Vascular Endothelial Growth Factor D and Intratumoral Lymphatics as Independent Prognostic Factors in Epithelial Ovarian Carcinoma

Authors


Abstract

Lymph node metastasis is an important prognostic indicator for disease progression and is crucial for therapeutic strategies of epithelial ovarian carcinoma. Vascular endothelial growth factor (VEGF)-D has been confirmed to have potent lymphangiogenic function in experimental models, but the role in the progression of human ovarian carcinoma remains presently controversial. The purpose of this study was to investigate the prognostic significance of VEGF-D and the presence of intratumoral lymphatics in patients with epithelial ovarian carcinoma. The VEGF-D expression was evaluated by immunohistochemistry in 78 specimens of epithelial ovarian carcinoma and tumoral lymphatic vessels were measured by D2-40. The expression of VEGF-D protein was detected in the cytoplasm of the tumor cells and in stroma occasionally. The high expression of VEGF-D was closely associated with the FIGO stage, intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Univariate and multivariate analysis indicated that VEGF-D, intratumoral lymphatics, and lymphatic invasion were independent prognostic factors for overall survival and disease-free survival in patients with epithelial ovarian carcinoma. We conclude that VEGF-D plays an essential role in tumoral lymphangiogenesis and lymphatic spread, VEGF-D expression, and the intratumoral lymphatics may be clinically useful indicators for prognostic evaluation in patients with epithelial ovarian carcinoma. Anat Rec, 2009. © 2009 Wiley-Liss, Inc.

Ovarian carcinoma represents the first significant cause of gynecological carcinoma-related death for women worldwide. Patients are always asymptomatic until the later stage of the disease and have the poorest prognosis among malignancies in the gynecologic field (Katso et al.,2000). Established prognostic parameters for epithelial ovarian carcinoma include surgical staging, histological subtype, histological grade, lymph node metastasis, and residual tumor after cytoreductive surgery (Yokoyama et al.,2003a), but there is a lack of clinically reliable molecular markers for evaluating prognosis in ovarian carcinoma. Thus, it is urgently required to identify extra prognostic indicators and to clarify the mechanism by which tumor spreads from the ovaries to regional lymph nodes.

It has long been known that some cancers metastasize via lymphatics to the regional lymph nodes (Sundar et al.,2006). Lymphangiogenesis is thought to promote the progression and lymph node metastasis of malignant tumors. Lymphangiogenesis, that is, the formation of new lymphatic vessels may play a role in this procedure (Skobe et al.,2001; Stacker et al.,2002; Von Marschall et al.,2005). One proposed mechanism is the induction of new lymphatic vessels by tumor or inflammatory cells. Formation of lymphatic vessel is regulated by the members of vascular endothelial growth factor (VEGF) family and their receptors (Shimizu et al.,2004). VEGF-C and VEGF-D have been identified to play an important role in the development of lymphatic vessels and promotion of lymphatic metastases by acting on VEGF receptor-3 (Kaipainen et al.,1995). VEGF-D expression was detected in colorectal (Funaki et al.,2003), ovarian (Yokoyama et al.,2003a), endometrial (Yokoyama et al.,2003b), and breast carcinomas (Nakamura et al.,2003), and melanoma (Liu et al.,2008). However, their effects in human cancers remain controversy. Some studies confirmed a significant association between VEGF-D expression and tumor lymphangiogenesis, lymph node metastasis and prognosis and others no relationships (Currie et al.,2004).

The aim of this study was to determine the expression levels of VEGF-D in tumor cells and to examine its association with peritumoral lymphatics, intratumoral lymphatics, and lymph node metastasis as well as to assess the utility as prognostic indicator. Additionally, the density and location of tumor lymphatic vessels were investigated, whether the presence of intratumoral lymphatic vessels correlated with lymph node involvement as well as patients clinical outcome were evaluated in patients with epithelial ovarian carcinoma.

MATERIALS AND METHODS

Patients and Tumor Samples

Paraffin-embedded stored specimens from 78 patients with primary epithelial ovarian carcinoma diagnosed for ovariectomy with standard lymph node dissection at Harbin Medical University Clinical Hospital (Harbin, China) between January 2001 and December 2002 were studied using immunohistochemistry. None of them had received preoperative chemotherapy. Formal consent was obtained from patients and approval of the study from the ethics committee. All patients were surgically staged in accordance with the International Federation of Gynecology and Obstetrics (FIGO) criteria. Table 1 summarized the most important clinicopathological characteristics of the investigated epithelial ovarian carcinoma.

Table 1. Clinicopathological characteristics of the patients
Total no.78
Age (years)
 Range34–72
 ≤4027
 >4051
Histological type
 Serous45
 Nonserous33
Histological grade
 Well25
 Moderate32
 Poor21
Lymph node metastasis (no. of patients)
 Negative35
 Positive43
FIGO stage (no. of patients)
 I13
 II21
 III25
 IV19
Residual disease
 ≤2 cm28
 >2 cm50

Immunohistochemistry

Immunohistochemical staining was performed using the streptavidin-peroxidase conjugate method. Briefly, 4 μm paraffin sections were deparaffinized in dimethylbenzene, rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min at room temperature. After a phosphate-buffered saline (PBS) rinse, the tissue sections were antigen-retrieved in 0.01 M citrate buffer (PH 6.0) at full pressure for 2 min. After rinsing with PBS, slides were incubated overnight at 4°C with VEGF-D polyclonal rabbit-anti-human antibody, 1:100 (Santa Cruz Biotechnology, USA); D2-40 monoclonal mouse-anti-human antibody, 1:200 (Santa Cruz Biotechnology, USA). D2-40, is a monoclonal antibody recognizing podoplanin, a 38 kD mucin-type transmembrane glycoprotein present on lymphatic endothelial cells, and is considered a reliable and selective marker of lymphatic endothelium. After a PBS rinse, slides were then incubated for 25 min at room temperature with polyHRP goat-anti-rabbit IgG and polyHRP rat-anti-mouse IgG, respectively (Zhongshan Biotechnology, Beijing, China). After rinsing with PBS, slides were stained with fresh 3,3′-diaminobenzidine (DAB) (Zhongshan Biotechnology, Beijing, China) according to the manufacturer's instructions and then counterstained in hematoxylin, dehydrated and mounted. Paraffin sections of normal human placenta were used as a positive control. Slides incubated without primary antibody were used as negative controls for VEGF-D staining.

Quantitative Analysis of Immunohistochemistry and Lymphatic Vessel Density

Two observers independently evaluated the results of immunohistochemical staining without knowledge of the clinical data of each patient. VEGF-D staining was assessed by estimating the percentage of tumor cells and divided tumors into four groups: (0%), negative; (<10%), mild; (10-50%), moderate; (>50%), strong; tumors were considered positive when they showed moderate or strong staining for VEGF-D and mild staining was regarded as negative expression (Umemoto et al.,2001).

To analyze tumoral lymphatic density, D2-40 antibody was used to detect lymphatic vessels. Only vessels with typical irregular morphology and a lumen that stained with D2-40 were considered lymphatic vessels. In each tumor section, five fields with the highest number of lymphatic vessels (hotspots) were evaluated and the average number was defined as lymphatic vessel density (LVD). This approach has been validated in published literature (Fox and Harris,2004).

Statistical Analysis

Each experiment was performed independently at least twice with similar results. One representative result for each experiment is presented here. All of the statistical analysis were performed using SPSS 15.0. Data were expressed as mean ± SD. The significance of the data was determined using the χ2-test. Correlations between VEGF-D expression and other tumor parameters were determined using Spearman's rank correlation coefficient. Survival rates were calculated starting from a month post-operation. Survival curves were drawn according to the Kaplan-Meier method, and differences were analyzed by the log-rank test. Univariate and multivariate analysis of prognostic factors were based on the Cox proportional hazards model. A value of P < 0.05 indicates statistical significance.

