypaque density gradient centrifugation (density 1.077 g/mol; Pharmacia, Freiburg, Germany) at 400 g for 30 min. In total, 8 × 106 MNC per patient were analyzed for the presence of cytokeratin (CK)-positive cells using the murine monoclonal antibody A45-B/B3, directed against a common epitope of CKpolypeptides, including the CK-heterodimers 8/18 and 8/19 (Micromet,
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Munich, Germany). Microscopic evaluation of the slides was carried out using the ARIOL-system (Applied Imaging), according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) evaluation criteria and the DTC-consensus [23,24]. Statistical analysis Association between VEGF-receptor expression and the patient's clinicopathological parameters, including the presence of DTC in the BM, was analyzed by the Fisher's exact test and additionally, in terms of an explorative analysis, by the proportional hazard model (Cox regression). Prognostic significance of VEGF-receptor immunoreactivity was described by Kaplan–Meier curves and the Log-rank (Mantel–Cox) test. A p-value less than 0.05 was considered statistically significant; all indicated p-values are two-tailed. Statistical analysis was performed using GraphPad Prism version 6.01 for Windows (GraphPad Software) and IBM SPSS Statistics version 21 for Windows. Results Positivity and immunostaining pattern of VEGF-receptor in primary ovarian cancer tissue Protein expression of VEGF-R1, VEGF-R2 and VEGF-R3 was evaluated in primary ovarian cancer tissue (n = 73) by immunohistochemistry. In 32/73 patients (44%), positivity for at least one of the three receptors was detected. Positivity of VEGF-R1 was observed in 25/73 patients (34%), whereas 13/73 patients (18%) were VEGF-R2 positive and 19/73 patients (26%) were positive for VEGF-R3. Positivity rates for the receptor combinations VEGF-R1 + 2, VEGF-R1 + 3 and VEGFR2 + 3 were 15%, 19% and 12%, respectively (Fig. 1). Subsequently, VEGF-receptor expression was further evaluated with regard to its immunostaining pattern in ovarian cancer tissue. On average, we were able to detect and assign two different immunostained compartments per analyzed tissue section. Altogether, in our entire patient cohort, we observed VEGF-receptor positivity in the vasculature (comprising endothelial cells or smooth muscle cells of the vascular wall), in lymph endothelial cells, across the stroma (comprising fibroblasts, inflammatory cells, fibro-muscular cells) and in malignant ovarian cancer cells (Fig. 2). VEGF-receptor immunoreactivity in the vasculature and in lymph endothelial cells appeared as sharply defined, moderate to locally intense staining (Fig. 2a–c), whereas VEGF-receptor
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positivity in stromal compartments emerged comparatively weaker, more diffuse and covered large tissue areas (Fig. 2d). Moreover, in only two patients, we detected VEGF-receptor immunoreactive patches of irregularly shaped and intensively stained tumor cells (Fig. 2e). VEGFR1, VEGF-R2 and VEGF-R3 expressions were predominantly observed in the vasculature and across the stroma, whereas immunoreactive lymph endothelial cells were exclusively detected in VEGF-R3 stained tissue sections (5%) and staining in malignant ovarian cancer cells was restricted to positivity for VEGF-R1 (4%) or VEGF-R3 (5%, Fig. 3). Correlation of VEGF-receptor expression with the patient's clinicopathological parameters In our patient cohort, residual tumor burden after surgery was an independent predictive factor for PFS (HR = 2.09, 95% CI = 1.08– 4.02, p = 0.028) as well as for overall survival (OS, HR = 2.63, 95% CI 1.36–5.09, p = 0.004). Moreover, tumor stage revealed to be an independent predictor for PFS (HR = 3.85, 95% CI 1.11–13.31, p = 0.033), whereas the presence of distant metastasis (FIGO IV) and tumor grading were independent predictors for OS (HR = 2.24, 95% CI = 1.08–4.64, p = 0.029 and HR = 2.22, 95% CI = 1.19–4.16, p = 0.012, respectively, data not shown). To interrogate clinical relevance of VEGF-receptor expression in primary ovarian cancer, immunohistochemical data on VEGF-receptor analysis were subsequently correlated with the patient's clinicopathological features (Table 2). For a subset of 68 patients before surgery and 34 patients after chemotherapy, we were able to analyze BM aspirations for the presence of DTC. Likewise, these data on tumor cell dissemination to the BM were correlated with VEGF-receptor immunoreactivity in the corresponding primary tumor. VEGF-R1, VEGF-R2 and VEGF-R3 receptor positivity, as well as positivity for the receptor combinations 1 + 2, 1 + 3 and 2 + 3, significantly associated with the histological subtype (p = 0.043, p = 0.002, p = 0.019, p = 0.001, p = 0.007, p = 0.001, respectively). Moreover, interestingly, total VEGFreceptor expression correlated with residual tumor after surgery as well as with the presence of DTC in the BM at primary diagnosis (p = 0.035, p = 0.023, respectively). Contrarily, no associations between VEGF-receptor immunostaining and tumor stage, nodal status, distant metastasis, tumor grading or platinum-resistance were observed. Prognostic significance of VEGF-receptor expression in ovarian cancer patients In the following, Kaplan–Meier analyses were performed, in order to describe prognostic significance of VEGF-R1, VEGF-R2 or VEGF-R3 expression as well as of combined receptor positivity (VEGF-R1 + 2, 1 + 3 and 2 + 3) for ovarian cancer. Interestingly, we revealed that VEGFR1 immunoreactivity in the primary tumor, at the time of primary diagnosis, significantly correlated with a decreased PFS (p = 0.026, Fig. 4a). Moreover, we observed a trend that VEGF-R1 positivity also indicated decreased OS, but, however, the difference in the survival curves did not reach statistical significance (p = 0.059, Fig. 4b). Discussion
Fig. 1. VEGF-receptor expression in primary ovarian cancer tissue. The bar chart depicts positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 in primary ovarian cancer tissue. Percentages were calculated for total VEGF-receptor expression (represented by observed positivity for at least one of the three receptors), for each receptor alone and for denoted receptor combinations, represented by positivity of two defined receptors, respectively.
In the present study, we systematically investigated expression, compartmental distribution and clinical relevance of the VEGFreceptor family in ovarian cancer and observed that total VEGFreceptor immunoreactivity associates with tumor cell spread to the BM. Moreover, as our key finding, we demonstrate prognostic capacity of VEGF-receptor expression and show that VEGF-R1 positivity indicates increased risk of recurrence. By allocating VEGF-receptor expression to the different histological compartments in the given tissue sections, we primarily observed VEGF-receptor immunoreactivity in the vasculature and across the stroma, whereas only a small proportion of patients showed VEGF-receptor
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Fig. 2. VEGF-receptor staining in different histological compartments. The figure shows representative examples for VEGF-receptor immunoreactivity, observed in different histological compartments of primary ovarian cancer tissue. Regions of interest are indicated by red arrowheads. VEGF-receptor positivity was observed a) in endothelial cells b) in the vascular wall c) in lymph endothelial cells d) across stromal cells and e) within patches of rounded and irregularly shaped malignant ovarian cancer cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
positive malignant tumor cells. Profound VEGF-receptor expression in the vasculature appears reasonable, because in a cancerous condition, angiogenic VEGF, e.g. derived from tumor cells, primarily acts on pre-existing vasculature and initiates vessel sprouting. Moreover, this finding is principally accordant with a recent investigation, reporting that in human solid cancers (including ovarian cancer), VEGF-R2 and VEGF-R3 are primarily localized in the tumor vasculature [25]. However, in this study, immunoreactivity was exclusively observed in the vascular endothelium, whereas in our investigation, VEGF-receptors were found not only in endothelial cells but also in mural cells, such as pericytes or smooth muscle cells of tumor associated blood vessels. This may be consistent with a previous observation that VEGF, produced by tumor cells, interacts with VEGF-R1 of vascular smooth muscle cells and increases their proliferative and migratory capacity, thereby recruiting also smooth muscle cells for tumor vessel formation [26].
