VEGFR‐2 expression in human melanoma: Revised assessment

Vascular endothelial growth factor (VEGF) is an angiogenic factor that also functions as an autocrine growth factor for VEGF receptor (VEGFR)‐2+ melanomas. In multiple studies, VEGFR‐2 was detected by immunostaining in 78–89% of human melanoma cells, suggesting that most patients with melanoma would benefit from anti‐VEGF therapy. Here, we evaluated 167 human melanoma specimens in a tissue microarray to verify the presence of VEGFR‐2, but found disparities in staining with commercial antibodies A‐3 and 55B11. Antibody A‐3 stained melanoma cells in 79% of specimens, consistent with published results; however, we noted extensive nonspecific staining of other cells such as smooth muscle and histiocytes. In contrast, antibody 55B11 stained melanoma cells in only 7% (95% confidence interval: 3.3–11.5) of specimens. As an internal positive control for VEGFR‐2 detection, vascular endothelial cells were stained with antibody 55B11 in all specimens. We compared VEGFR‐2+ and VEGFR‐2− melanoma cell lines by immunoblotting and immunohistochemistry after small interfering RNA (siRNA) knockdown and transient overexpression of VEGFR‐2 to validate antibody specificity. Immunoblotting revealed that A‐3 primarily cross‐reacted with several proteins in both cell lines and these were unaffected by siRNA knockdown of VEGFR‐2. In contrast, 55B11 staining of VEGFR‐2+ cells was mostly eliminated by siRNA knockdown of VEGFR‐2 and increased in VEGFR‐2− melanoma cell lines following transfection to express ectopic VEGFR‐2. Our results show that relatively few melanoma cells (