High specificity and sensitivity of NRAS Q61R immunohistochemistry (IHC) in melanomas

To the Editor: BRAF and NRAS are the most frequently mutated genes in melanoma (40%-50% and 20% of cases, respectively).1Jakob J.A. Bassett Jr., R.L. Ng C.S. et al.NRAS mutation status is an independent prognostic factor in metastatic melanoma.Cancer. 2012; 118: 4014-4023Crossref PubMed Scopus (501) Google Scholar Determining BRAF and NRAS tumor status is mandatory for patient treatment with targeted therapies and is commonly achieved by DNA sequencing. Immunohistochemistry (IHC) has recently appeared as a reliable surrogate technique for molecular BRAF V600E mutation screening.2Just P.A. Audebourg A. Pasmant E. et al.Immunohistochemistry versus next-generation sequencing for the routine detection of BRAF V600E mutation in melanomas.Hum Pathol. 2014; 45: 1983-1984Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Ilie et al3Ilie M. Long-Mira E. Funck-Brentano E. et al.Immunohistochemistry as a potential tool for routine detection of the NRAS Q61R mutation in patients with metastatic melanoma.J Am Acad Dermatol. 2015; 72: 786-793Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar in this journal, and others,4Massi D. Simi L. Sensi E. et al.Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.Mod Pathol. 2015; 28: 487-497Crossref PubMed Scopus (50) Google Scholar reported that IHC had 100% specificity and sensitivity for detecting the most frequent NRAS mutation, Q61R. We aimed to confirm these results on a different IHC platform (Bond-III, Leica Biosystems, Nussloch, Germany) in a selected series of 70 stage-IV melanomas previously screened by targeted next-generation sequencing for presence of NRAS and BRAF mutations.2Just P.A. Audebourg A. Pasmant E. et al.Immunohistochemistry versus next-generation sequencing for the routine detection of BRAF V600E mutation in melanomas.Hum Pathol. 2014; 45: 1983-1984Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar After antigen retrieval (100°C for 60 minutes + 12 minutes' cooling with ER2 solution), slides were incubated with the anti-NRAS Q61R antibody, clone SP174 (1:100 dilution, SpringBio Science, Pleasanton, CA) for 20 minutes at 37°C and revealed using the Bond Polymer Refine Red detection kit (Leica Biosystems). Of the 22 of 70 cases with NRAS Q61R mutation, all but 1 displayed a moderate to strong cytoplasmic staining in more than 80% of tumor cells (Fig 1). The 1 remaining case showed heterogenous staining, with strongly stained areas adjacent to faintly stained areas. Tumor cellularity was estimated at ∼80% for this case, whereas NRAS Q61R mutation allelic ratio was estimated at ∼25% using next-generation sequencing. Faintly stained areas may therefore represent a tumor subclone without NRAS Q61R mutation. The remaining 48 cases (10 NRAS Q61L, 9 NRAS Q61K, 2 NRAS Q61H, 13 BRAF V600E, 1 BRAF V600K, and 13 cases with no NRAS or BRAF mutation) showed no staining. We confirmed 100% specificity and sensitivity of IHC for the diagnosis of NRAS Q61R mutation in melanoma. We often noticed a moderate to strong staining in endothelial cells (Fig 1), not described in previous studies.3Ilie M. Long-Mira E. Funck-Brentano E. et al.Immunohistochemistry as a potential tool for routine detection of the NRAS Q61R mutation in patients with metastatic melanoma.J Am Acad Dermatol. 2015; 72: 786-793Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 4Massi D. Simi L. Sensi E. et al.Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.Mod Pathol. 2015; 28: 487-497Crossref PubMed Scopus (50) Google Scholar This endothelial staining might be due to cross-reactivity with endothelial or serum proteins. Of note, anti-BRAF V600E antibody has been previously shown to cross-react with axonemal dynein of ciliated cells.5Jones R.T. Abedalthagafi M.S. Brahmandam M. et al.Cross-reactivity of the BRAF VE1 antibody with epitopes in axonemal dyneins leads to staining of cilia.Mod Pathol. 2015; 28: 596-606Crossref PubMed Scopus (44) Google Scholar We also noticed a dramatic loss of antigenicity when slides were cut several days before IHC was performed. We therefore highly recommend performing NRAS Q61R IHC on freshly cut slides. BRAF V600E and NRAS Q61R IHC are superior to sequencing in terms of cost, rapidity, and tissue sparing. Furthermore, IHC may be more relevant than molecular techniques in tumors with very low cellularity, or high clonal heterogeneity. A strong and diffuse staining is specifically associated with the presence of NRAS Q61R or BRAF V600E mutations and molecular validation may not be mandatory in such cases. However, quality assurance and certification programs of oncogenetic testing require procedures to control genotype and patient/result matching. In our hospital, both IHC and next-generation sequencing analysis are performed in parallel on each sample to confirm the genotype and to detect potential false results of either technique, as described by Massi et al4Massi D. Simi L. Sensi E. et al.Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.Mod Pathol. 2015; 28: 487-497Crossref PubMed Scopus (50) Google Scholar for NRAS sequencing or by us for BRAF V600E IHC.2Just P.A. Audebourg A. Pasmant E. et al.Immunohistochemistry versus next-generation sequencing for the routine detection of BRAF V600E mutation in melanomas.Hum Pathol. 2014; 45: 1983-1984Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar