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Clinical Cancer Research Vol. 11, 8364-8371, December 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Peritumoral Lymphatic Vessel Density and Vascular Endothelial Growth Factor C Expression in Early-Stage Squamous Cell Carcinoma of the Uterine Cervix

Zoltan Gombos1, Xiaowei Xu1, Christina S. Chu2, Paul J. Zhang1 and Geza Acs1

Authors' Affiliations: 1 Department of Pathology and Laboratory Medicine and 2 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania

Requests for reprints: Geza Acs, 6 Founders Pavilion, 3400 Spruce Street, Philadelphia, PA 19104. Phone: 215-662-6503; Fax: 215-349-5910; E-mail: geza{at}mail.med.upenn.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Lymphatic invasion and nodal metastasis plays a major role in the spread of cervical cancer; however, little is known about the mechanisms whereby tumor cells enter the lymphatic system.

Experimental Design: We examined the intra- and peritumoral lymphatic vessel density (LVD) using D2-40 immunohistochemistry in 111 cervical squamous cell carcinomas and correlated them with vascular endothelial growth factor (VEGF)-C expression, clinicopathologic tumor features, and outcome.

Results: Compared with benign cervix, intratumoral and peritumoral LVD was significantly increased (P < 0.0001). Peritumoral LVD was significantly higher than intratumoral LVD (P = 0.009). High peritumoral, but not intratumoral, LVD showed significant correlation with high tumor stage, lymphatic invasion, and nodal metastasis. VEGF-C showed increased expression at the invasive edge compared with the center of tumors (P < 0.0001) and correlated with high peritumoral LVD, lymphatic invasion, and nodal metastasis. High peritumoral LVD and VEGF-C expression at the invasive edge of tumors were associated with poor overall and recurrence-free survival in univariate analysis. In multivariate analysis, peritumoral LVD was the only independent term predictive of overall survival.

Conclusions: Our findings suggest a potential role for VEGF-C in tumor-induced lymphangiogenesis represented by high peritumoral LVD, which may be one of the mechanisms leading to lymphatic invasion and metastatic spread. High peritumoral LVD may be an independent prognostic factor in early-stage cervical cancer.

Tumor cell dissemination is mediated by several mechanisms, including local invasion, lymphatic or hematogenous spread, and direct seeding of body cavities or surfaces. In most solid tumors, including cervical cancer, the spread of cancer cells via the lymphatics to regional lymph nodes is an important early event in metastatic disease (1, 2). Indeed, nodal metastasis is a key factor in the staging of human cancers, an important prognostic indicator for outcome, and forms the basis of treatment of regional lymph nodes by surgery and radiation (3).

Previous studies have established the role of angiogenesis in tumor growth and hematogenous spread (4). However, despite the major role for the lymphatics in the initial spread of cancers, little is known about the mechanisms whereby tumor cells enter the lymphatic system (5, 6) and whether nodal metastasis is dependent on tumor-induced lymphangiogenesis or invasion of preexisting lymphatic vessels (5).

In recent years, several markers specific for lymphatic endothelium, including vascular endothelial growth factor (VEGF) receptor 3, the transmembrane proteins LYVE-1 and podoplanin, and the transcription factor Prox-1 (2), have been used to evaluate intratumoral lymphatic vessels in solid tumors (7–12). Intratumoral lymphatic endothelial cells were shown to be capable of proliferation, suggesting de novo lymphangiogenesis (7, 10). These studies also showed a correlation between intratumoral lymphatic vessel density (LVD) and nodal metastasis in some tumors (8) but not in others (7, 9–11, 13, 14).

The recently developed monoclonal antibody D2-40 detects a fixation-resistant epitope on podoplanin (15, 16). It was shown to be a selective marker for lymphatic endothelium allowing the specific identification of lymphatic vessels in formalin-fixed, paraffin-embedded tissue and the study of LVD in solid tumors (14, 16–18).

