Quantitative expression of VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 in melanoma tissue microarrays

Abstract

Angiogenesis is required for progression and metastasis of melanoma. Analysis of angiogenic molecules in benign and malignant tissues may allow identification of markers useful for prediction of sensitivity to antiangiogenic agents. We hypothesized that differential expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, VEGF-R2, and VEGF-R3 would be higher in melanomas than nevi and higher in advanced melanoma. Using automated quantitative analysis, we quantified VEGF, -R1, -R2 and -R3 expression in melanoma tissue microarrays composed of 540 nevi and 468 melanoma specimens (198 primaries, 270 metastases). VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 expression was significantly higher in melanomas than nevi by unpaired t tests (P < .0001). VEGF-R2 expression was higher in metastatic specimens (P < .0001), but VEGF-R3 expression was higher in primaries (P < .0001). VEGF was coexpressed with all 3 receptors when assessed by Spearman's rank correlation. VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 expression is higher in melanomas than nevi. Higher expression of VEGF-R2 was found in metastases versus primaries, supporting the idea that selection for an angiogenic phenotype in metastatic melanoma is conferred via up-regulation of VEGF-R2. However, higher expression of VEGF-R3 was seen on primary lesions, potentially implicating this receptor in initiation of lymphatic tumor spread. Clinical trials using antiangiogenic agents in melanoma should include correlative assays of VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 as biomarkers of response to therapy, preferably using quantitative methods such as automated quantitative analysis. Such assessments could assist with evaluation of these molecules as therapeutic targets in melanoma, ultimately facilitating improved selection of patients for treatment.

Figure 1

Expression of VEGF, VEGF-R1, VEGF-R2 and VEGF-R3 in melanoma cell lines. Protein was extracted from YUSAC, YUSOC, YUMAC, MEL501, YUFIC and YUROB melanoma cell lines and was subjected SDS-PAGE and Western blot analysis to detect VEGF (A), VEGF-R1 (B), VEGF-R2 (C) and VEGF-R3 (D).

Figure 2

Regression plots for scores from the two sets of melanoma arrays for A) VEGF, B)VEGF-R1, C)VEGF-R2 and D)VEGF-R3. The y axis represents AQUA scores from one slide and the x axis from the second slide. Each array contains histospots from the same patients, taken from different areas of the tumor.

Figure 3

Cytoplasmic VEGF, VEGF-R1, VEGF-R2 and VEGF-R3 expression in melanoma histospots using S100 to define the tumor mask (green), 4,6-diamidine-2-phenylindole to define the nuclear compartment (blue), and Cy5 (red) for the target. Merged images displayed in the upper outer quadrants of panels A,B,C, and D show the amount of VEGF, VEGF-R1, VEGF-R2 and VEGF-R3, respectively, within the nuclear compartment and within the cytoplasmic compartment within the tumor mask at 10× magnification. Upper inner quadrants of panels A,B,C, and D show expression of VEGF, VEGF-R1, VEGF-R2 and VEGF-R3 at 60× magnification

Figure 4

Expression of VEGF, VEGF-R1, VEGF-R2 and VEGF-R3 (P < 0.0001) was significantly higher in malignant than benign tissues. Expression of VEGF-R2 (P < 0.0001) was substantially higher in metastatic than primary specimens. Conversely, expression of VEGF-R3 (P < 0.0001) was more pronounced in primary versus metastatic spots.