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Clinical Cancer Research Vol. 11, 7344-7353, October 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Lymphangiogenesis Correlates with Lymph Node Metastasis, Prognosis, and Angiogenic Phenotype in Human Non–Small Cell Lung Cancer

Ferenc Renyi-Vamos1,2,4, Jozsef Tovari5, Janos Fillinger3, Jozsef Timar1,5, Sandor Paku6, Istvan Kenessey5, Gyula Ostoros1, Laszlo Agocs2, Ibolya Soltesz3 and Balazs Dome1,5

Authors' Affiliations: Departments of 1 Tumor Biology, 2 Thoracic Surgery, and 3 Pathology, National Koranyi Institute of Pulmonology; 4 Department of Surgery and 5 Tumor Progression, National Institute of Oncology; and 6 Department of Molecular Pathology, Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary

Requests for reprints: Balazs Dome, Department of Tumor Biology, National Institute of Pulmonology, Piheno. u. 1., Budapest H-1529, Hungary. Phone: 36-1391-3210; Fax: 36-1391-3223; E-mail: domeb{at}yahoo.com.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Recent experimental studies have revealed that lymphangiogenesis plays an important role in cancer progression, but its clinical significance in the case of non-small cell lung cancer (NSCLC) remains unclear. Our aim was to assess the lymphangiogenesis of human NSCLC, and to correlate this with angiogenic phenotype (angiogenic versus nonangiogenic growth pattern) and clinical behavior.

Experimental Design: One hundred and three patients with NSCLC and complete follow-up information were included. Tumor samples were immunostained for vascular endothelial growth factor-C (VEGF-C), the lymphatic endothelial markers, LYVE-1 and D2-40/Podoplanin, and the panvascular marker, CD31. Lymphatic vessel density (LVD) and perimeters were evaluated within the tumor and peritumorally.

Results: LVDs at the tumor periphery were significantly higher in lymph node metastatic tumors (P < 0.005) and high LVDs correlated with poor overall survival (P < 0.001). However, this tendency proved to be significant only in the angiogenic tumor group (P < 0.001). Although 68% of the patients with nonangiogenic tumors had lymph node metastasis (P = 0.0048 versus angiogenic tumors), in the patient group with nonangiogenic NSCLCs, there was no information from the LVDs in any investigated tumor area (P > 0.05). In contrast to angiogenic tumors, which had actively sprouting lymphatics in all of the investigated tumor areas, nonangiogenic tumors showed no Ki67 staining intratumorally.

Conclusions: Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in NSCLC. Moreover, it also provides the first evidence that nonangiogenic NSCLCs mainly co-opt host tissue lymphatics during their growth, in contrast to most of the angiogenic tumors, which expand with concomitant lymphangiogenesis.

Non–small cell lung cancer (NSCLC) is well-known for its ability to involve regional lymph nodes even at the early stages of tumor growth. Therefore, "N status" is a major determinant for the staging and clinical management of lung cancer (1). However, little is known about the lymphatic system in human NSCLC, and the relationship of the lymphatic system to nodal metastasis and prognosis.

Although research of tumor-induced lymphangiogenesis has been eclipsed by the greater emphasis laid on the mechanisms of tumor vascularization, recent experimental evidence suggests that tumors can provoke lymphatic capillary growth by the production of lymphangiogenic factors such as that found in members of the vascular endothelial growth factor (VEGF) family, and that the activity of tumor-induced lymphangiogenesis is directly correlated with the extent of tumor spread to regional lymph nodes (2–4). However, it is still uncertain whether lymphatic spread is achieved through the formation and invasion of new lymphatics (tumor-induced lymphangiogenesis via sprouting) or whether tumors acquire their lymphatic vessels by co-option, as it was described recently by our group and other observers during the development of tumor blood vasculature (5, 6). In the case of NSCLC, a putatively nonangiogenic growth pattern was observed (7). In this "alveolar type" of growth, tumor cells fill the alveoli, entrapping, but not destroying the alveolar walls with the co-opted capillaries. Furthermore, in the tumor cell nests circumscribed by the alveolar walls, no neoangiogenesis is present.