Research during recent years has also provided a better understanding of the molecular mechanisms underlying the development and maintenance of lymphatic vessels and their role in various pathologic conditions (2, 19). Two lymphangiogenic growth factors, named VEGF-C and VEGF-D, which signal through VEGF receptor 3, have been discovered and at least VEGF-C was shown to be essential for the development of lymphatic vessels in embryos (20, 21). In experimental tumors, VEGF-C and VEGF-D expression has been shown to induce lymphangiogenesis and correlate with lymphatic invasion and nodal metastasis (22–25). VEGF-C expression has been reported in several types of human cancer (7, 8, 10, 26–28). These studies suggested a correlation between VEGF-C expression in primary tumors and nodal metastasis (29).

In the current study, we examined the intra- and peritumoral LVD using D2-40 immunohistochemistry in a series of early-stage invasive squamous cell carcinomas of the cervix and correlated them with VEGF-C expression, clinicopathologic features, and outcome of the tumors.


   Materials and Methods
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Results
 Discussion
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Clinical samples and clinical data. One hundred eleven cases of radical hysterectomies (n = 104) and cone biopsies (n = 7) done for invasive cervical squamous cell carcinoma were selected from the files of the University of Pennsylvania Medical Center. H&E-stained slides were reviewed and the diagnoses were confirmed. In addition to the invasive carcinoma, carcinoma in situ and benign squamous mucosa were present in the representative sections selected for the study in 49 and 102 cases, respectively. Staging was defined according to the Fédération Internationale des Gynaecologistes et Obstetristes (FIGO) clinical staging system (30). Tumor grade was determined according to established criteria (31). The clinicopathologic features of the tumors are summarized in Table 1. No microinvasive carcinomas were included in the study. Pelvic/paraaortic lymph node dissection was done in 101 (91.0%) cases; the median number of lymph nodes per case examined was 24 (range, 1-82). Lymph node metastasis was present in 32 (31.7%) cases; the median number of positive lymph nodes was 2 (range, 1-7). Primary treatment was surgical in all cases; 38 patients received adjuvant treatment, consisting of radiation in 30 and combination radiation and chemotherapy in 8 cases, respectively. Follow-up of patients was done on the basis of information reported in the clinical histories. We considered as uncensored only records of patients who died of disease; we considered as censored records of all patients who were alive at follow-up or patients who died of a cause not related to the disease. Study protocols were approved by the University of Pennsylvania Institutional Review Board.


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  		Table 1. Summary of clinicopathologic features (n = 111)

 
Immunohistochemistry. Immunohistochemical assays were done on formalin-fixed, paraffin-embedded sections. For D2-40 and VEGF-C immunostaining, slides were boiled in 1x EDTA (LabVision, Fremont, CA) or 0.01 mol/L sodium citrate buffer (pH 6.0) for 20 minutes, respectively. Slides were incubated with the D2-40 (mouse monoclonal, 1:25 dilution; Signet Laboratories, Dedham, MA) and VEGF-C (rabbit polyclonal, 1:30 dilution; Zymed Laboratories, South San Francisco, CA) antibodies for 1 hour on room temperature. Immunohistochemical staining was done on a DAKOCytomation Autostainer using the EnVision+ horseradish peroxidase 3,3'-diaminobenzidine system (DAKOCytomation, Carpinteria, CA) according to the recommendations of the manufacturer. A negative control was done in each case by omission of the primary antibody. For D2-40 and VEGF-C, slides of normal human tonsil and colonic adenocarcinoma, known to show strong VEGF-C immunoreactivity, were used as positive controls, respectively.

