To the Editors: Melanoma is not a single disease, but rather a heterogeneous group of neoplasms whose clinical–histopathological features, biologic behaviors, and responses to therapy are related to patterns of somatic mutations in specific oncogenes.1 Studies have identified that BRAF and NRAS mutational status correlate with both primary tumor characteristics and prognosis in patients with metastatic disease.2–7 BRAFV600E is the most commonly mutated oncogene identified in melanoma (∼50% of cases) and the target for Food and Drug Administration–approved therapy (ie, vemurafenib) in patients with metastatic disease.1 Immunohistochemistry, using novel mutant protein-specific antibody, has been reported to be highly sensitive and specific for detection of the clinically important BRAFV600E mutation in melanoma.8,9 Accordingly, investigators have proposed the use of immunohistochemistry as an initial screening tool, a supplemental assay, or possibly a replacement test to conventional BRAF mutation analysis by molecular methods.8,9 Anti-BRAFV600E mutant antibodies (Spring Bioscience, Pleasanton, CA) show strong cytoplasmic reactivity in melanomas with this mutation (Figs. 1A, B). Anti-BRAFV600K mutant and anti-BRAFV600D mutant antibodies have also recently become available (NewEast Biosciences, King of Prussia, PA).FIGURE 1: Invasive melanoma (A) demonstrating tumor cell reactivity for BRAFV600E mutant protein-specific antibody by immunohistochemistry, with cytoplasmic-restricted staining pattern (B). Invasive melanoma (C) demonstrating tumor cell reactivity for NRASQ61R mutant protein-specific antibody by immunohistochemistry, with predominantly membranous and weaker cytoplasmic staining pattern (D).Activating mutations in NRAS have been identified in 0%–50% of primary and metastatic melanoma tumor samples2,3; although, more typically in the range of 15%–20%.4–7,10NRAS and BRAF mutations are reported to be mutually exclusive at the single cell level; however, rare tumors characterized by both mutations have been described.4–6,10 The most common NRAS mutations in melanoma are observed in exon 2 at codon 61 (>80% of all mutations in this gene).2,4–6,10,11 The Q61R mutation, which leads to a glutamine to arginine substitution (c.182A > G), has been reported in 5%–11% of melanomas and accounts for ∼35%–40% of all NRAS mutations in these tumors.4–6 Other codon 61 mutations include Q61K, Q61L, and Q61H that result in glutamine to lysine, leucine, and histidine substitutions, respectively.4–6NRAS mutations have also been infrequently reported in exon 1 at codons 12 and 13 in melanoma.4,5 Mutations in the Q61 residue lock the RAS protein in a GTP-bound state with resultant continuous activation of its downstream effectors via the Ras-Raf-MEK-ERK, Ras-PI3K-Akt, and RAL pathways and thereby promoting cellular proliferation, invasion, and survival.10,11 Similar to BRAFV600E, mutant NRAS has been investigated as a potential therapeutic target in advanced melanoma.10,11 By immunohistochemistry, anti-NRASQ61R mutant protein-specific antibody (Spring Bioscience) shows a predominantly membranous and weaker cytoplasmic profile, indicative of the cellular localization of this antigen (Figs. 1C, D). This antibody has been found to be specific for Q61R mutations in melanoma (detected by molecular analysis), with no cross-reactivity for other Q61 mutations identified to date, specifically Q61K and Q61H (personal observations). Immunohistochemistry is faster, less expensive, and technically simpler than currently available molecular technologies. In addition, results are potentially quantifiable and can be simultaneously correlated with the cytoarchitectural features of a particular tumor. As mutant oncogene-specific antibodies become more widely available, it is likely that immunohistochemical studies will be used to determine the translational expression of mutated oncogenes [ie, protein presence, level, location, and/or pattern (ie, homogeneous vs. heterogeneous distribution)] and its significance in different types of melanoma samples. Such assays would likely enhance current molecular testing efforts to investigate associations between specific oncogene mutations and both distinct clinical–histopathological features of melanoma and patient outcomes. In addition, supplemental testing for mutant protein expression could have a potential role in guiding the selection of targeted therapy and predicting treatment response in patients with metastatic disease. Immunohistochemistry may have important clinical utility in tumors with mutational heterogeneity and/or in those instances where the limits of sensitivity (range of ∼5%–20% at present) of current molecular methods are approached,8 such as micrometastatic deposits in which only rare or scattered melanoma cells are present within a larger nontumor cell background population.