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From the Cutaneous Biology Research Center and Department of Dermatology,* Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts; the Departments of Pathology and Biostatistics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; the Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; the Institute of Molecular Medicine,¶ John Radcliffe Hospital, Headington, Oxford, United Kingdom; and the Department of Dermatology,|| University of Tübingen Medical School, Tübingen, Germany
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Abstract |
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Malignant melanomas metastasize via blood and lymphatic vessels, and the induction of tumor angiogenesis provides a possible explanation for how tumor cells escape their original site by invading the newly formed vascular bed.10 Tumor vascularization has been observed in human melanoma both experimentally11,12 and clinically.13 The importance of tumor angiogenesis for the prognosis of primary malignant melanomas of the skin, however, has remained controversial.14 Whereas several studies found an inverse correlation of tumor microvessel density with disease-free and overall survival,15,16 other reports did not detect any significant differences of tumor microvessel density between metastasizing and nonmetastasizing melanomas.17 Therefore, the potential prognostic value of tumor vascularization in malignant melanoma remains at present unclear.
Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, even at the early stages of tumor invasion. Hence lymph node metastasis, as determined by the analysis of sentinel lymph nodes, is a major determinant for the staging and clinical management of melanoma.5,18 In contrast to the extensive studies on melanoma-associated angiogenesis, little is known about the mechanisms by which melanoma cells gain entry into the lymphatic system. Dilated tumor-associated lymphatic vessels, sometimes containing tumor cells, have been observed by routine histology and by electron microscopy in cutaneous melanoma.19,20 However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear, and the very existence of melanoma-associated lymphangiogenesis has even been questioned.21 Recent experimental evidence strongly suggests that tumors can actively induce lymphangiogenesis via production of lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-C and VEGF-D, and that the extent of tumor lymphangiogenesis is directly correlated with the extent of experimental metastatic tumor spread to regional lymph nodes.22,23 We have previously reported that VEGF-C is expressed in several human melanoma cell lines in vitro;24 however, because of the lack of specific markers for the lymphatic endothelium in human cancers, the importance of tumor-associated lymphangiogenesis for melanoma progression has remained unclear. The recent discovery of the lymphatic endothelial hyaluronan receptor-1 (LYVE-1) as a specific marker for normal25 and tumor-associated lymphatic vessels26 has now provided the tool for a detailed analysis of tumor lymphangiogenesis in melanoma.
In the present study, we investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using tumor samples obtained from clinically and histologically matched primary cutaneous malignant melanomas from patients who developed early lymph node metastasis and from patients who remained metastasis-free. Our results reveal, for the first time, a higher incidence of intra- and peritumoral lymphangiogenesis in metastatic melanoma, as compared with nonmetastatic melanomas. Moreover, multivariate proportional hazards analysis identified peritumoral lymphatic vascular density as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.
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Materials and Methods |
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Patients were identified retrospectively through review of survival data from the German Melanoma Registry. Of a total of 1050 patients with primary cutaneous malignant melanoma, 19 patients with nonmetastatic primary melanoma (mean disease-free follow-up of 6.5 years; range, 34 to 119 months) were closely matched with a group of 18 patients who had documented early (<1 year) lymph node metastasis (Table 1)
. The two groups were matched for gender, age at diagnosis, tumor thickness, Clark’s level of invasion, histological type, and presence of ulceration. Exclusion criteria were acrolentiginous melanoma and adjuvant therapy after the surgical excision of the primary tumor. The diagnosis of melanoma, the tumor thickness, and the level of tumor invasion were reconfirmed by two pathologists (SSD and MCM). Additional parameters such as the frequency of mitoses, tumor regression, vascular invasion, and microsatellites were also evaluated. Peritumoral inflammation was evaluated as absent, present (nonbrisk), and brisk.27
The degree of inflammation was further graded as low (
50% of tumor area) or high (>50% of tumor area).