RESULTS

Expression of VEGF-D in Epithelial Ovarian Carcinoma

Positive immunostaining for VEGF-D was observed in the cytoplasm of the tumor cells, the peritumoral stroma occasionally stained positively for VEGF-D. The results were shown in Fig. 1. Fourty-six of 78 (58.9%) expressed VEGF-D moderately or strongly. 83.7% and 28.6% of metastatic cases and nonmetastatic cases, respectively, showed positive staining for VEGF-D. The incidence of VEGF-D detected in lymph node metastatic carcinoma was significantly higher than that detected in nonmetastatic carcinoma (Table 3; Spearman's rank test, P < 0.001).

Figure 1.

Immunohistochemical staining for vascular endothelial growth factor (VEGF)-D in epithelial ovarian carcinoma. VEGF-D was mainly expressed in the cytoplasm of tumor cells. Positive (A) and negative staining (B) of VEGF-D protein in tumor cells. Original magnifications: ×400.

Expression of D2-40 in Epithelial Ovarian Carcinoma

D2-40 marked thin-walled vessels without recognizable muscle layer or erythrocyte corresponded to lymphatic vessels (Fig. 2). Mean LVD for VEGF-D positive and negative samples were 10.8/mm2 (SD, 1.6) and 6.6/mm2 (SD, 1.2), respectively. Comparison of LVD in VEGF-D positive samples versus VEGF-D negative samples revealed a significant increase of LVD (P < 0.001) in VEGF-D positive carcinoma. Peritumoral and intratumoral lymphatics were detected in epithelial ovarian carcinoma, peritumoral lymphatics were present within stroma around tumor cell nests (Fig. 2A), intratumoral lymphatics were defined as those located within the tumor mass (Fig. 2B). Intratumoral D2-40-expressing lymphatics were detected in 48 epithelial ovarian carcinomas. Moreover, the lymphatic invasion by tumor cells were examined in 49 ovarian carcinomas (Fig. 2B–D), high presence of intratumoral lymphatics was associated with lymphatic invasion (44/48), and lymphatic invasion was more frequent in lymph node metastatic tumors (39/43), there was a clear association between the intratumoral lymphatics, the presence of lymphatic vessels invasion and lymph node metastatic status (P < 0.001, Table 2). The association between intratumoral lymphatics, lymphatic invasion, and metastatic features suggested that lymphangiogenesis might promote malignant progression of epithelial ovarian carcinoma.

Figure 2.

Lymphatic vessels were stained with D2-40. Peritumoral lymphatic vessels (A, arrows); Intratumoral lymphatics (B, arrows); (B–D) lymphatic invasion by tumor cells in epithelial ovarian carcinoma (arrows). Original magnifications: ×200 (A, B); ×400 (C, D).

Table 2. Association between lymphatic invasion and the presence of intratumoral lymphatic vessel as well as lymph node metastasis in epithelial ovarian carcinoma
GroupNo. of patientsLymphatic vessel invasion (no. %)P value
Intratumoral lymphatics
 Negative305 (16.6%)<0.001
 Positive4844 (91.6%)
Lymph node metastasis
 Negative3510 (28.6%)<0.001
 Positive4339 (90.7%)

VEGF-D Expression and Clinicopathological Variables

VEGF-D protein expression was compared with each clinicopathological variable (Table 3). Compared with patients who did not express VEGF-D, those positive for VEGF-D was related with the following characteristics: tumor LVD, the presence of intratumoral lymphatics, lymphatic vessel invasion by tumor cell, and the status of lymph node metastasis as well as FIGO stage (Table 3). By contrast, expression of VEGF-D was not significantly associated with age, histological type, histological grade, tumor size, and residual disease.

Table 3. Relation between VEGF-D expression and clinicopathological factors in epithelial ovarian carcinoma
FactorsNo. of patientsNo. (%) of VEGF-D positive casesP value
  1. NS, not significant.