Our hypothesis was that similarities might exist between hem- and lymphangiogenesis in NSCLC. Therefore, the objective of the current study was to clarify the role of lymphatics in the progression of lung tumors. We have analyzed how NSCLC acquires its lymphatic network and investigated whether the extent of lymphangiogenesis may be related to the angiogenic phenotype (angiogenic versus nonangiogenic) and/or to the risk of lymph node metastasis and to patient survival, using tumor samples obtained from NSCLC patients.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Clinical data. A total of 103 patients with histopathologically defined NSCLC treated at the Department of Surgery, National Institute of Pulmonology, Budapest from January 1994 to December 1997 were included in the study. There were 62 male and 41 female patients with a median age of 63 years (range, 38-76 years; Table 1). Formalin-fixed, paraffin-embedded NSCLC samples were retrieved from the files of our pathology department, with the approval of the Ethics Committee of the National Institute of Pulmonology, Budapest, and in accordance with the ethical standards prescribed by the Helsinki Declaration of the World Medical Association. Tissue blocks containing a representative fraction of the tumor and the tumor-lung parenchyma interface were used. All samples were obtained after elective surgery. No adjuvant therapy was given prior to surgery. Histologic diagnosis and N stage were determined on H&E stained sections. There were 53 squamous cell carcinomas, 42 adenocarcinomas, and 8 large cell carcinomas. Samples of tumor-free lung parenchyma were also obtained either from lung tissue distant from the tumor (two samples) or from patients operated on because of emphysema (two samples). The cases were staged according to operative and pathologic findings based on the American Joint Committee on Cancer/Unio Internationale Contra Cancrum tumor-node-metastasis classification (8). Following Pezzella et al. (7), the tumors were also classified as angiogenic (showing the destruction of normal lung tissue and the production of newly formed blood vessels and stroma) or nonangiogenic (lacking parenchymal destruction and neoangiogenesis) carcinomas.


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  		Table 1. Correlation of clinicopathologic features and expression of VEGF-C in patients with NSCLC

 
Characterization of the lymphatic system by immunohistochemistry. Immunohistochemical stainings were done on samples fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections (5 µm) were dewaxed and rehydrated. For light microscopy, peroxidase was quenched with methanol and 3% H2O2 for 15 minutes. Antigen retrieval was done in 0.1 mol/L citrate buffer (pH 6) in an 800 W microwave for 15 minutes. After washing in PBS, the following primary antibodies were used: rabbit polyclonal anti-human LYVE-1 (1:100; Reliatech, Braunschweig, Germany), mouse monoclonal anti-human D2-40 (1:100; DakoCytomation, Carpinteria, CA), mouse monoclonal anti-human CD31 (1:40; DakoCytomation), polyclonal rabbit anti-human VEGF-C (1:100, Zymed, San Francisco, CA), rabbit monoclonal anti-human Ki67 (1:200, Lab Vision, Suffolk, United Kingdom). Normal mouse and rabbit IgGs were substituted for primary antibodies as negative controls (same concentrations as the test antibodies).

For immunofluorescent staining, after washing in PBS, slices were incubated simultaneously with the appropriate secondary antibodies (FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rabbit IgG, rhodamine-conjugated goat anti-mouse IgG, and rhodamine-conjugated goat anti-rabbit IgG, 1:50; all from Jackson ImmunoResearch Inc., West Grove, PA) with nuclear staining with TOTO-3 (1:1,000, Molecular Probes, Eugene, OR).

For light microscopy, sections were first incubated with LYVE-1 or D2-40 antibodies and then developed using the horseradish peroxidase Envision System (DakoCytomation). Slides were then washed in PBS and incubated with antibodies to anti-human CD31, anti-human Ki67, and anti-human VEGF-C, followed by development with AP (DakoCytomation, EnVision System) conjugate. Finally, counterstaining was done using methylene green.

For VEGF-C status, samples were divided into positive or negative groups, with a cutoff value based on the findings of previous reports (9, 10). When ≥30% of the cancer cells in a given specimen were positively stained for VEGF-C, the sample was classified as VEGF-C-positive, and when <30% of the cells were stained, the sample was classified as VEGF-C-negative.

Sections were examined using a Nikon Eclipse 80i microscope and digital images were captured using either a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI) or the Bio-Rad MRC-1024 confocal laser-scanning microscopy system (Bio-Rad, Richmond, CA).

Computer-assisted morphometric analysis of the lymphatic network. Morphometric variables were determined by labeling of lymphatic vessels with anti-human LYVE-1 and anti-human D2-40/podoplanin. Three different tumoral and peritumoral regions were assessed separately for each section. These were (a) the tumor center, (b) the tumor periphery—a 1-mm-wide band of tumor immediately adjacent to the invasive edge, and (c) the peritumoral host tissue—a 1-mm-wide band of host connective tissue immediately adjacent to the tumor periphery. Three sections per tumor were analyzed using CUE-2 computerized image analysis system (consisting of special software, image processor, digital camera, and video monitor; Olympus, Tokyo, Japan) as described previously (11). To increase the contrast between dark brown from lymphatic capillaries (obtained by diamino benzidine staining) and green from the nuclei (methylene green), a 436-nm filter was used in the microscope. The image analysis (12) was done using the computer: images were captured with the microscope coupled to the video camera and digitalized using an internal frame-grabbing board and IBM computer (IBM Computer, Inc., Armonk, New York). This procedure consisted of converting the captured image in points or pixels according to the gray tone. In cases where interference was observed with other dark material, such as anthracotic pigment or horn material, this was corrected interactively. Once the computer selected these areas of similar color (within a set of tolerance and after eliminating the background), these regions were filled in black and the image was converted to a gray scale. The computerized system measured the following in each of the 5 to 10 fields of 20x objective/section/region: LVD (lymph vessel density; the number of lymphatics per square millimeter derived from image analysis) and lymphatic vessel perimeter. Finally, results from the different peri- and intratumoral regions were integrated into one single result of each variable for a single sample.

Statistical analysis. Categorical data were compared by the {chi}2 or Fishers' exact probability test. Continuous data were compared with Student's t test if the sample distribution was normal or with Mann-Whitney U test if the sample distribution was asymmetrical. The relationship between lymph vessel variables and lymph node status was analyzed by one-way ANOVA, followed by the Neuman-Keuls test. Overall survival analyses were done using the Kaplan-Meier method. Overall survival intervals were determined as the time period from initial diagnosis to the time of death. The comparison between survival functions for different strata was assessed with the log-rank statistic. Multivariate analysis of prognostic factors was done using Cox's regression model. Differences were considered significant when P < 0.05. All statistical analyses were done using Statistica 6.0 (StatSoft Inc. Tulsa, OK) software program.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Identification of lymphatic vessels in normal human lung tissue: Lyve-1 and D2-40/podoplanin antibody specificity. In the normal lung parenchyma, LYVE-1 (a lymph-specific receptor for hyaluronan; ref. 13) and D2-40 (specific for the lymphatic marker podoplanin; ref. 14) revealed a similar pattern. Positive staining was seen in thin-walled lymphatic vessels devoid of RBCs (Fig. 1A). These vessels were associated mainly with arterioles and venules. The distinction between blood and lymphatic vessels was further corroborated by double immunostaining for LYVE-1 or D2-40 and the panendothelial marker CD31. LYVE-1– and D2-40-positive lymphatic vessels were weakly CD31-positive. On the contrary, erythrocyte-containing CD31-positive blood vessels were LYVE-1- and D2-40-negative (Fig. 1A).



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  		Fig. 1. Lymphatic capillaries of normal lung parenchyma and NSCLC tissue. A, immunostaining of lymphatic (LYVE-1; brown, arrowhead) and erythrocyte-containing blood vessel (CD31; pink, arrow) capillaries in normal lung tissue. The LYVE-1-positive lymphatic vessel is weakly CD31-positive. The blood vessel is negative for LYVE-1. B, double immunostaining of NSCLC tumor periphery for LYVE-1 and D2-40. LYVE-1 (brown) seemed to stain only a subset of lymphatics when compared with D2-40 (pink). From the three lymphatic capillaries visible (arrows) only one (asterisk) is positive for LYVE-1. C, positive staining for Ki67 (pink) is seen in the nuclei of tumor cells as well as of lymphatic capillary (brown) endothelial cells (arrow). Magnification, x200 (A) and x400 (B, and D).

