title: NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.
abstract: BACKGROUND
  The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies in the theranostic mutation screening of melanomas.
  
  
  METHODS
  142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry.
  
  
  RESULTS
  23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS (Q61R) and 23 BRAF (V600E) -mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS (Q61L) , NRAS (Q61K) , BRAF (V600K) and BRAF (V600R) mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity.
  
  
  CONCLUSIONS
  In our study, we showed that combining immunohistochemistry analysis targeting NRAS(Q61R) and BRAF(V600E) proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.
authors.affiliation: Inserm, U1078, Brest, F-29200, France. arnaud.uguen@chu-brest.fr.|CHRU Brest, Service d'anatomie et cytologie pathologiques, Brest, F-29220, France. arnaud.uguen@chu-brest.fr.|Université Européenne de Bretagne, Rennes, France. arnaud.uguen@chu-brest.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Uguen
authors.affiliation: CHRU Brest, Service d'anatomie et cytologie pathologiques, Brest, F-29220, France. matthieu.talagas@chu-brest.fr.|Université Européenne de Bretagne, Rennes, France. matthieu.talagas@chu-brest.fr.|Faculté de Médecine et des Sciences de la Santé Université de Brest, EA4685, Brest, F-29200, France. matthieu.talagas@chu-brest.fr.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Talagas
authors.affiliation: CHRU Brest, Service d'anatomie et cytologie pathologiques, Brest, F-29220, France. sebastian.costa@chu-brest.fr.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Costa
authors.affiliation: CHRU Brest, Service d'anatomie et cytologie pathologiques, Brest, F-29220, France. laura.samaison@gmail.com.|Université Européenne de Bretagne, Rennes, France. laura.samaison@gmail.com.|Faculté de Médecine et des Sciences de la Santé Université de Brest, EA4685, Brest, F-29200, France. laura.samaison@gmail.com.
authors.forename: Laura
authors.initials: L
authors.lastname: Samaison
authors.affiliation: CHRU Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Brest, F-29220, France. laure.paule@chu-brest.fr.
authors.forename: Laure
authors.initials: L
authors.lastname: Paule
authors.affiliation: Inserm, CIC1412, Brest, F-29200, France. zarrin.alavi@chu-brest.fr.
authors.forename: Zarrin
authors.initials: Z
authors.lastname: Alavi
authors.affiliation: Inserm, U1078, Brest, F-29200, France. marc.debraekeleer@chu-brest.fr.|Université Européenne de Bretagne, Rennes, France. marc.debraekeleer@chu-brest.fr.|CHRU Brest, Laboratoire de Cytogénétique et Biologie de la Reproduction, Brest, F-29220, France. marc.debraekeleer@chu-brest.fr.
authors.forename: Marc
authors.initials: M
authors.lastname: De Braekeleer
authors.affiliation: Inserm, U1078, Brest, F-29200, France. cedric.lemarechal@chu-brest.fr.|Université Européenne de Bretagne, Rennes, France. cedric.lemarechal@chu-brest.fr.|CHRU Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Brest, F-29220, France. cedric.lemarechal@chu-brest.fr.|Department of Molecular Genetics and Histocompatibily, University Hospital Morvan, 2, Avenue Foch 29609, Brest, France. cedric.lemarechal@chu-brest.fr.
authors.forename: Cédric
authors.initials: C
authors.lastname: Le Marechal
authors.affiliation: CHRU Brest, Service d'anatomie et cytologie pathologiques, Brest, F-29220, France. pascale.marcorelles@chu-brest.fr.|Université Européenne de Bretagne, Rennes, France. pascale.marcorelles@chu-brest.fr.|Faculté de Médecine et des Sciences de la Santé Université de Brest, EA4685, Brest, F-29200, France. pascale.marcorelles@chu-brest.fr.|Department of Pathology, University Hospital Morvan, 2, Avenue Foch 29609, Brest, France. pascale.marcorelles@chu-brest.fr.
authors.forename: Pascale
authors.initials: P
authors.lastname: Marcorelles
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008545:Melanoma / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D008565:Membrane Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human