title: VEGFR-2 expression in human melanoma: revised assessment.
abstract: 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 (<10%) express detectable levels of VEGFR-2, and therefore, the majority of patients with melanoma are unlikely to benefit from antiproliferative effects of anti-VEGF therapy.
authors.affiliation: Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA, USA. klr5w@virginia.edu
authors.forename: Kerrington R
authors.initials: KR
authors.lastname: Molhoek
authors.forename: Gulsun
authors.initials: G
authors.lastname: Erdag
authors.forename: J K
authors.initials: JK
authors.lastname: Rasamny
authors.forename: Cheryl
authors.initials: C
authors.lastname: Murphy
authors.forename: Donna
authors.initials: D
authors.lastname: Deacon
authors.forename: James W
authors.initials: JW
authors.lastname: Patterson
authors.forename: Craig L
authors.initials: CL
authors.lastname: Slingluff
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D045744:Cell Line, Tumor; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D009362:Neoplasm Metastasis; D046888:Tissue Array Analysis; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D040301:Vascular Endothelial Growth Factor Receptor-2