Cytoplasmic and/or membrane VEGF-C immunoreactivity in tumor cells was evaluated semiquantitatively on a four-tiered scale. The percentages of weakly, moderately, and strongly staining cells were determined and a score was calculated as follows: score (maximum of 300) = sum of 1 x percentage of weak, 2 x percentage of moderate, and 3 x percentage of strong staining (18). Because the invasive edge of tumors is the site where lymphatic invasion is likely to occur (24), it was suggested that VEGF-C expression in the marginal portion of tumors may be more important than that of the central portion (11). Thus, we evaluated VEGF-C immunoreactivity separately at the marginal portion (defined as tumor cells located within 2 mm of the external edge) and in the center (the rest of the tumor) of the tumors (11). High VEGF-C expression in the marginal and central portions of tumors was defined as scores higher than the respective median values for all tumors. Immunohistochemical stains were evaluated independently by two pathologists (Z.G. and G.A.).

Intratumoral and peritumoral LVD were determined by the hotspot method as previously described (4). Briefly, slides were scanned at low power and intra- and peritumoral areas with the highest density of D2-40 positive vessels were identified. Intratumoral lymphatic vessels were defined as those located within the tumor mass; most of such lymphatic vessels were present within stroma in between tumor cell nests. Peritumoral lymphatic vessels were those located outside of the tumor mass but within 2 mm from the tumor. For benign epithelia and carcinomas in situ, stromal areas located within 2 mm of the underlying basement membranes showing the highest density of D2-40 positive lymphatic vessels were evaluated in a similar fashion. In all cases, LVD was determined by counting the number of D2-40-positive vessels in five high-power fields in the selected areas by two independent pathologists (G.A. and X.X.) and the mean values of vessel counts in the total 10 high-power fields were obtained. High intra- and peritumoral LVD was defined as those higher than the respective median values for all tumors.

Statistical analysis. Median LVD values and VEGF-C immunostaining levels were compared using the Mann-Whitney test or the Kruskal-Wallis one-way ANOVA by ranks followed by Dunn's multiple comparison test, where appropriate. Corresponding median intra- and peritumoral LVD values and VEGF-C expression levels in the marginal and central portions of tumors were compared using the Wilcoxon signed-rank test. High versus low immunostaining levels and LVD values in carcinomas were compared using unpaired t test and {chi}2 test, where appropriate. The correlation between VEGF-C expression levels and LVD values was estimated using the Spearman rank correlation test. Survival curves were plotted using the method of Kaplan and Meier and compared using the log-rank test. A Cox proportional hazards model was used to assess the effect of tumor variables on survival. Two-sided P < 0.05 was considered significant.


   Results
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Discussion
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Lymphatic vessel density. The D2-40 antibody strongly stained irregular vessels devoid of RBC corresponding to lymph vessels (16–18) whereas blood vessels showed no immunolabeling (Fig. 1A). The D2-40-positive lymph vessels were unevenly distributed throughout the tumors. The majority of intratumoral lymph vessels were small and collapsed (Fig. 1B). In contrast, lymphatic vessels located at the invasive edge of tumors were often enlarged and dilated (Fig. 1C). Lymphatic vessels associated with carcinomas in situ were small, irregular, and collapsed, contrasting the open lymphatics of benign cervical stroma. As expected, D2-40 immunostaining also highlighted the presence of lymphatic invasion usually present at the periphery of tumors (Fig. 1C).



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  		Fig. 1. A, the D2-40 antibody strongly and specifically stains lymphatic endothelial cells and highlights lymphatic invasion. Vascular endothelial cells show no immunostaining (arrow). B, intratumoral lymphatic vessels were small and collapsed. C, lymphatic vessels located at the invasive edge of tumors were often enlarged and dilated. D to F, LVD is significantly increased in stroma associated with in situ (E) and invasive carcinomas (F) compared with benign squamous mucosa (D). Note that LVD within carcinomas is significantly less compared with peritumoral areas. (Immunohistochemical stains for D2-40 with hematoxylin counterstain.) G, comparison of LVD in stroma associated with benign cervix, carcinoma in situ, and intra- and peritumoral regions. Lines, median LVD values expressed as mean vessel counts per high-power field [carcinoma in situ (CIS); *, P < 0.05; ***, P < 0.0001, Kruskal-Wallis test]. H to I, comparison of peritumor LVD in cervical squamous cell carcinomas associated with the absence or presence of lymphatic invasion (H) and nodal metastasis (I). Lines, median LVD values expressed as mean vessel counts per high-power field (**, P < 0.01; ***, P < 0.0001, Mann-Whitney test).