Age (years)
 >405128 (54.9%)NS
 ≤402718 (66.7%)
Histological type
 Serous4527 (60.0%)NS
 Nonserous3319 (57.6%)
Histological grade
 Well2510 (40.0%)NS
 Moderate3223 (71.9%)
 Poor2113 (61.9%)
Tumor size
 ≤4 cm3721 (56.8%)NS
 >4 cm4125 (61.0%)
Lymphatic vessel density (LVD)
 ≤6245 (20.8%)<0.001
 >65441 (75.9%)
Intratumoral lymphatics
 Absence303 (10.0%)<0.001
 Presence4843 (89.6%)
Lymphatic vessel invasion
 Absence291 (3.4%)<0.001
 Presence4945 (91.8%)
Lymph node metastasis
 Negative3510 (28.6%)<0.001
 Positive4336 (83.7%)
FIGO stage
 I/II3411 (32.4%)<0.001
 III/IV4435 (79.5%)
Residual disease
 ≤2 cm2813 (46.4%)NS
 >2 cm5023 (46.0%)

Univariate and Multivariate Analysis for Risk Factors in Ovarian Carcinoma

Univariate analysis for overall survival revealed seven significant variables: FIGO stage, histological grade, VEGF-D expression, the presence of intratumoral lymphatics, lymphatic invasion, lymph node metastatic status, and residual disease (Table 4) in the Cox regression model. Multivariate analysis for overall survival identified four significant variables: FIGO stage, VEGF-D expression, intratumoral lymphatics, and lymphatic invasion (Table 4). Univariate and multivariate analysis for disease-free survival identified the same significant variables as the analysis for overall survival (Table 5). The multivariate Cox hazard ratio model identified FIGO stage (overall survival: P = 0.011, hazard ratio 11.750, 95% confidence interval 1.772–77.909; disease-free survival: P = 0.025, hazard ratio 7.290, 95% confidence interval 1.291–41.176), expression of VEGF-D (overall survival: P = 0.010, hazard ratio 24.721, 95% confidence interval 2.172–281.347; disease-free survival: P = 0.032, hazard ratio 12.537, 95% confidence interval 1.247–126.052) and intratumoral lymphatics (overall survival: P = 0.001, hazard ratio 6.114, 95% confidence interval 2.121–17.622; disease-free survival: P = 0.008, hazard ratio 3.839, 95% confidence interval 1.421–10.371) as well as lymphatic invasion (overall survival: P = 0.005, hazard ratio 17.339, 95% confidence interval 2.355–127.640; disease-free survival: P = 0.003, hazard ratio 10.925, 95% confidence interval 2.260–52.816) as independent prognostic factors for both overall survival and disease-free survival for patients with epithelial ovarian carcinoma in the present study (Tables 4 and 5).

Table 4. Univariate and multivariate analysis for the effect of different risk factors on overall survival in 78 patients with epithelial ovarian carcinoma
VariablesUnivariate analysis for overall survivalMultivariate analysis for overall survival
Hazard ratio95% CIPHazard ratio95% CIP
Age (>40 vs. ≤40 years)0.8630.499–1.4920.598   
FIGO stage(III/IV vs. I/II)24.9158.497–73.057<0.00111.7501.772–77.9090.011
Histological type (serous vs. nonserous)0.6690.387–1.1570.150   
Histological grade (poor/moderate vs. well)18.6247.484–46.346<0.0010.5960.214–1.6650.324
VEGF-D (positive vs. negative)105.42416.673–666.602<0.00124.7212.172–281.3470.010
Intratumoral lymphatics (presence vs. absence)12.9435.918–28.309<0.0016.1142.121–17.6220.001
Lymphatic invasion (presence vs. absence)49.08311.198–215.1340.01317.3392.355–127.640.005
Lymph node metastasis (yes vs. no)6.1063.181–11.718<0.0011.4580.680–3.1260.333
Residual disease (>2 cm vs. ≤2 cm)14.2776.122–33.293<0.0010.1780.290–1.0900.062
Table 5. Univariate and multivariate analysis for the effect of different risk factors on disease-free survival in 78 patients with epithelial ovarian carcinoma
VariablesUnivariate analysis for disease-free survivalMultivariate analysis for disease-free survival
Hazard ratio95% CIPHazard ratio95% CIP
Age (>40 vs. ≤40 years)0.8570.502–1.4630.572   
FIGO Stage (III/IV vs. I/II)21.4258.232–55.763<0.0017.2901.291–41.1760.025
Histological type (serous vs. nonserous)0.7190.423–1.2230.224   
Histological grade (poor/moderate vs. well)20.1398.055–50.348<0.0010.9040.303–2.6960.857
VEGF-D (positive vs. negative)124.60316.305–952.221<0.00112.5371.247–126.0520.032
Intratumoral lymphatics (presence vs. absence)10.2124.980–20.939<0.0013.8391.421–10.3710.008
Lymphatic invasion (presence vs. absence)30.17110.161–89.583<0.00110.9252.260–52.8160.003
Lymph node metastasis (yes vs. no)5.8183.102–10.910<0.0011.5290.713–3.2800.275
Residual disease (>2 cm vs. ≤2 cm)13.1226.006–28.669<0.0010.3150.067–1.4850.144

