Original contributionQuantitative expression of VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 in melanoma tissue microarrays☆
Introduction
Melanoma is a relatively chemotherapy resistant disease that is associated with an extremely poor prognosis once systemic metastases develop. An estimated 62 480 new melanomas will be diagnosed in the United States in 2008, and an estimated 8420 people in the United States will die of the disease. For patients with metastases, the median survival is in the range of 9 to 12 months [1]. No therapy has been proven to prolong survival in patients with melanoma in randomized clinical trials, underscoring the need for the development of more effective treatments.
Angiogenesis has been recognized as an important process for the growth and invasion of malignant tumors, including melanoma. The vascular endothelial growth factor (VEGF) pathway plays a critical role in angiogenesis. This pathway has been the focus of much attention with the development of bevacizumab (Avastin, Genentech, San Francisco, CA), an antibody targeted against VEGF that has been shown to prolong progression free survival in kidney and breast cancers and overall survival in lung and colon cancers [2], [3]. The VEGF family consists of five isoforms, VEGF-A (known as VEGF), VEGF-B, VEGF-C, VEGF-D, and placental growth factor. VEGF-A, also known as vascular permeability factor or VEGF, was described as a potent endothelial cell mitogen which stimulates the proliferation and migration of endothelial cells. VEGF signals primarily through receptor tyrosine kinases VEGF receptor 1 (VEGF-R1, flt-1) and 2 (VEGF-R2, flk2/kdr). While VEGF-R1 has a much greater affinity for VEGF, ligand mediated autophosphorylation is weaker than that initiated by ligand binding to VEGF-R2. It has been postulated that VEGF-R1 may play a decoy role in VEGF signaling by regulating VEGF through decreased binding to VEGF-R2, and that the majority of effects of VEGF in malignancy are mediated through VEGF-R2. VEGF-C and -D primarily signal through VEGF-R3; this pathway is thought to be essential for the development of lymphatic vasculature and for normal and tumor lymphangiogenesis [4].
Several studies have examined the expression of members of the VEGF signaling pathway in melanoma. Secretion of VEGF occurs during progression of early cutaneous melanocytic lesions, with low VEGF expression in benign nevi increasing significantly in dysplastic nevi and more so in malignant melanoma [5]. The transition of melanomas from the radial to the aggressive vertical growth phases is also marked by increased VEGF production [6]. Several groups have reported results of VEGF expression assessed by immunohistochemical staining in cohorts of melanoma tumor tissue [7], [8], [9], [10], [11], [12]. While most of these studies showed increased VEGF expression in malignant versus benign tumors, with higher expression associated with more invasive phenotypes, prevalence of tumors expressing VEGF varied widely, and no clear relationship with prognosis has been verified. In these tissues VEGF has traditionally been thought to signal in a paracrine fashion, recruiting endothelial cells for the formation of neovasculature to nourish growing tumors. However, previous identification of VEGF receptors in melanoma cell lines suggests VEGF may also signal in melanoma through an autocrine loop, although reports conflict on its presence [13], [14]. Small studies have assessed the presence of VEGF-R1 and R2 on human melanoma tissue [7], [15], and their results confirm the presence of these receptors in human melanomas as well as a possible relationship of VEGF-R2 expression with thicker and more invasive tumors.
Recognized for its distinct role in lymphangiogenesis, VEGF-R3 expression in malignant melanocytes is a focus of rising interest. VEGF-R3 expression has been seen on both tumor cells as well as blood vessels and lymphatics [16], where in benign melanocytic lesions, it has been shown to be confined to lymphatic vessels only [17], implicating a role in metastatic spread. Mouawad et al. recently reported high levels of VEGFR-3 in melanoma tumor tissue, accompanied by significantly higher pre-treatment serum levels of VEGFR-3 in melanoma patients. Interestingly, median serum VEGFR-3 levels were increased in patients with high tumour burden as well as in non-responders compared to responders, and low VEGFR-3 related positively to disease free survival, underscoring the potential prognostic implications for expression of this receptor [18].
Given the probable diverse roles of VEGF and its receptors in promoting melanoma growth and metastases, we hypothesized that in melanoma, VEGF signaling may contribute substantially to tumor growth and metastasis. We thus sought to characterize expression of VEGF and its receptors VEGF-R1, VEGF-R2, and VEGF-R3 on a large number of human melanoma specimens and benign nevi and to correlate expression with survival, disease stage, age, gender, and the presence of known histologic prognostic factors such as Clark's level, Breslow depth, and the presence of ulceration or tumor infiltrating lymphocytes. We expected that higher expression levels of these molecules would be seen in malignant melanomas compared with benign nevi and that differential expression would be seen in primary and metastatic subsets. We were further interested in examining potential coexpression among the VEGF ligand and its receptors within melanomas. To accomplish this, we employed tissue microarrays of melanoma specimens using a newly developed, automated method of analysis (AQUA for Automated, QUantitative Analysis). This method provides precise, reproducible measurement of antigen levels, free of the subjectivity associated with pathologist-based scoring employed in traditional immunohistochemistry studies [19]. AQUA analysis provides continuous output scores, as opposed to arbitrary nominal scores obtained with pathologist-based "by-eye" scoring of 0, 1, 2, or 3 or "positive" and "negative." This is particularly important when therapeutic decisions are made based on immunohistochemistry under nonstandardized conditions. This method has been validated [19], has been proven to be more accurate than pathologist-based scoring of DAB stain, and has been used in several prior melanoma studies as previously described [20]. Recent clinical trial designs, including melanoma trials, have planned for the incorporation of AQUA analysis of tissues from cancer patients treated with targeted therapies, with the goal of identifying markers that might predict response to treatment [21]. We sought to use AQUA to examine a large historical cohort of 468 melanomas and 540 nevi. No previous studies to our knowledge have examined the expression of VEGF and its three major receptors in cohorts of clinical specimens using an automated method of expression analysis. Our results indicate higher expression of VEGF and all 3 of the receptors in melanomas compared with nevi. The expression of VEGF-R2 was higher in metastatic than in primary melanomas; however, for VEGF-R3, higher expression was seen in primary lesions.
Section snippets
Cell lines and western blots
YUSAC, YUSOC, YUMAC, YUFIC, and YUROB are cell lines derived from tumors of patients treated at Yale University. The MEL501 cell line was obtained from Dr Steven Rosenberg at the Surgery Branch, National Cancer Institute, Bethesda, MD. Proteins from lysates were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blotting was performed by standard methods using antibodies to VEGF (sc-152, Santa Cruz Biotechnology, Santa Cruz, CA; 1:500), VEGF-R1 (sc-316, Santa Cruz
Results
Lysates from a panel of melanoma cell lines were probed for VEGF, VEGF-R1, VEGF-R2, and VEGF-R3, and dominant bands were seen at 21 KD, 180 KD, 200 KD and 150 KD, consistent with reports in the literature [22]. The intensity of the bands was more variable for VEGF-R1 and -R3, as shown in Fig. 1.
To assess for intra-tumor heterogeneity, two separate arrays, each containing a core from a different area of the tumor for each patient, were used to evaluate the expression of each marker. None of the
Discussion
The purpose of this study was to quantitatively assess the expression of VEGF and its receptors using an objective, automated method and to characterize differences in expression between melanomas and nevi. Furthermore, we evaluated associations with clinical and pathologic variables, and assessed co-expression among VEGF and its receptors. Expression of VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 was significantly higher in malignant compared to benign specimens. When comparing unmatched (from
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Supported by NIH grant CA115756-01 (to H Kluger).