title: Dual NRASQ61R and BRAFV600E mutation-specific immunohistochemistry completes molecular screening in melanoma samples in a routine practice.
abstract: NRAS and BRAF mutational status has become mandatory to treat patients with metastatic melanomas. Mutation-specific immunohistochemistry (IHC) can help analyze challenging tumor samples. We report our experience integrating NRASQ61R (SP174) and BRAFV600E (VE1) IHC in routine practice in a cancer molecular genetic platform. All samples screened for BRAF and NRAS mutations during the year 2014 were analyzed by IHC and pyrosequencing, with an independent analysis of the 2 methods. Cases with first-line discordant results benefited from a complementary second-round IHC and next-generation sequencing (NGS) with a final interpretation taking into account the results of pyrosequencing, IHC, NGS, and quantification of the tumor cells. We analyzed 111 consecutive formalin-fixed and paraffin-embedded melanoma samples from 101 patients. Twenty-two and 11 samples were concordant for BRAFV600E and NRASQ61R mutations, respectively. Second-round analyses of 9 discordant and 1 molecularly inconclusive samples allowed conclusion in 4 further mutated samples (2 BRAFV600E and 2 NRASQ61R). A sample remained NRASQ61R IHC negative but NRASQ61R mutated with molecular methods. Overall, BRAFV600 and NRASQ61 mutation frequencies were 31.7% and 30.7%, respectively. When compared to molecular results, the sensitivity and specificity of IHC were 100% for BRAFV600E IHC and 92.3% and 98.9% for NRASQ61R IHC, respectively. IHC interpretation required a more stringent cutoff for BRAFV600E IHC than NRASQ61R to minimize false results. We conclude that NRASQ61R and BRAFV600E IHC coupled with NGS allow detection of mutations in melanoma challenging samples.
authors: INSERM, U1078, Brest, F-29200 France; Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France; Université Européenne de Bretagne, Brest, F-29200 France. Electronic address: arnaud.uguen@chu-brest.fr.
authors: Arnaud
authors: A
authors: Uguen
authors: INSERM, U1078, Brest, F-29200 France; Université Européenne de Bretagne, Brest, F-29200 France; CHRU Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Brest, F-29220 France. Electronic address: paul.gueguen@chu-brest.fr.
authors: Paul
authors: P
authors: Guéguen
authors: Service de Dermatologie, CHRU Brest, Brest, F-29220 France.
authors: Delphine
authors: D
authors: Legoupil
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France. Electronic address: stephanie.bouvier4@gmail.com.
authors: Stéphanie
authors: S
authors: Bouvier
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France. Electronic address: sebastian.costa@chu-brest.fr.
authors: Sebastian
authors: S
authors: Costa
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France. Electronic address: sandrine.duigou@chu-brest.fr.
authors: Sandrine
authors: S
authors: Duigou
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France. Electronic address: gilles.lemasson@chu-brest.fr.
authors: Gilles
authors: G
authors: Lemasson
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France. Electronic address: francoise.lede@chu-brest.fr.
authors: Françoise
authors: F
authors: Ledé
authors: Institut de Cancérologie et Hématologie, CHRU Brest, Brest, F-29220 France. Electronic address: bruno.sassolas@chu-brest.fr.
authors: Bruno
authors: B
authors: Sassolas
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France; Université Européenne de Bretagne, Brest, F-29200 France; Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, F-29200 France. Electronic address: matthieu.talagas@chu-brest.fr.
authors: Matthieu
authors: M
authors: Talagas
authors: INSERM, U1078, Brest, F-29200 France; Université Européenne de Bretagne, Brest, F-29200 France; CHRU Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Brest, F-29220 France. Electronic address: claude.ferec@chu-brest.fr.
authors: Claude
authors: C
authors: Férec
authors: INSERM, U1078, Brest, F-29200 France; Université Européenne de Bretagne, Brest, F-29200 France; CHRU Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Brest, F-29220 France. Electronic address: cedric.lemarechal@chu-brest.fr.
authors: Cédric
authors: C
authors: Le Maréchal
authors: INSERM, U1078, Brest, F-29200 France; Université Européenne de Bretagne, Brest, F-29200 France; Service de Cytogénétique et Biologie de la Reproduction, CHRU Brest, Brest, F-29220 France. Electronic address: marc.debraekeleer@chu-brest.fr.
authors: Marc
authors: M
authors: De Braekeleer
authors: Service d'Anatomie et Cytologie Pathologiques, CHRU Brest, Brest, F-29220 France; Université Européenne de Bretagne, Brest, F-29200 France; Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, F-29200 France. Electronic address: pascale.marcorelles@chu-brest.fr.
authors: Pascale
authors: P
authors: Marcorelles
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D055088:Early Detection of Cancer; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008545:Melanoma / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D012878:Skin Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human
keywords: BRAF; Immunohistochemistry; Melanoma; Molecular analysis; NRAS