 
Characterization of lymph vessels in human non–small cell lung cancer tissue. To characterize lymphatic capillaries in NSCLC, we carried out immunostaining with antibodies to LYVE-1 and D2-40. With both antibodies, positive lymph vessels were always seen in the peritumoral lung and at the tumor periphery (Fig. 1B). With respect to LYVE-1 immunoreactivity, central tumor areas seemed devoid of immunoreactive vessels, except for nonangiogenic tumors growing without host tissue destruction, where LYVE-1-expressing vessels were preferentially associated with co-opted intratumoral blood vessels (Fig. 2A and B). These incorporated lung lymphatics were positive for D2-40 as well. With respect to D2-40/podoplanin immunoreactivity, examination of the central tumor areas in destructively growing angiogenic tumors revealed that a minority of lymphatic vessels were in fact dispersed between the tumor cells in close contact with them. In these angiogenic tumors, D2-40+ lymphatics were randomly distributed within the tumor mass.



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  		Fig. 2. Distribution of lymphatic capillaries and examples of immunostaining for VEGF-C in human NSCLC according to angiogenic phenotype. A, in destructively growing angiogenic NSCLCs showing robust angiogenesis and high numbers of intratumoral blood vessels (CD31; red fluorescence), lymphatics were randomly distributed within the periphery of the tumor mass (LYVE-1; green fluorescence). B, in nonangiogenic NSCLCs, tumor cells filled the alveoli without the destruction of the alveolar walls, containing the incorporated blood (CD31; red fluorescence) and lymphatic (LYVE-1; green fluorescence) host tissue capillaries. C, diffuse VEGF-C expression (red fluorescence) of cancer cells in a destructively growing angiogenic tumor. D, focal VEGF-C expression (red fluorescence) of tumor cells in a nonangiogenic tumor. Arrows, (C and D) point at lymph vessels (D2-40; green fluorescence) with VEGF-C expressing endothelial cells within the tumors. Nuclear staining (A-D) was applied with TOTO-3 iodide (blue fluorescence). Magnification, x200 (A-D).

 
Lymphatic vessel density and perimeter in human non-small cell lung cancer. Because LVDs and lymph vessel perimeters of N1 and N2 tumor groups did not differ from each other significantly (P > 0.1 for all analyses, data not shown), node-positive and node-negative groups were established. Based on both LYVE-1 and D2-40 staining, in all the applied categories, LVD was highest in the peritumoral host tissue and the tumor periphery was more vascular than the tumor center (Fig. 3A). Although evaluation of the LVDs by LYVE-1 or D2-40 reactivity in the peritumoral host tissue and in the tumor center of NSCLCs revealed only a slight association with the presence of lymph node metastases (P > 0.05), assessment of tumor periphery LVDs indicated a significant increase in the cases of both LYVE-1 and D2-40 in the lymph node–metastatic compared with the –nonmetastatic tumors (from 4.8 to 9.9/mm2 in case of LYVE-1 and from 5.8 to 11.3/mm2 in case of D2-40; P <0.005; Fig. 3A).



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  		Fig. 3. Morphometry of tumor-associated intra- and peritumoral lymphatic vessels based on both LYVE-1 and D2-40 immunostaining in nonmetastatic (N0) versus lymph node metastatic (N+) NSCLCs. LVDs are mean lymph vessel counts per square millimeter. Perimeters are expressed in microns. Data are mean ± SE. A, LVD scores of the entire patient population; *, P < 0.005 (versus nonmetastatic groups), one-way ANOVA, followed by Neuman-Keuls multiple comparison test; n = 103. B, LVD scores of nonangiogenic tumors; n = 19. C, LVD scores of angiogenic tumors; *, P < 0.005 (versus nonmetastatic groups), one-way ANOVA, followed by Neuman-Keuls multiple comparison test; n = 84. D, lymph vessel perimeters of the entire patient population; n = 103.

