Tumor Lymphangiogenesis
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64328-3
ISSN1525-2191
AutoresSoheil S. Dadras, Thomas A. Paul, Jennifer Bertoncini, Lawrence F. Brown, Alona Muzikansky, David G. Jackson, Ulf Ellwanger, Claus Garbe, Martín C. Mihm, Michael Detmar,
Tópico(s)Dermatologic Treatments and Research
ResumoMalignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. 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 double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma. Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. 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 double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma. Malignant melanoma of the skin is a common and frequently lethal neoplasm with increasing worldwide incidence.1Landis SH Murray T Bolden S Wingo PA Cancer statistics, 1998.CA Cancer J Clin. 1998; 48: 6-29Crossref PubMed Scopus (2449) Google Scholar In 1998, 42,000 new cases of cutaneous malignant melanoma and 7300 related deaths were reported in the United States.1Landis SH Murray T Bolden S Wingo PA Cancer statistics, 1998.CA Cancer J Clin. 1998; 48: 6-29Crossref PubMed Scopus (2449) Google Scholar Complete surgical excision with wide margins represents the therapy of choice for primary melanomas. The vertical thickness2Breslow A Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma.Ann Surg. 1970; 172: 902-908Crossref PubMed Scopus (1881) Google Scholar and the anatomical level of invasion (Clark level)3Clark Jr, WH From L Bernardino EA Mihm MC The histogenesis and biologic behavior of primary human malignant melanomas of the skin.Cancer Res. 1969; 29: 705-727PubMed Google Scholar of the primary tumor are the most valuable prognostic indicators for the metastatic risk of cutaneous melanoma.4Balch CM Soong SJ Milton GW Shaw HM McGovern VJ Murad TM McCarthy WH Maddox WA A comparison of prognostic factors and surgical results in 1,786 patients with localized (stage I) melanoma treated in Alabama, USA, and New South Wales, Australia.Ann Surg. 1982; 196: 677-684Crossref PubMed Scopus (303) Google Scholar, 5Balch CM Buzaid AC Soong SJ Atkins MB Cascinelli N Coit DG Fleming ID Gershenwald JE Houghton Jr, A Kirkwood JM McMasters KM Mihm MF Morton DL Reintgen DS Ross MI Sober A Thompson JA Thompson JF Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma.J Clin Oncol. 2001; 19: 3635-3648Crossref PubMed Scopus (2247) Google Scholar, 6Barnhill RL Fine JA Roush GC Berwick M Predicting five-year outcome for patients with cutaneous melanoma in a population-based study.Cancer. 1996; 78: 427-432Crossref PubMed Scopus (246) Google Scholar, 7Vollmer RT Seigler HF Using a continuous transformation of the Breslow thickness for prognosis in cutaneous melanoma.Am J Clin Pathol. 2001; 115: 205-212Crossref PubMed Scopus (17) Google Scholar However, it is still difficult to predict the outcome after excision of the primary tumor, in particular in thin melanomas.8Blessing K McLaren KM McLean A Davidson P Thin malignant melanomas (less than 1.5 mm) with metastasis: a histological study and survival analysis.Histopathology. 1990; 17: 389-395Crossref PubMed Scopus (71) Google Scholar, 9Guitart J Lowe L Piepkorn M Prieto VG Rabkin MS Ronan SG Shea CR Tron VA White W Barnhill RL Histological characteristics of metastasizing thin melanomas: a case-control study of 43 cases.Arch Dermatol. 2002; 138: 603-608Crossref PubMed Google Scholar Hence novel indicators for the prognostic risk of metastatic melanoma spread are urgently needed. 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.10Weidner N Semple JP Welch WR Folkman J Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma.N Engl J Med. 1991; 324: 1-8Crossref PubMed Scopus (5298) Google Scholar Tumor vascularization has been observed in human melanoma both experimentally11Warren BA Shubik P The growth of the blood supply to melanoma transplants in the hamster cheek pouch.Lab Invest. 1966; 15: 464-478PubMed Google Scholar, 12Hubler Jr, WR Wolf Jr, JE Melanoma. Tumor angiogenesis and human neoplasia.Cancer. 1976; 38: 187-192Crossref PubMed Scopus (31) Google Scholar and clinically.13Barnhill RL Fandrey K Levy MA Mihm Jr, MC Hyman B Angiogenesis and tumor progression of melanoma. Quantification of vascularity in melanocytic nevi and cutaneous malignant melanoma.Lab Invest. 