BRAFV600E and NRASQ61L/Q61R mutation analysis in metastatic melanoma using immunohistochemistry : a study of 754 cases highlighting potential pitfalls and guidelines for interpretation and reporting

Background and aims: BRAF or NRAS mutations occur in approximately 60% of cutaneous melanomas, and the identification of such mutations underpins the appropriate selection of patients who may benefit from BRAF and MEK inhibitor targeted therapies. The utility of immunohistochemistry (IHC) to detect NRASQ61L mutations is currently unknown. This study sought to assess the sensitivity and specificity of anti-BRAFV600E (VE1), anti-NRASQ61R (SP174) and anti-NRASQ61L (26193) antibodies for mutation detection in a large series of cases. Methods and results: Mutation status was determined using the OncoCarta assay in 754 cutaneous melanomas. IHC with the anti-BRAFV600E antibody was performed in all cases, and the anti-NRASQ61R and anti-NRASQ61L antibodies were assessed in a subset of 302 samples utilizing tissue microarrays. The staining with the anti-BRAFV600E and anti-NRASQ61R antibodies was diffuse, homogeneous and cytoplasmic. The anti-NRASQ61L antibody displayed variable intensity staining, ranging from weak to strong in NRASQ61L mutant tumours. The sensitivity and specificity for anti-BRAFV600E was 100 and 99.3%, anti-NRASQ61R was 100 and 100% and anti-NRASQ61L was 82.6 and 96.2%, respectively. Conclusions: The use of IHC is a fast, efficient and cost-effective method to identify single specific mutations in melanoma patients. BRAFV600E and NRASQ61R antibodies have high sensitivity and specificity; however, the NRASQ61L antibody appears less sensitive. IHC can help to facilitate the timely, appropriate selection and treatment of metastatic melanoma patients with targeted therapies. Detection of melanoma-associated mutations by IHC may also provide evidence for a diagnosis of melanoma in metastatic undifferentiated neoplasms lacking expression of melanoma antigens.