title: BRAF(V600E) and NRAS(Q61L/Q61R) mutation analysis in metastatic melanoma using immunohistochemistry: a study of 754 cases highlighting potential pitfalls and guidelines for interpretation and reporting.
abstract: OBJECTIVE
  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 NRAS(Q61L) mutations is currently unknown. This study sought to assess the sensitivity and specificity of anti-BRAF(V600E) (VE1), anti-NRAS(Q61R) (SP174) and anti-NRAS(Q61L) (26193) antibodies for mutation detection in a large series of cases.
  
  
  RESULTS
  Mutation status was determined using the OncoCarta assay in 754 cutaneous melanomas. IHC with the anti-BRAF(V600E) antibody was performed in all cases, and the anti-NRAS(Q61R) and anti-NRAS(Q61L) antibodies were assessed in a subset of 302 samples utilizing tissue microarrays. The staining with the anti-BRAF(V600E) and anti-NRAS(Q61R) antibodies was diffuse, homogeneous and cytoplasmic. The anti-NRAS(Q61L) antibody displayed variable intensity staining, ranging from weak to strong in NRAS(Q61L) mutant tumours. The sensitivity and specificity for anti-BRAF(V600E) was 100 and 99.3%, anti-NRAS(Q61R) was 100 and 100% and anti-NRAS(Q61L) 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. BRAF(V600E) and NRAS(Q61R) antibodies have high sensitivity and specificity; however, the NRAS(Q61L) 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.
authors.affiliation: Melanoma Institute Australia, North Sydney, NSW, Australia.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.
authors.forename: Hojabr
authors.initials: H
authors.lastname: Kakavand
authors.affiliation: Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.|Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Emily
authors.initials: E
authors.lastname: Walker
authors.affiliation: Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Trina
authors.initials: T
authors.lastname: Lum
authors.affiliation: Melanoma Institute Australia, North Sydney, NSW, Australia.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.
authors.forename: James S
authors.initials: JS
authors.lastname: Wilmott
authors.affiliation: Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Christina I
authors.initials: CI
authors.lastname: Selinger
authors.affiliation: Melanoma Institute Australia, North Sydney, NSW, Australia.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Smith
authors.affiliation: Melanoma Institute Australia, North Sydney, NSW, Australia.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.
authors.forename: Robyn P M
authors.initials: RP
authors.lastname: Saw
authors.affiliation: Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.|Medical Genomics, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Bing
authors.initials: B
authors.lastname: Yu
authors.affiliation: Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.|Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Cooper
authors.affiliation: Melanoma Institute Australia, North Sydney, NSW, Australia.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.|Melanoma Oncology, Royal North Shore and Mater Hospital, Sydney, NSW, Australia.
authors.forename: Georgina V
authors.initials: GV
authors.lastname: Long
authors.affiliation: Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia.|Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
authors.forename: Sandra A
authors.initials: SA
authors.lastname: O'Toole
authors.affiliation: Melanoma Institute Australia, North Sydney, NSW, Australia. richard.scolyer@sswahs.nsw.gov.au.|Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia. richard.scolyer@sswahs.nsw.gov.au.|Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia. richard.scolyer@sswahs.nsw.gov.au.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Scolyer
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards*; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods / Q000592:standards; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000556:secondary; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms; D046888:Tissue Array Analysis; D000096142:Cutaneous Malignant Melanoma
chemical_list: D008565:Membrane Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human
keywords: BRAF; NRAS; diagnosis; immunohistochemistry; melanoma