 
Compared with benign cervical mucosa, intra- and peritumoral LVD and LVD associated with carcinomas in situ were significantly increased (P < 0.0001, Kruskal-Wallis test; Fig. 1D-G). In addition, peritumoral LVD was significantly higher compared with intratumoral LVD in the corresponding tumors (P = 0.009, Wilcoxon signed-rank test).

Intra- and peritumoral LVD showed no correlation with patient age, tumor size, and depth of invasion of carcinomas. Carcinomas associated with high peritumoral LVD tended to be larger in size [3.43 ± 0.30 versus 2.75 ± 0.23 cm (mean ± SE)] but this difference did not reach statistical significance (P = 0.0713, unpaired t test). Intratumoral LVD showed no correlation with tumor grade or FIGO stage; tumors associated with lymphatic invasion and nodal metastases showed higher intratumoral LVD but the difference did not reach statistical significance (Table 2). High peritumoral LVD showed significant correlation with higher tumor stage and the presence of lymphatic invasion and nodal metastasis (Table 2; Fig. 1H and I).


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  		Table 2. Correlation of intra- and peritumor LVD with clinicopathologic tumor features

 
Vascular endothelial growth factor C expression. Immunohistochemical expression of VEGF-C was heterogeneous within tumors; however, all but one tumor showed at least focal weak VEGF-C immunoreactivity. The expression levels of VEGF-C were significantly higher in the marginal portions of carcinomas compared with the central regions (median immunostaining scores were 160 and 70, respectively; P < 0.0001, Wilcoxon signed-rank test; Fig. 2A-D). Tumor cell groups within lymphatic spaces usually showed strong VEGF-C immunoreactivity (data not shown).



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  		Fig. 2. Immunohistochemical expression of VEGF-C in invasive cervical squamous cell carcinoma. A, expression of VEGF-C is heterogeneous with frequent overexpression at the invasive edge (right) compared with the central portion of tumors (left). B, weak VEGF-C immunoreactivity in the central portion, contrasting with strong immunoreactivity in the marginal portion (C) of invasive squamous cell carcinoma. (Immunohistochemical stains for VEGF-C with hematoxylin counterstain.) D, comparison of immunohistochemical expression of VEGF-C in the central and marginal portions of invasive cervical squamous cell carcinomas. Lines, median VEGF-C immunostaining score values (*, P < 0.0001, Wilcoxon signed-rank test). E to F, comparison of VEGF-C expression levels at the marginal portions of invasive cervical squamous cell carcinomas associated with the absence or presence of lymphatic invasion (E) and lymph node metastasis (F). Lines, median VEGF-C immunostaining score values (*, P < 0.05; ***, P < 0.0001, Mann-Whitney test). G, level of immunohistochemical expression of VEGF-C at the marginal portions of cervical squamous cell carcinomas shows a highly significant correlation with peritumor LVD (r = 0.7269, P < 0.0001, Spearman test). Line, calculated regression line.

 
VEGF-C expression in the central and marginal portions of carcinomas showed no correlation with patient age, depth of invasion, tumor grade, and FIGO stage. VEGF-C expression in the central portions of tumors also did not correlate with tumor size, lymphatic invasion, and nodal metastasis. However, high VEGF-C expression levels at the marginal portions of tumors were significantly correlated with larger tumor size [3.58 ± 0.31 versus 2.56 ± 0.27 cm (mean ± SE); P = 0.0141, unpaired t test], the presence of lymphatic invasion, and nodal metastasis (Table 3; Fig. 2E and F).