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Paraffin sections (6 µm thickness) were dewaxed, hydrated, and treated with 0.01% protease XXIV (Sigma, St. Louis, MO) in phosphate-buffered saline for 20 minutes at 37°C. Sections were double-stained using a rabbit polyclonal antibody against human LYVE-1 (1:600)25
and a mouse monoclonal anti-human CD31 antibody (1:40; DAKO, Carpinteria, CA), followed by incubation with the respective secondary antibodies that were labeled with either Texas Red (1:50) or with fluorescein isothiocyanate (1:50) (Jackson ImmunoResearch, West Grove, PA) as previously described.24
Cell nuclei were counterstained with Hoechst bisbenzimide (Sigma) at 20 µg/ml. Additional immunohistochemical stains were performed using affinity-purified rabbit polyclonal antibodies against human VEGF-C (C-terminus; Zymed, San Francisco, CA) or against human LYVE-1, followed by incubation with conjugated rabbit anti-human immunoglobulin (1:200), using the 3-amino-9-ethylcabazole peroxidase substrate kit (Vector Laboratories, Burlingame, CA). To detect proliferating cells, the Zymed proliferating cell nuclear antigen (PCNA) 3,3'-diaminobenzidine staining kit was used as previously described.28
For specificity controls, either the secondary antibody was omitted or the primary anti-VEGF-C antibody was preincubated with a 40-fold molar excess of recombinant human VEGF-C (a generous gift from Dr. K. Alitalo, University of Helsinki, Finland). In situ hybridization was performed as previously described,29
using a riboprobe for human VEGF that detects all known VEGF splice variants29
or a 808-bp human VEGF-C riboprobe described previously.30
Transcription reactions were performed using the Riboprobe Gemini II kit (Promega, Madison, WI) in the presence of [
-35S]UTP. For autoradiography, slides were coated with NTB2 film emulsion and exposed for 4 weeks. Sections were examined using a Nikon E-600 microscope (Nikon, Melville, NY) and digital images were captured using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Computer-Assisted Morphometric Vessel Analysis
To analyze the lymphatic and blood vessel density and size within and surrounding the 37 primary melanomas, we performed double-immunofluorescence stains for CD31 and LYVE-1. Sections were examined using a Nikon E-600 microscope and digital images were captured using a SPOT digital camera. For each tumor section, three fields with the highest lymphatic vascular density (hot spots) were evaluated at x100 magnification. Digital images of tumor-associated lymphatic vessels and blood vessels were captured in the same field. Peritumoral lymphatic vessels were defined as LYVE-1-positive vessels within an area of 100 µm from the tumor border. Intratumoral lymphatic vessels were defined as LYVE-1-positive vessels located within the tumor mass and not confined by invagination of normal tissue. Tumor borders were determined on serial sections using Hoechst nuclear stains and hematoxylin and eosin stains. Morphometric analyses of lymphatic vessels and of blood vessels were performed using the IP-Lab software (Scanalytics, Fairfax, VA) to determine the vessel number per mm2, the average vessel size, and the relative tumor area occupied by vessels as described.31 The relative lymphatic vascular area was determined in the peritumoral area and was calculated as the area covered by lymphatic vessels divided by the total area examined times 100, expressed in percent.
Statistical Analyses
The unpaired Student’s t-test was used to determine the statistical significance (P value) of the mean for all vascular parameters. The chi-square square test was used to evaluate differences in the frequency of intratumoral lymphatic vessels. Disease-free survival and overall survival intervals were determined as the time period from initial diagnosis to the time of first metastasis or the time of death. Patients with no events (ie, no lymph node metastasis) were censored and the disease-free interval for these patients was the same as their overall follow-up time. Disease-free survival and overall survival analyses were performed using the Kaplan-Meier method. The comparison between survival functions for different strata was assessed with the log-rank statistic. Univariate and multivariate analyses of prognostic factors were based on the Cox proportional hazards model with model selection based on the backwards elimination process.