1992; 67: 331-337PubMed Google Scholar The importance of tumor angiogenesis for the prognosis of primary malignant melanomas of the skin, however, has remained controversial.14Streit M, Detmar M: Melanoma angiogenesis, lymphangiogenesis and metastasis. Oncogene (in press)Google Scholar Whereas several studies found an inverse correlation of tumor microvessel density with disease-free and overall survival,15Srivastava A Laidler P Davies RP Horgan K Hughes LE The prognostic significance of tumor vascularity in intermediate-thickness (0.76–4.0 mm thick) skin melanoma. A quantitative histologic study.Am J Pathol. 1988; 133: 419-423PubMed Google Scholar, 16Straume O Salvesen HB Akslen LA Angiogenesis is prognostically important in vertical growth phase melanomas.Int J Oncol. 1999; 15: 595-599PubMed Google Scholar other reports did not detect any significant differences of tumor microvessel density between metastasizing and nonmetastasizing melanomas.17Busam KJ Berwick M Blessing K Fandrey K Kang S Karaoli T Fine J Cochran AJ White WL Rivers J Tumor vascularity is not a prognostic factor for malignant melanoma of the skin.Am J Pathol. 1995; 147: 1049-1056PubMed Google Scholar 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.5Balch CM Buzaid AC Soong SJ Atkins MB Cascinelli N Coit DG Fleming ID Gershenwald JE Houghton Jr, A Kirkwood JM McMasters KM Mihm MF Morton DL Reintgen DS Ross MI Sober A Thompson JA Thompson JF Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma.J Clin Oncol. 2001; 19: 3635-3648Crossref PubMed Scopus (2247) Google Scholar, 18Gershenwald JE Thompson W Mansfield PF Lee JE Colome MI Tseng CH Lee JJ Balch CM Reintgen DS Ross MI Multi-institutional melanoma lymphatic mapping experience: the prognostic value of sentinel lymph node status in 612 stage I or II melanoma patients.J Clin Oncol. 1999; 17: 976-983Crossref PubMed Google Scholar 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.19Deutsch A Lubach D Nissen S Neukam D Ultrastructural studies on the invasion of melanomas in initial lymphatics of human skin.J Invest Dermatol. 1992; 98: 64-67Crossref PubMed Scopus (12) Google Scholar, 20Kashani-Sabet M Sagebiel RW Ferreira CM Nosrati M Miller III, JR Vascular involvement in the prognosis of primary cutaneous melanoma.Arch Dermatol. 2001; 137: 1169-1173Crossref PubMed Scopus (95) Google Scholar 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.21de Waal RM van Altena MC Erhard H Weidle UH Nooijen PT Ruiter DJ Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.Am J Pathol. 1997; 150: 1951-1957PubMed Google Scholar 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.22Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1472) Google Scholar, 23Stacker SA Caesar C Baldwin ME Thornton GE Williams RA Prevo R Jackson DG Nishikawa S Kubo H Achen MG VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat Med. 2001; 7: 186-191Crossref PubMed Scopus (1049) Google Scholar We have previously reported that VEGF-C is expressed in several human melanoma cell lines in vitro;24Skobe M Hamberg LM Hawighorst T Schirner M Wolf GL Alitalo K Detmar M Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.Am J Pathol. 2001; 159: 893-903Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar 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 normal25Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1268) Google Scholar and tumor-associated lymphatic vessels26Beasley NJ Prevo R Banerji S Leek RD Moore J van Trappen P Cox G Harris AL Jackson DG Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer.Cancer Res. 2002; 62: 1315-1320PubMed Google Scholar 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. 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 ( 50% of tumor area).Table 1Clinical and Pathological Characteristics of Patients with MelanomaCategoryNonmetastaticMetastaticNumber of patients1918 Male1110 Female88Time of lymph node metastasis (months) Mean—24.4 Range—5–69Time of visceral organ metastasis (months) Mean—38.0 Range—14–76Age at diagnosis (years) Mean53.854.9 Range21–7923–76Histologic type Superficial spreading1414 Nodular44 Lentigo maligna10Breslow thickness (mm) 6.044 1–61113 030Regression Present42 Absent1515Peritumoral inflammation Absent67 Present99 Brisk42 Open table in a new tab 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)25Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1268) Google Scholar 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.24Skobe M Hamberg LM Hawighorst T Schirner M Wolf GL Alitalo K Detmar M Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.Am J Pathol. 