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  		Table 3. Correlation of VEGF-C expression levels with clinicopathologic tumor features

 
We found no correlation between VEGF-C expression levels in the central regions and intra- or peritumoral LVD or between VEGF-C expression at the marginal regions and intratumoral LVD (P > 0.05, Spearman test). In contrast, a highly significant positive correlation was found between VEGF-C expression at the marginal regions of tumors and peritumoral LVD (r = 0.7269, P < 0.0001, Spearman test; Fig. 2G).

Survival analysis. During the follow-up interval, tumor recurrence was observed in 19 (17%) cases and 8 (7%) patients died of disease. The median time to death for the uncensored subgroup was 21.1 (range, 10.0-44.8) months whereas the median follow-up of censored patients was 24.9 (range, 0-180.6) months. The median time to tumor recurrence was 13.6 (range, 4.1-69.3) months. In univariate analysis, peritumoral LVD (P = 0.0006), VEGF-C expression in the marginal tumor regions (P = 0.013), nodal metastasis (P = 0.016), FIGO stage (P = 0.021), and lymphatic invasion (P = 0.032) were associated with poor overall survival (Fig. 3A and B). Peritumoral LVD (P < 0.0001), VEGF-C expression in he marginal tumor regions (P = 0.0002), lymphatic invasion (P = 0.012), nodal metastasis (P = 0.023), and depth of invasion (P = 0.029) showed significant association with recurrence-free survival (Fig. 3C and D). Tumor size, grade, intratumoral LVD, and VEGF-C expression in the central portions of tumors showed no correlation with either overall or recurrence-free survival. Backward elimination by Cox regression led to a model with one independent term predictive of overall survival [peritumoral LVD (P < 0.021)] and three independent terms predictive of recurrence-free survival [peritumoral LVD (P = 0.004), presence of lymphatic invasion (P = 0.024), and nodal metastasis (P = 0.047)].



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  		Fig. 3. Kaplan-Meier disease-related overall survival (A and B) and recurrence-free survival (C and D) curves stratified for low versus high peritumor LVD (A and C) and VEGF-C expression in the marginal portions of tumors (B and D) in invasive cervical squamous cell carcinomas.

 

   Discussion
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 Results
 Discussion
References
 
Malignant tumors may give rise to nodal metastases via several mechanisms, including invasion into intratumoral lymph vessels, preexisting lymphatics located at the tumor periphery, or tumor-induced growth of new lymphatics (2). The relative importance of these mechanisms is unclear and it may vary in different types of cancer. Recent evidence for the de novo formation of lymphatic capillaries has raised the possibility that tumor cells can contribute actively to lymphatic dissemination through the induction of lymphangiogenesis (22).

There has been considerable debate about the functional significance of intratumoral lymphatics and many investigators suggested that tumors do not possess a lymphatic supply (32). In addition, in cases where intratumoral lymphatics have been detected, these have been reported to be nonfunctional based on the results of dye uptake measurements (24). Although some investigators found intratumoral lymphatic vessels to be absent in tumors (13, 27, 33), others have observed intratumoral lymphatics in melanomas (9, 10), breast (34), head and neck (7, 8, 14, 35) and pancreatic carcinomas (11), and islet cell tumors (12). Our results are in agreement with the latter studies and contrast a prior study on cervical cancer (27): We found that intratumoral lymphatic vessels are present in cervical cancers and they are unevenly distributed throughout the tumors with a significantly higher density compared with normal cervical tissue. Similar to previous studies, we found that intratumoral lymphatics were small and flattened with a close lumen (12, 24), contrasting the widely open lymphatics in peritumoral regions. These findings suggest that the detected intratumoral lymphatics were newly developed rather than entrapped preexisting vessels (12). Similar to prior studies (27, 36), we found an increased density of open, dilated lymphatic vessels at the periphery of tumors, especially when lymphatic vessel invasion was present.

Prior studies (28) suggested that in cervical carcinogenesis a potential switch to the lymphangiogenic phenotype occurs at the stage of cervical intraepithelial neoplasia grade 3. Our results also indicate an increased density of lymphatic vessels associated with severe dysplasia/carcinoma in situ and suggest an essential role for lymphangiogenesis in the progression to invasive behavior in cervical carcinogenesis.