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Results |
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We closely matched two cohorts of patients with metastatic (n = 18) or with nonmetastatic (n = 19) primary cutaneous malignant melanoma for age, gender, tumor type, thickness, invasion level, and presence of ulceration (Table 1)
. Histological analysis of hematoxylin and eosin-stained paraffin sections revealed that additional prognostic parameters such as mitotic activity, peritumoral inflammation, or regression also showed comparable distribution in both groups (Table 1)
. Next, melanoma-associated lymphatic and blood vessels were simultaneously visualized in all primary melanomas, using immunofluorescence double stains for the lymphatic vessel marker LYVE-1 and for the panvascular marker CD31. Whereas CD31-positive/LYVE-1-negative blood vessels were homogeneously distributed throughout the tumors, CD31-positive/LYVE-1-positive lymphatic vessels were found in prominent hotspots both within and around primary cutaneous melanomas (Figure 1
; A to F). Such hotspots were seen in thick melanomas (Figure 1
; A to C) as well as in thin melanomas (Figure 1
; D to F). Foci of intratumoral lymphatic vessels were preferentially localized near the tumor border in metastatic melanomas (Figures 1E and 2A)
. In all cases, peritumoral lymphatic vessels with frequently open lumina were found within a distance of 100 µm from the tumor border (Figures 1E and 2A)
. Intratumoral lymphatic vessels frequently exhibited a thin-walled, basket-like morphology (Figure 2B)
resembling that seen in blood vessel networks during VEGF-induced angiogenesis.32
Importantly, intratumoral lymphatics were found more frequently in metastatic melanomas (77.8% of all cases) than in nonmetastatic melanomas (36.8%, P = 0.01, chi-square test; Figure 2D
). Pigmented tumor cells within LYVE-1-positive intratumoral lymphatics were found in 2 of 18 (11%) metastatic melanomas (Figure 2, E and F)
, whereas no intralymphatic tumor cells were detected in nonmetastatic melanomas. Differential immunostains for LYVE-1 and for PCNA revealed PCNA-positive nuclei in the majority of melanoma cells and in several LYVE-1-positive lymphatic endothelial cells (Figure 2C)
, confirming the occurrence of active intratumoral lymphangiogenesis in primary human melanomas.
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Metastatic melanomas exhibited more hotspots of intratumoral and peritumoral LYVE-1-positive lymphatic vessels than nonmetastatic tumors (Figure 1B)
, whereas the extent of blood vascularization was uniform throughout both metastatic and nonmetastatic tumors. The density of both lymphatic and blood vessels was higher in melanomas than in neighboring normal human skin (data not shown). Computer-assisted morphometric analysis confirmed a comparable blood vascular density, average blood vessel size, and tumor area covered by blood vessels in metastatic and nonmetastatic melanomas (Figure 3
; A to C). In contrast, the number of peritumoral lymphatic vessels was significantly increased in metastatic melanomas (16.5 ± 1.62 vessels/mm2) as compared with nonmetastatic melanomas (9.1 ± 0.76 vessels/mm2, P = 0.0002; Figure 3D
). Moreover, the average lymphatic vessel size was significantly larger in metastatic melanomas (1773.0 ± 280.9 µm2) than in nonmetastatic melanomas (908.6 ± 86.2 µm2, P = 0.0048; Figure 3E
). The relative peritumoral area covered by lymphatic vessels was threefold higher in metastatic melanomas (2.25 ± 0.27%) than in nonmetastatic melanomas (0.75 ± 0.07%, P < 0.0001; Figure 3F
).
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Because VEGF-C expression has been recently linked to tumor lymphangiogenesis in experimental models, we next analyzed VEGF-C protein expression in all 37 primary melanomas by in situ hybridization and by immunohistochemistry. By in situ hybridization, we detected focal, low-level tumor cell expression of VEGF-C mRNA (Figure 4
; A to C) in 44% of the metastatic melanomas and in 44% of the nonmetastatic melanomas. VEGF-C mRNA expression was frequently also detected in epidermal keratinocytes overlying the tumors and in peritumoral stromal cells (Figure 4D)
. Moreover, mRNA expression of the angiogenesis factor VEGF was detected in 33.3% of the metastatic and in 30% of the nonmetastatic melanomas (data not shown). Focal cytoplasmic VEGF-C protein expression (Figure 5, A and B)
was found more frequently in tumor cells of metastatic melanomas (50.0%) than in nonmetastatic melanomas (31.6%); however, these differences did not reach statistically significant levels (P > 0.05). VEGF-C expression was also detected in peritumoral dermal fibroblasts near the invasive edge of metastatic (55.6%) (Figure 5C)
and of nonmetastatic melanomas (31.6%, P = 0.14).