 
According to previous observers (7, 15), based on CD31 immunoreactivity, NSCLCs were separated into angiogenic (84 cases; Fig. 2A) and nonangiogenic (19 cases; Fig. 2B) tumors. Despite the finding that out of the 19 patients who had nonangiogenic tumor, 13 had lymph node metastasis (68% versus 33.3% in the angiogenic tumor group; P = 0.0048), and that patients with node-positive nonangiogenic cancer tended to have high tumor periphery LVD more often than those with node-negative disease, in the group of nonangiogenic tumors, there was no statistically significant information from the LVD scores in any investigated part of the tumors (P > 0.05 for all analyses; Fig. 3B).

In the group of angiogenic tumors, tumor periphery LVD evaluated by both LYVE-1 and D2-40 immunostaining was significantly increased in lymph node metastatic NSCLCs as compared with nonmetastatic NSCLCs (from 4.5 to 10.8/mm2 in the case of LYVE-1 and from 5.6 to 11.9/mm2 in the case of D2-40; P < 0.005; Fig. 3C). Although quantification of LVDs by both the LYVE-1 and the D2-40 antibodies revealed a correlation between peritumoral LVDs and lymph node metastases in angiogenic tumors, this tendency proved to be statistically nonsignificant (P > 0.05; Fig. 3C). No correlation was present between tumor center LVD and N status in the angiogenic tumor group in the case of either LYVE-1 or D2-40 (Fig. 3C).

Lymphatic vessel perimeters were lowest in the tumor center, and there was no difference in this respect between the various categories (Fig. 3D). However, there was no significant association between the lymphatic perimeters in any portion of the tumor and clinicopathologic factors. Finally, there was no significant association between lymph vessel densities or perimeters and sex, histology, T- and pathologic stage, grade of differentiation, or necrosis (P > 0.1 for all analyses; data not shown).

Patient survival. Because lymph node metastatic NSCLCs were characterized by a significant increase in tumor periphery LVD, we next used Kaplan-Meier analysis to calculate the overall survival rate for patients with low and high tumor periphery LVD (Fig. 4). This classification was based on the median values of LVDs in our patient population. We found that a high level of lymphangiogenesis in the peripheral portions of the tumors was a significant prognostic factor for reduced overall survival. The 5-year survival rates of patients with high LYVE-1-LVD and patients with low LYVE-1-LVD were 23.3% and 68.7%, respectively (P < 0.001, log-rank test; Fig. 4A). The 5-year survival rates of patients with high versus low D2-40-LVD were 30% and 67.2%, respectively (P < 0.001, log-rank test; Fig. 4A). However, if we classified the tumors according to their angiogenic phenotype, this tendency proved to be significant only in the angiogenic tumor group (P < 0.001 in case of both Lyve-1 and D2-40, log-rank test; Fig. 4B), and there was no statistically significant information from the LVDs of nonangiogenic tumors (P values in case of Lyve-1 and D2-40 were 0.59 and 0.98, respectively, log-rank test; Fig. 4C).



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  		Fig. 4. A, Kaplan-Meier curves for the overall survival of the entire patient population with NSCLC, according to tumor periphery LVDs as determined with LYVE-1 and D2-40 staining. B, Kaplan-Meier curves for the overall survival of the patient population with angiogenic NSCLC, according to tumor periphery LVDs as determined with LYVE-1 and D2-40 staining. C, Kaplan-Meier curves for the overall survival of the patient population with nonangiogenic NSCLC, according to tumor periphery LVDs as determined with LYVE-1 and D2-40 staining. D, Kaplan-Meier curves for the overall survival of patients with NSCLC, according to the angiogenic phenotype.