Patients with VEGF-D-positive tumors were found to have significantly shorter survival times and reduced disease-free survival when compared with those with VEGF-D-negative tumors (P < 0.001, Fig. 3A), whereas patients with the presence of intratumoral lymphatics and lymphatic invasion also showed poorer overall survival and reduced disease-free survival when compared with those with the absence of intratumoral lymphatics and lymphatic invasion tumors (P<0.001, Fig. 3B,C).

Figure 3.

Kaplan-Meier survival analysis of overall survival and disease-free survival depending on VEGF-D expression, the presence of intratumoral lymphatic vessel and lymphatic invasion in epithelial ovarian carcinoma. Expression of VEGF-D (A), the presence of intratumoral lymphatics (B), and lymphatic invasion (C) exhibited significantly poorer overall survival and decreased disease-free survival than those with negative tumors (P < 0.001, log-rank test).

DISCUSSION

The present report studied the expression of VEGF-D in epithelial ovarian carcinoma and examined the tumoral lymphatics. The results showed that increased levels of VEGF-D protein were significantly related to tumor LVD, intratumoral lymphatics, lymphatics invasion, and lymph node metastasis as well as FIGO stage in epithelial ovarian carcinoma. These results suggested that VEGF-D might play an important role in tumor lymphangiogenesis and lymphatic spread. Moreover, we also investigated the relation between the expression of VEGF-D and the clinicopathological status as well as the prognostic significance of VEGF-D for epithelial ovarian carcinoma.

Recent studies have reported similar results concerning the relation between VEGF-D detection, lymphatic spread, clinical parameters, and outcome. High levels of VEGF-D expression correlated with lymph node metastasis in colorectal carcinoma (White et al.,2002), lung adenocarcinoma (Niki et al.,2000), and head and neck squamous cell carcinoma (O-charoenrat et al.,2001). However, no association between the presence of VEGF-D and lymphatic spread was found in colorectal carcinoma (George et al.,2001) and in breast cancer (Mohammed et al.,2007). These discrepancies may depend on the subjective judgement of immunohistochemical results or the existence of biases, or maybe because of the different carcinoma explored. So some complementary assays, such as quantitative RT-PCR, are necessary to cross-evidence the “expression level”. In addition, good study design and scientific statistical analysis are also needed to avoid biases for more accurate and realistic results (McShane et al.,2006; Kyzas et al.,2005b,2007a,b).