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We next studied whether the degree of lymphangiogenesis in metastatic melanomas was correlated with the degree of peritumoral inflammation, as assessed by routine histology. We found that the lymphatic vascular area was significantly increased (P = 0.005) in melanomas with high-grade nonbrisk inflammation (3.15 ± 0.10%), as compared with melanomas with low-grade inflammation (1.18 ± 0.10%). LYVE-1-positive macrophages near dilated, LYVE-1-positive lymphatic vessels were frequently found in metastatic melanomas with high-grade nonbrisk inflammation (Figure 5E)
. VEGF-C-expressing inflammatory cells were detected in 3 of 18 (16.7%) metastatic melanomas, but not in nonmetastatic tumors. The peritumoral mononuclear inflammatory cells, mostly located near the invasive tumor edge, expressed VEGF-C in a cytoplasmic granular pattern (Figure 5F)
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Lymphangiogenesis Is a Novel Prognostic Parameter for Melanoma Metastasis and Survival
Because metastatic melanomas were characterized by a significant increase of lymphatic vessel density and lymphatic vessel size, we next investigated whether the degree of tumor lymphangiogenesis might serve as a new prognostic parameter for the risk of melanoma metastasis. A univariate proportional hazard analysis revealed that the presence of intratumoral lymphatic vessels and a higher level of peritumoral lymphatic vascular area were significantly associated with a more rapid development of lymph node metastasis (Table 2)
. A multivariate proportional hazard analysis, using backward elimination of all other variables, revealed that only the level of peritumoral lymphatic area was an independent predictor of the time to lymph node metastasis. An additional univariate proportional hazard analysis for the effect of different risk factors on overall survival identified the presence of intratumoral lymphatics and the extent of peritumoral lymphatic vascularization as the only two parameters that were significantly associated with reduced overall survival (Table 3)
. Because both groups of metastatic and nonmetastatic melanomas were exactly matched for tumor thickness, no prognostic significance could be assigned to this parameter by the hazard analyses.
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1.0%), medium (1.0% to 1.5%), or high (>1.5%) peritumoral lymphatic vascular area (Figure 6
; B to D). This classification was based on the distribution of lymphatic vascular areas in our patient population (Figure 6A)
. We found that a high level of tumor lymphangiogenesis was a significant prognostic factor for reduced overall survival (Figure 6B
; P < 0.0001) and for reduced disease-free survival (Figure 6C
; P < 0.0001). The estimates for 3, 5, and 10 years for each stratum were calculated with pair-wise comparisons, which were all significant (P < 0.05) for both overall and disease-free survival. Importantly, the detection of intratumoral lymphatic vessels was significantly associated with a poorer disease-free survival, as compared with melanomas without intratumoral lymphatics (Figure 6D
; P = 0.0177).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In our present study, using double immunostains for CD31 and LYVE-1, we detected both peri- and intratumoral lymphatic vessels in a large number of primary cutaneous melanomas. Although intratumoral lymphatics have been proposed to be nonfunctional in experimental mouse tumor models,39,40 univariate proportional hazard analysis revealed that the presence of intratumoral lymphatics was a significant risk factor for the development of lymph node metastasis in patients with cutaneous melanoma. Not only did intratumoral lymphatics occur at a significantly higher incidence in metastatic melanoma, but their presence was also related to a significantly increased risk of lymph node metastasis. These results suggest that intratumoral lymphatics play a clinically significant role as conduits for the metastasis of cutaneous melanoma, proposing an active role of lymphangiogenesis as evidenced by hot spots of lymphatic vessels within and surrounding metastatic melanomas. In contrast, a traditional view asserts that lymphatic endothelium plays a passive role during tumor metastasis and that lymphatic invasion only occurs via infiltrating tumor cells invading pre-existing lymphatic vessels.41 However, several studies in animal tumor models provide direct experimental evidence that increased tumor lymphangiogenesis promotes lymphatic tumor spread to regional lymph nodes.22,23,42-44 Taken together, the results obtained in animal tumor models and in human tumors26 support the recently proposed concept of an active lymphangiogenesis model for tumor metastasis.45