2001; 159: 893-903Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar 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.28Streit M Riccardi L Velasco P Brown LF Hawighorst T Bornstein P Detmar M Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.Proc Natl Acad Sci USA. 1999; 96: 14888-14893Crossref PubMed Scopus (252) Google Scholar 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,29Detmar M Brown LF Claffey KP Yeo K-T Kocher O Jackman RW Berse B Dvorak HF Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (641) Google Scholar using a riboprobe for human VEGF that detects all known VEGF splice variants29Detmar M Brown LF Claffey KP Yeo K-T Kocher O Jackman RW Berse B Dvorak HF Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (641) Google Scholar or a 808-bp human VEGF-C riboprobe described previously.30Skobe M Brown LF Tognazzi K Ganju RK Dezube BJ Alitalo K Detmar M Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.J Invest Dermatol. 1999; 113: 1047-1053Crossref PubMed Scopus (106) Google Scholar Transcription reactions were performed using the Riboprobe Gemini II kit (Promega, Madison, WI) in the presence of [α-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). 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 ×100 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.31Detmar M Velasco P Richard L Claffey KP Streit M Riccardi L Skobe M Brown LF Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas.Am J Pathol. 2000; 156: 159-167Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar 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. 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. 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 (Figure 1, Figure 2). In all cases, peritumoral lymphatic vessels with frequently open lumina were found within a distance of 100 μm from the tumor border (Figure 1, Figure 2). Intratumoral lymphatic vessels frequently exhibited a thin-walled, basket-like morphology (Figure 2B) resembling that seen in blood vessel networks during VEGF-induced angiogenesis.32Pettersson A Nagy JA Brown LF Sundberg C Morgan E Jungles S Carter R Krieger JE Manseau EJ Harvey VS Eckelhoefer IA Feng D Dvorak AM Mulligan RC Dvorak HF Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.Lab Invest. 2000; 80: 99-115Crossref PubMed Scopus (360) Google Scholar 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.Figure 2Higher frequency of intratumoral lymphangiogenesis in metastatic melanomas. A: Immunofluorescent stain for LYVE-1 (green) depicts thick-walled peritumoral (arrows) and thin-walled intratumoral lymphatics (arrowheads) in a thin melanoma (1.05 mm). The tumor border is indicated by a dotted line. Blood vessels (asterisks) are negative for LYVE-1. B: Higher magnification of intratumoral lymphatics reveals thin-walled, basket-like morphology. C: Double immunostain for LYVE-1 (red) and PCNA (brown) reveals an intratumoral lymphatic vessel with proliferating lymphatic endothelial cells (arrows) and adjacent melanoma cells (arrowhead). D: Significantly increased frequency of detectable intratumoral lymphatic vessels in metastatic melanomas (M; n = 18), as compared with nonmetastatic (NM; n = 19) tumors (mean ± SEM, chi-square test, P = 0.01). E: Detection of melanin-containing tumor cells within an intratumoral lymphatic vessel. H&E stain. F: Immunofluorescent stain of a serial section for LYVE-1 (green) and CD31 (red) confirms that tumor cells are located within a lymphatic vessel. Cell nuclei are counterstained blue with Hoechst (B and F). Original magnifications: ×200 (A); ×400 (B, C, E, F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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). 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).Figure 5VEGF-C protein expression in cutaneous melanomas. A: Immunoperoxidase staining for VEGF-C (red) demonstrates focal expression in the cytoplasm of melanoma cells in one tumor nest but not in the adjacent nests (asterisks). B: Serial section negative control with omission of secondary antibody. C: Immunoperoxidase staining for VEGF-C (red) demonstrates VEGF-C expression by peritumoral fibroblasts (rectangle) at the invasive edge of a metastatic melanoma (top left) in the reticular dermis. D: H&E stain of the invasive tumor edge of a metastasizing cutaneous melanoma reveals mononuclear inflammation (asterisks) near dilated lymphatic vessel (arrow). E: Immunoperoxidase staining of a serial section for LYVE-1 (red) decorates dilated peritumoral lymphatics (arrow) and adjacent mononuclear infiltrate. Blood vessels are LYVE-1-negative (arrowheads). F: Immunoperoxidase staining for VEGF-C (red) stains the cytoplasm of peritumoral mononuclear cells in a granular pattern. Original magnifications: ×200 (A–D); ×400 (E); and ×600 (F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We
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