 
As nonangiogenic tumor phenotype was found to be related to the risk of lymph node metastasis, we also analyzed the overall survival of our patient population according to the angiogenic phenotype of their tumor, and found that patients with nonangiogenic tumors have a significantly poorer survival than those with angiogenic tumors. The 5-year survival rates of patients with nonangiogenic versus angiogenic tumors were 26.3% and 54.8%, respectively (P = 0.032, log-rank test; Fig. 4D).

Although we failed to show prognostic information from the level of lymphangiogenesis (LVD scores) in nonangiogenic tumors, analysis of the above data by Cox's regression model confirmed that nonangiogenic phenotype is an adverse prognostic factor for overall survival (P = 0.044; Table 3). Multivariate analysis (including standard prognostic variables such as tumor extension, lymph node status, and patient age) also showed that tumor periphery LVDs predicted outcome independent of other variables (Lyve-1, P = 0.007; D2-40, P = 0.008; Table 3). Further prognostic factors related to poor survival were tumor stage and lymph node metastasis. In case of patients with angiogenic tumors, a high level of lymphangiogenesis in the peripheral portions of the tumors proved to be an independent unfavorable prognostic factor as well (Lyve-1, P < 0.001; D2-40, P = 0.002; Table 3).


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  		Table 3. Multivariate analysis of various prognostic factors in patients with NSCLC

 
Lymphatic proliferation. To explore the proliferation status of the lymphatic network in NSCLC, we carried out double immunostaining with antibodies against D2-40/podoplanin and the proliferation-associated Ki67 nuclear protein (Fig. 1C). The labeling index of lymphatic endothelial cells within the intra- and peritumoral and normal lymphatic vessel populations was determined by counting the labeled as well as all the endothelial cell nuclei (100/studied area) along the lymphatic capillary tubes. The results corroborated Ki67 nuclear staining in lymph vessel endothelial cells and, as anticipated, in the cancer cells themselves (Fig. 1C). The endothelial cell labeling index of lymphatic vessels was always the highest peritumorally and was always lower in the tumor center than in the periphery (Table 2). The most extreme situation was that of the nonangiogenic tumor group, where a moderate number of dividing lymphatic endothelial cell nuclei were observed peritumorally, but no Ki67 staining was observed intratumorally (Table 2). This evidence suggests that the intratumoral lymphatics in nonangiogenic human NSCLCs are indeed preexisting lymphatics that have simply been co-opted and entrapped by the growing tumor mass. In contrast, assessment of the proliferation status of lymphatic endothelial cells of tumors exhibiting an angiogenic growth pattern has shown that lymphatic endothelium can actively divide in all of the investigated areas (Table 2).


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  		Table 2. Comparison of lymphatic endothelial cell labeling indexes of the human NSCLC according to the angiogenic phenotype, based on Ki-67 and D2-40 double labeling of lymphatic capillaries

 
Vascular endothelial growth factor-C expression in human non–small cell lung cancer. Of 103 cases with NSCLC, 56 cases were positive for VEGF-C. No staining was observed in the normal lung tissue. The staining pattern was either diffuse (mainly in angiogenic growth type; Fig. 2C) or focal (mainly in nonangiogenic growth type; Fig. 2D). VEGFC was significantly more frequently expressed in angiogenic carcinomas when compared with nonangiogenic tumors (P = 0.0065; Table 1). However, no significant associations with age, gender, histologic type, tumor differentiation, tumor (T) status, lymph node (N) status, smoking history, or disease stage were detected (Table 1). Furthermore, there was no significant correlation between the intensity of VEGF-C expression and the lymphatic endothelial cell labeling index or LVDs as evaluated by podoplanin and LYVE-1 immunostaining (P > 0.05 for all analyses listed above, data not shown). Accordingly, we were unable to detect the prognostic role of VEGF-C expression in a multivariate analysis (Table 3).