Although some studies suggested a role for intratumoral lymphatics in tumor dissemination and others showed high peritumoral LVD to be a favorable prognostic factor in other tumors (7, 8, 10, 14, 33, 36), most available data indicate a stronger correlation between peritumoral lymphangiogenesis and tumor aggressiveness (9, 13, 14, 27). Using an experimental system, Padera et al. (24) showed metastatic spread despite no detectable intratumoral lymphatic vessels and proposed that functional lymphatics at the tumor margin are sufficient for promoting metastasis by offering a larger area for tumor cell escape. Our results also suggest that high peritumoral, but not intratumoral, LVD is associated with aggressive behavior in cervical squamous cell carcinomas: High peritumoral LVD seemed to be a highly significant factor predicting poor overall and recurrence-free survival in both univariate and multivariate analyses in our cohort of patients. High peritumoral LVD was also associated with the presence of nodal metastases and aggressive behavior in several, but not all, clinical studies as well (7, 10, 13, 27, 34, 36). The contradicting results about the role of peritumoral lymphatics in tumor progression may be due to differences in patient selection, methodology, and/or the types of tumors included in the analyses. It might also reflect the fact that tumor lymphangiogenesis and lymphatic metastasis are complex mechanisms which can differ significantly in tumors of different types or anatomic locations (35).

The possibility that VEGF-C might promote tumor lymphangiogenesis has received much attention in recent years. Despite ample experimental evidence of an association between VEGF-C expression and lymphangiogenesis (22, 23, 37), this relationship is less conspicuous in human tumors where there is as yet little evidence for direct lymphangiogenesis. In recent years, a number of clinical studies have reported VEGF-C expression in human tumors and illustrated a significant association between VEGF-C levels of primary tumors and lymph node metastasis (23, 29, 38, 39).

Similar to previous studies (28), we detected VEGF-C expression in all but one tumor in our current series. VEGF-C immunoreactivity was increased at the periphery of cervical cancers consistent with up-regulation of VEGF-C expression at the invasive edge of the tumors as reported in breast and pancreatic carcinomas (11, 34). Similar to other reports (12, 40), we found a highly significant correlation between VEGF-C expression in the marginal portions of tumors and peritumor LVD, suggesting that VEGF-C expressed by tumor cells at the invasive edge may play a significant role in tumor-induced lymphangiogenesis in the stromal tissue immediately confronting tumor invasion. In cervical carcinoma, peritumoral LVD was also reported to correlate with the density of VEGF-C-expressing peritumoral macrophages (27).

In addition to peritumoral LVD, we found that high VEGF-C expression at the marginal, but not the central, portions of tumors was associated with lymphatic invasion and nodal metastasis and correlated with poor overall and recurrence-free survival in univariate analysis. In a recent study of pancreatic carcinomas, high VEGF-C expression at the marginal portion of the tumors was also associated with lymphatic invasion and nodal metastases (11).

In summary, among the histopathologic factors evaluated in this series of early-stage cervical squamous cell carcinoma, peritumoral LVD, VEGF-C expression at the invasive tumor edge, nodal metastasis, FIGO stage, lymphatic invasion, and depth of invasion were shown to have significant association with overall and/or recurrence-free survival whereas high peritumoral LVD, presence of lymphatic invasion, and nodal metastasis were independently predictive of poor overall and/or shorter recurrence-free survival. Our results suggest that high VEGF-C expression by tumor cells at the invasive edge of cervical cancers may induce lymphangiogenesis in peritumoral regions and contribute to high peritumoral LVD, leading to increased aggressiveness, lymphatic invasion, and metastatic spread. Our data also suggest that tumor cells at the invasive front of solid tumors may have some biological properties that distinguish them from those in the central portions of the tumor and may play a significant role in tumor progression.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 8/05; revised 8/25/05; accepted 9/ 6/05.


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