In our study, VEGF-D was associated with a significantly shorter overall survival and disease-free survival. However, histological type or histological grade and lymph node metastasis did not show impact on overall survival and disease-free survival. When all of the significant prognostic factors were taken into account simultaneously in a Cox proportional hazards model, VEGF-D was an independent prognostic indicator for overall survival and disease-free survival. Therefore, the expression of VEGF-D was found to be an independent prognostic factor in ovarian carcinoma. Yokoyama et al. (2003a) reported that VEGF-D is an independent prognostic factor in ovarian carcinoma. Nevertheless, a mechanistic explanation for the prognostic independence of VEGF-D remains unclear. The determination of the presence of VEGF-D in combination with other prognostic factors may enhance the identification of ovarian carcinoma patients who are at risk for poor outcome. Although a large scale study are necessary to establish the usefulness of VEGF-D expression as a prognostic factor, identification of a correlation between the presence of VEGF-D and patient survival as well as lymphatic metastasis in different carcinoma types might lead to a novel therapeutic approach to prevent tumor progression in many carcinomas.

In the present study, we investigated LVD, the presence of intratumoral lymphatics, and lymphatic invasion to understand the prognostic significance of the tumoral lymphatic vessels in ovarian cancer. Lymphatic vessels play an essential role in the maintenance of tissue homeostasis and in the transport of immune cells, but they also serve as the primary conduit for malignant tumor cell metastasis to regional lymph nodes (Schacht et al.,2005). In recent years, specific lymphatic endothelial markers have been discovered, such as LYVE-1 (Jackson et al.,2001), Prox-1 (Wilting et al.,2002), and D2-40 (Sangoi et al.,2008), which are able to stain lymphatic endothelial cells in tumors; however, LYVE-1 is not restricted to the lymphatic vessels, it also expressed in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis (Mouta Carreira et al.,2001). The recently discovered marker D2-40 was found to be highly specific for lymphatic endothelial cells and has been used to evaluate intratumoral lymphatics (Longatto Filho et al.,2007; Sangoi et al.,2008). Intratumoral lymphatic endothelial cells were shown to be capable of proliferation, suggesting de novo lymphangiogenesis. In squamous cell carcinomas, intratumoral lymph vessel density has been shown to correlate with lymph node metastasis (Sipos et al.,2004). However, no evidence of intratumoral lymphatics was found in ovarian (Birner et al.,2000), breast (Williams et al.,2003), or cervical carcinoma (Schoppmann et al.,2002).

The identification of immunohistochemical markers that can reliably distinguish lymphatic vessels from blood vessels in tissue sections has yielded new insight into the mechanisms of metastasis (Sundar et al.,2006). But it still remained controversial whether the degree of intra- or peritumoral lymphatics might serve as a prognostic indicator of tumor progression. Many investigators have suggested that tumors do not possess a lymphatic supply. In addition, in cases where intratumoral lymphatics have been detected, these have been reported to be nonfunctional (Padera et al.,2002). Although we cannot comment on the function of lymphatic vessels in ovarian carcinoma, the strong implication is that they are involved in lymphatic invasion and lymph node metastasis. Same results showed in head and neck squamous carcinoma (Kyzas et al.,2005a). The observations that the presence of intratumoral lymphatics was associated with lymphatic invasion and metastatic tumor features indicate that intratumoral lymphatics may promote the malignant progression of ovarian carcinoma. Our results also showed that intratumoral lymphatics as an independent prognostic indicator.

Lymphatic invasion of tumors has been clearly documented to be a reliable prognostic variable predicting nodal metastasis and survival in breast, cervix cancer, and impacts on decision making for therapy in testicular cancer (Sundar et al.,2006). In our study, we observed that lymphatic invasion was present in most of the samples. Together with the fact that epithelial ovarian carcinoma metastasize frequently to lymph nodes, these findings support the hypothesis that tumor lymphangiogenesis may be a fundamental pathway for neoplasia dissemination in epithelial ovarian carcinoma.

In conclusion, our study demonstrated that the expression of VEGF-D and the presence of intratumoral lymphatics are independent indicator for overall survival and disease-free survival in patients with epithelial ovarian carcinoma. Moreover, expression of VEGF-D is closely correlated with tumoral lymphatic density and lymphatic spread. Thus the assessment of VEGF-D expression may further enhance the accuracy of prognosis in patients with epithelial ovarian carcinoma.

Acknowledgements

The authors thank Jing Zhao for her assistance in providing tissue samples and Fei Su for statistical design for this study.

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