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
NSCLC generally gives rise to lymph node metastases, and the degree of locoregional lymph node involvement is one of the most important prognostic factors in this tumor type. However, our knowledge of the lymphatic system of lung tumors lags far behind that of the vascular system, due largely to the lack of specific markers for the lymphatic endothelium in human cancers. The recent discovery of the lymphatic endothelial hyaluronan receptor-1 (LYVE-1; ref. 16) and the antibody directed against the M2A antigen (the monoclonal mouse anti-D2-40, which was recently proven to be specific for human podoplanin; ref. 14) as specific markers for normal and tumor-associated lymphatics have now provided tools for a detailed analysis of lymphangiogenesis in human NSCLC.

Investigating the distribution of lymphatic capillaries in human NSCLC using antibodies against the above antigens, we found a decrease in LVD as we moved from peritumoral lung tissue towards the tumor center, regardless of tumor size or histologic type. This in part accords with the findings in a previous paper, in which the authors failed to detect LYVE-1+ lymphatics within the main bulk of NSCLC tissue (17). It is important to note, however, that in contrast with the observation of Koukourakis et al. (17), who found lymphatics only at the tumor periphery and in the peritumoral lung, our measurement did not yield such an extreme result. In the normal parenchyma, LYVE-1 and podoplanin showed a similar staining pattern. Although we occasionally found them in association with respiratory bronchioles, the peribronchial lymphatics typically started at the level of the terminal bronchioles, whereas lymphatics in the alveolar walls were never observed. Inspection of NSCLC tissue revealed that, in accordance with findings in a report by others on human pancreatic endocrine tumors (18), LYVE-1 seemed to stain only a subset of lymphatics when compared with podoplanin. Surprisingly, the categorization of NSCLCs according to their angiogenic phenotype (7) indicated that this feature was more apparent in destructively growing angiogenic tumors in which LYVE-1+ lymphatics were detected exclusively at the tumor periphery and in the peritumoral host tissue, whereas podoplanin-positive lymphatics could be identified in the central tumor areas in addition to the tumor margin. In contrast, a significant proportion of nonangiogenic NSCLCs contained lymphatics positive for LYVE-1 (and also for podoplanin) both in the tumor center and at the tumor periphery. The reasons for the discrepancy between LYVE-1 and podoplanin staining in the main bulk of angiogenic NSCLCs are not fully understood. The simple collapse of lymphatics can hardly explain the selective loss of LYVE-1, because even collapsed lymphatic capillaries would be expected to retain the expression of LYVE-1 in the endothelium. Because the central areas of angiogenic tumors showed significantly lower LVD scores (based on both LYVE-1 and podoplanin staining) as compared with the tumor periphery and the peritumoral host tissue, it is possible that the relatively higher expression of podoplanin in these tumors is the result of completely destructive growth, during which the loss of hyaluronan receptors occurs earlier in the degenerative walls of the co-opted host lymphatics than the loss of the mucin-type glycoprotein podoplanin. Alternatively, because intratumoral lymphatic endothelial cell proliferation was seen exclusively in LYVE-1-negative/podoplanin-positive lymphatics of angiogenic tumors, the newly formed lymph vessels may express podoplanin earlier than LYVE-1. Furthermore, the observation that nonangiogenic tumors exhibited lymphangiogenesis only peritumorally, taken together with the finding that the arrangement of intratumoral lymph vessels were similar to those in the normal lung tissue, allows us to conclude that these tumors grow by engulfing and exploiting the lymph vessels present in the normal lung parenchyma.

A variety of lymphatic markers evaluated from the primary tumor have shown some association with the onset of lymphatic metastases in human gastric (19), breast (20), and head and neck squamous cell carcinomas (21), and in melanoma (22). However, only two recent studies have addressed the presence of lymphangiogenesis in human NSCLC (17, 23), and no data have been available on the influence of lymph vessel density on lymphatic metastasis capacity and prognosis. In this study, we present the novel finding that although in both N0 and N+ categories, the peritumoral lung contained more lymph vessels than the investigated tumor areas, morphometry showed a significant increase in tumor periphery (but not tumor center) LVD in the case of lymph node metastatic tumors. Furthermore, it becomes clear that lymph vessels may be present in the central areas of NSCLC, but the density of lymph vessels at the tumor periphery may have a far greater clinical significance. High peripheral lymph vessel counts were associated not only with a high number of metastatic lymph nodes at the time of the diagnosis but with a poor overall survival. However, this tendency proved to be significant only in the angiogenic tumor group, suggesting that in the main bulk of angiogenic NSCLCs, the mechanical forces induced by the growing tumor mass and the constantly invading cancer cells compress and destroy lymphatic capillaries, rendering them nonfunctional, and furthermore, that in angiogenic NSCLC, the site of lymphatic invasion does not necessarily correspond to the area of the highest LVD, especially when the "hotspot" is located outside the tumor mass. In conclusion, the lymph vessels of the tumor periphery are more important in the metastatic process than the peritumoral lymph vessel network (corresponding to the "lymphatic hotspots") or the demolished and therefore nonfunctional lymphatics of the tumor center in angiogenic NSCLCs.