We have previously reported that the lymphangiogenic factor VEGF-C is expressed by several melanoma cell lines in vitro and that overexpression of recombinant VEGF-C in human melanoma cell lines promoted tumor lymphangiogenesis after xenotransplantation in nude mice.24 In the human melanomas studied in the present investigation, however, we only detected low-level and heterogeneous expression of VEGF-C by tumor cells that did not significantly correlate with the metastatic potential of the primary tumors. Similarly, only weak VEGF-C protein has been recently found in metastatic human squamous cell carcinomas of the head and neck,26 whereas other studies found a correlation between the occurrence of tumor metastasis and tumor expression of VEGF-C mRNA in a number of epithelial cancers.46 We also found detectable VEGF expression in only a minority of all melanomas examined, confirming previous studies reporting that VEGF was only expressed in some cases of primary melanomas.47,48 Taken together, these results indicate that tumor-derived VEGF and VEGF-C likely do not represent the major source of angiogenic or lymphangiogenic activity in cutaneous melanomas. Therefore, it is tempting to speculate that the relative low levels of VEGF-C expression by melanoma cells are complimented by nontumoral, stromal sources such as dermal fibroblasts and peritumoral macrophages, as demonstrated in our study. Moreover, recent evidence indicates that peritumoral inflammation and VEGF-C production by inflammatory cells might also contribute to the induction of lymphangiogenesis.24,49 Although we were unable to detect any expression of VEGF-D,50 another known lymphangiogenic factor, in the 37 cases studied (data not shown), lymphatic vessel growth might be stimulated by other, yet unknown growth factors. The recent establishment of specific human lymphatic endothelial cell cultures51-53 and the development of in vivo lymphangiogenesis assays54 now provide the experimental tools for future in vitro and in vivo studies for the identification and characterization of novel lymphangiogenesis factors that might include members of the fibroblast growth factor family.54
Surprisingly, we did not detect any significant differences in tumor angiogenesis between the metastatic and nonmetastatic matched cutaneous melanomas. The similarity in tumor angiogenesis may be because of the comparably weak levels of VEGF mRNA expression in both metastatic and nonmetastatic melanomas in our study that did not include cases with early organ metastasis. Moreover, both groups were exactly matched for tumor thickness, thereby excluding differences of local tumor progression that has been previously correlated with increased vascularity.13,16,55,56 A recently published prospective study in 417 cutaneous melanoma patients found that tumor vascularity, as assessed on routine histological stains, was the most important determinant of overall survival.20 In contrast, other investigators failed to detect any correlation between melanoma vascularization and prognosis. Using morphometric analysis of Ulex europaeus type I lectin-labeled sections obtained from 86 melanomas with no evidence of recurrence after a minimum follow-up period of 5 years and of 21 cases with locoregional recurrence and/or metastasis, one study found that tumor recurrence could not be predicted by any of the derived vascular parameters (vascular length, surface, and volume density) either independently or together with other histological and clinical features.57 In another study of 60 cases of metastasizing and nonmetastasizing cutaneous melanomas that were matched for tumor thickness, age, sex, and anatomical site, there was no significant difference in the number of microvessels or in the pattern of vascular microarchitecture between metastasizing and nonmetastasizing tumors.17 The reported increase of VEGF protein expression during the transition from horizontal growth to the vertical melanoma growth phase55,56 suggests that angiogenesis may be important for promoting primary tumor growth whereas it seems to play a minor role if any in promoting lymphatic melanoma metastasis.
In summary, our study demonstrates, for the first time, the presence of intra- and peritumoral lymphangiogenesis in primary human cutaneous melanomas. It also provides the first evidence that the extent of tumor lymphangiogenesis may be related to the risk of lymph node metastasis as well as patient survival, and that peritumoral lymphatic vascular density might serve as a novel prognostic indicator for the risk of lymph node metastasis of human cutaneous melanomas. The feasibility of CD31/LYVE-1 double immunostains on routine paraffin sections eliminates the need for fresh-frozen tumor samples and will greatly facilitate larger, prospective, multi-institutional clinical trials that are needed to further validate the prognostic value of tumor lymphangiogenesis for metastasis and patient survival of primary malignant melanomas of the skin and, potentially, of other human malignancies.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health/National Cancer Institute (grants CA69184, CA86410, and CA92644 to M. D., and pathology training grant 5T32CA09216 to S. S. D.), the Susan G. Komen Breast Cancer Foundation (to M. D.), the American Cancer Society (program project grant 99-23901 to M. D.), the Deutsche Forschungsgemeinschaft (to T. P), the Association for International Cancer Research (grants 99-250 and 00-311to D. G. J.), and the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement (to M. D.).
Accepted for publication March 7, 2003.
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