Fig. 3. Compartmental distribution of VEGF-receptor immunostaining. The bar chart depicts compartmental distribution of VEGF-receptor immunoreactivity in primary ovarian cancer tissue. In patients with positive VEGF-receptor staining, immunoreactivity could be allocated to the vasculature (comprising endothelial cells or smooth muscle cells of the vascular wall), to the stroma, to lymph endothelial cells or to malignant ovarian cancer cells.
The coverage of endothelial cells by smooth muscle cells from outside, in turn, might result in stabilization and persistence of tumor vessels, leading to improved tumor perfusion and to resistance against antiangiogenic drugs [27,28]. Moreover, in only two patients, we found ovarian cancer cells, being intensively immunoreactive for VEGF-R1 and VEGF-R3, respectively. Interestingly, VEGF-receptor positivity seems to be of variable occurrence in ovarian cancer, as another study reported on complete VEGF-R2 absence in epithelial cells of normal ovaries but observed VEGF-R2 positive malignant ovarian tumor cells in 75% of analyzed invasive cancer specimen [29]. Moreover, we frequently detected VEGF-receptor immunoreactive stromal cells across the analyzed tumor tissues. These observations may be explained by the fact that the VEGF-receptor axis, besides its profound angiogenic activity, may likewise be involved in pro-tumorigenic signaling, taking place in the tumor cell itself in terms of autocrine loops, or in terms of a cross-talk with tumorassociated stromal cells [29–31]. Since anti-angiogenic treatment has been implemented into first line treatment of ovarian cancer [13,14], a variety of studies intended to discover novel biomarkers for stratifying response to bevacizumab, by assessing e.g. micro-vessel density, components of the VEGF/VEGFreceptor signaling network, circulating endothelial cells, or genetic polymorphisms [9]. However, despite these efforts, there is no validated biomarker available at present, which could predict a patient's response to anti-angiogenic treatment [32]. Moreover, a variety of other studies investigated components of the VEGF/VEGF-receptor signaling pathway and primarily focused on clinical relevance of VEGF for ovarian cancer, independently from the context of anti-angiogenic treatment. In this regard, it was observed that VEGF is highly abundant in tumor tissue as well as in the circulation and is of prognostic significance [11,12,18, 19]. Complementarily to these previous findings, our results suggest clinical relevance of VEGF-receptor expression in primary ovarian cancer: We demonstrated that total VEGF-receptor positivity is associated with residual tumor load after primary surgery, which is principally accordant to another study, showing that VEGF-R3 overexpression in ovarian cancer paralleled debulking status [33]. Given that residual tumor burden is one of the most relevant prognostic factors for ovarian
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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Table 2 Clinical relevance of VEGF-receptor expression in ovarian cancer.
Tumor stage Nodal status Distant metastasis Grading Histology Residual tumor Platinum resistance DTCa before surgery DTC after chemotherapy
VEGF-R1
VEGF-R2
p p p p p p p p p
p p p p p p p p p
= 0.613 N 0.999 = 0.118 = 0.744 = 0.043 = 0.054 = 0.309 = 0.296 N 0.999
N 0.999 N 0.999 N 0.999 N 0.999 = 0.002 = 0.763 N 0.999 = 0.754 = 0.682
VEGF-R3
Total VEGF-R
VEGF-R 1 + 2
p p p p p p p p p
p p p p p p p p p
p p p p p p p p p
= 0.876 N 0.999 = 0.087 = 0.887 = 0.019 = 0.060 = 0.445 = 0.401 N 0.999
= 0.496 N 0.999 = 0.064 = 0.613 = 0.096 = 0.035 = 0.526 = 0.023 = 0.502
N 0.999 N 0.999 N 0.999 = 0.822 = 0.001 = 0.748 N 0.999 N 0.999 = 0.682
VEGF-R 1 + 3 p p p p p p p p p
N 0.999 = 0.715 = 0.258 = 0.715 = 0.007 = 0.074 = 0.203 N 0.999 = 0.693
VEGF-R 2 + 3 p p p p p p p p p
= 0.749 = 0.647 N 0.999 = 0.468 = 0.001 = 0.729 = 0.615 = 0.715 N 0.999
The present table shows a correlation of VEGF-receptor expression and the patient’s clinicopathological variables, including platinum-sensitivity status and the presence of disseminated tumor cells in the bone marrow. p-Values were calculated by the Fisher's exact test, a p-value b0.05 was considered statistically significant. a DTC, disseminated tumor cells in the bone marrow.