Although nonangiogenic growth type was associated with a significant risk for the development of lymph node metastasis and to a shorter survival in patients with NSCLC, we failed to show prognostic information from the LVD scores in any investigated part of these tumors. The putative nonangiogenic tumors are highly aggressive (7, 24), and co-opted lung lymphatics could still play a key role in addition to preexisting blood capillaries. The normal lymphatics incorporated into the tumor could be more effective than the newly formed vessels, because in nonangiogenic NSCLCs, lymph drains directly into the incorporated host lymphatic network, whereas in angiogenic cancers, the lymph is drained into the lymphatics of the surrounding normal tissue in peripheral areas only. In that way, lymphatic dissemination of cancer cells may occur via the preexisting lung lymphatics within the entire mass of nonangiogenic cancers.

Although in various malignant tumors, including NSCLC, VEGF-C expression has been reported to be significantly correlated with lymph node metastasis (25–30), and we found elevated VEGF-C immunoreactivity in NSCLC as compared with that in normal lung tissue, there was no obvious association between expression of VEGF-C and LVDs, lymph node metastasis, or patients' survival [in accordance with previous results from Arinaga et al. (31) and Ogawa et al. (32)]. Moreover, we failed to detect an association between VEGF-C expression and the lymphatic endothelial cell labeling index. It is difficult to conclude, therefore, that lymphangiogenesis in human NSCLC is a result of VEGF-C action, as recent studies using experimental tumor models have found (33, 34). Our results rather suggest that VEGF-C most probably acts as a survival factor in NSCLC. This idea was further corroborated by the observation that in nonangiogenic tumors with no evidence of intratumoral lymph vessel sprouting, the expression of VEGF-C was restricted to the direct vicinity of co-opted host lymphatics. However, chances are that as in tumor-induced angiogenesis, where the interaction of multiple cytokines controls tumor vascularization, the dynamic balance of several lymphangiogenic growth factors is also likely to determine the activity of lymphangiogenesis.

In conclusion, NSCLC metastasis to lymph nodes is a key event in disease outcome, and is frequently used as a prognostic factor. Our study shows, for the first time, that lymphangiogenesis occurs exclusively in the angiogenic growth type of human NSCLCs, and that LVD is correlated to clinical behavior and to lymph node status only in this growth type of NSCLCs. However, it also provides the first evidence that the risk of lymph node metastasis as well as a shorter survival was more likely to occur in the patient population with nonangiogenic tumors, and that these tumors mainly co-opt host tissue lymphatics during their growth, in contrast to most of the angiogenic ones, which expand with concomitant lymphangiogenesis. The latter suggests that the co-opted host lymphatics of human NSCLC are more important in the metastatic process than the newly formed ones. This assumption, however, would need further experimental and clinical support.


   Acknowledgments
 
We thank Anna Tamasi and Piroska Horvath, for the excellent technical assistance, and Andrea Ladanyi for critical review of the manuscript.


   Footnotes
 
Grant support: Ministry of Education, Hungary (NKFP-1/48/2001) and National Research Foundation (OTKA-D048519, OTKA-F046501, OTKA-F60158, and OTKA-TS49887).

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.

Note: F. Renyi-Vamos and J. Tovari contributed equally to this work.

Received 5/15/05; revised 7/12/05; accepted 7/28/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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