Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions.

BRAF(V600E) is the most common mutation in cutaneous melanoma and has become the target of treatment for patients with metastatic melanoma. A number of methods are currently available to determine mutation status. Recently, a monoclonal antibody (VE1) against mutant BRAF(V600E) was generated. Its use permits assessment of the mutant protein expression throughout a tumor sample and may allow faster and cheaper determination of the mutation status in selected cases. However, for BRAF(V600E) protein expression analysis to be of clinical value, high sensitivity and specificity of the antibody is a prerequisite. In this study we analyzed 44 metastatic melanoma samples with a known BRAF(V600E) mutation status with immunohistochemical expression of the BRAF(V600E) protein. None of the 22 tumors that lacked the BRAF(V600E) mutation labeled with the antibody VE1. This set of VE1-immunonegative tumors included 4 metastatic lesions with the BRAF(V600E) mutation. All 22 tumor samples that were known to carry the BRAF mutation were immunoreactive with VE1. Sixteen of them stained strongly and homogenously throughout the tumor sample. However, 6 tumor samples contained both BRAF(V600E)-immunopositive and BRAF(V600E)-immunonegative cell populations. When the BRAF status was compared with immunoreactivity for melanocyte differentiation antigens, no significant difference in the expression of melan-A, microphthalmia transcription factor, gp100, or tyrosinase was found between mutant and wild-type tumors. In addition to metastatic lesions, we also examined 20 primary melanomas for the expression of BRAF(V600E). Seven of 10 superficial spreading melanomas were immunoreactive with the antibody VE1. Five tumors were strongly and homogenously immunoreactive. In 2 primary tumors the staining was focal, involving only a subpopulation of the tumor. None of the nonsuperficial spreading melanomas was immunoreactive. In 7 primary tumors the mutation status could be analyzed: only tumors carrying the BRAF(V600E) mutation were immunoreactive with VE1. The high specificity and sensitivity of VE1 for the detection of mutant BRAF(V600E) suggests a valuable reagent for clinical purposes. Heterogeneity in BRAF expression may be relevant for treatment response to BRAF inhibitors.