cancer [3,4], our finding is of clinical interest and might imply that ovarian tumors with elevated VEGF-receptor expression and profound angiogenic capacity, may provide complex growth patterns, leading to insufficient debulking. For ovarian cancer, tumor cell spread to blood and BM has already been demonstrated to be a negative prognostic factor [34–36]. However, to the best of our knowledge, this is the first study, showing a relationship between VEGF-receptor expression in the primary tumor and the presence of DTC in the BM. Considering that VEGF-signaling contributes to intra-peritoneal dissemination and ascites formation [7], this finding appears reasonable and thus VEGFreceptor status may constitute a potential molecular biomarker to estimate tumor cell spread at the time of primary diagnosis. Finally, we reported that VEGF-R1 positivity indicates increased risk of recurrence. So far, prognostic significance of the VEGF-receptor family has rarely and incompletely been studied for ovarian cancer, with partly controversial results [33,37]. However, prognostic significance of VEGF-R1, our key finding, has never been described for ovarian cancer, yet. From a functional perspective, it was previously shown that VEGF-R1, in contrast to VEGF-R2 and VEGF-R3, is not required as a signaling receptor for endothelial cells and instead captures VEGF in order to spatially control VEGF-R2 signaling in terms of a negative regulator of angiogenesis [38]. Contrarily, other reports showed that VEGF-R1 signaling can modulate growth of Ras/MAPK-pathway driven
mouse tumor models or can promote migration and invasion of cancer cell lines [39,40]. Given these observations and also considering the role of VEGF-R1 in pro-angiogenic support of smooth muscle cell proliferation [26], we may hypothesize a pro-tumorigenic role of VEGF-R1 expression, being associated with tumor aggressiveness and high risk of recurrence. However, at the first glance, it might appear counterintuitive that only VEGF-R1 positivity is prognostically relevant, whereas VEGFR-2 and VEGFR-3 are prognostically non-informative, albeit obviously expressed across the analyzed cancer tissues. However, given the complex network of VEGF-receptors in terms of pro-tumorigenic and angiogenic signaling as well as its interaction with other signaling pathways, it may be possible that expression of VEGF-R2 or VEGF-R3 is collinear to tumor progression, but not necessarily associated with prognostic capacity. Conclusively, to the best of our knowledge, this is the first report suggesting that VEGF-receptor expression status in primary ovarian cancer tissue can serve as molecular biomarker for monitoring tumor cell spread to the BM and recurrence risk. Nevertheless, the present study is of explorative character, due to the limited total number of patients and, moreover, collinearity of VEGF-receptor expression with established risk factors of ovarian cancer, at least to a certain extent, cannot completely be ruled out at present. However, only very little is known about VEGF-receptor expression and its clinical relevance for
Fig. 4. Prognostic relevance of VEGF-receptor expression in ovarian cancer. Kaplan–Meier plots were drawn in order to estimate prognostic significance of VEGF-receptor expression in primary ovarian cancer tissue with regard to a) PFS and b) OS. Top curves refer to patients, being negative for VEGF-receptor expression, bottom curves refer to patient with VEGFreceptor positive primary tumor sections.
Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574
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ovarian cancer so far. Therefore, we suppose the present study to be a relevant cornerstone, raising hypotheses for further investigation, such as the role of VEGF-receptor expression as a potential companion diagnostic for neoadjuvant treatment or clinical significance of soluble VEGF-R1 in the patient's blood. Conflict of interest statement The authors declare that no conflict of interest exists.
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Please cite this article as: Wimberger P, et al, Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.574