Heterogeneity of BRAF, NRAS, and TERT Promoter Mutational Status in Multiple Melanomas and Association with MC1R Genotype

Data on somatic heterogeneity and germline–somatic interaction in multiple primary melanoma (MPM) patients are limited. We investigated the mutational status of BRAF, NRAS, and TERT promoter genes in 97 melanomas of 44 MPM patients and compared molecular and immunohistochemical findings. We further evaluated the association of somatic alterations with the germline MC1R genotype. Mutations in BRAF gene were identified in 41.2% (40/97) of melanomas, in NRAS in 2.1% (2/97), and in TERT promoter in 19.6% (19/97). Distribution of BRAF mutations did not differ across multiple melanomas (P = 0.85), whereas TERT promoter changes decreased from first to subsequent melanomas (P = 0.04). Intrapatient discrepancy of BRAF mutations among multiple tumors was detected in 14 of 44 MPM patients (32%) and of BRAF/NRAS/TERT promoter genes in 20 of 44 (45%). We observed a high rate of agreement between allele-specific TaqMan assay and immunohistochemistry in BRAFV600E detection (κ = 0.83, P < 0.01) with 86 of 97 melanomas (88.7%) presenting similar BRAF status. Germline MC1R variants were identified in 81.4% (35/43) of MPM patients with no association of MC1R genotype with somatic mutations or with intrapatient concordance of somatic mutational profile. Our results support the genetic diversity of multiple melanomas and show that somatic heterogeneity is not influenced by inherited MC1R variants. Immunohistochemistry may be useful as an initial screening test. Data on somatic heterogeneity and germline–somatic interaction in multiple primary melanoma (MPM) patients are limited. We investigated the mutational status of BRAF, NRAS, and TERT promoter genes in 97 melanomas of 44 MPM patients and compared molecular and immunohistochemical findings. We further evaluated the association of somatic alterations with the germline MC1R genotype. Mutations in BRAF gene were identified in 41.2% (40/97) of melanomas, in NRAS in 2.1% (2/97), and in TERT promoter in 19.6% (19/97). Distribution of BRAF mutations did not differ across multiple melanomas (P = 0.85), whereas TERT promoter changes decreased from first to subsequent melanomas (P = 0.04). Intrapatient discrepancy of BRAF mutations among multiple tumors was detected in 14 of 44 MPM patients (32%) and of BRAF/NRAS/TERT promoter genes in 20 of 44 (45%). We observed a high rate of agreement between allele-specific TaqMan assay and immunohistochemistry in BRAFV600E detection (κ = 0.83, P < 0.01) with 86 of 97 melanomas (88.7%) presenting similar BRAF status. Germline MC1R variants were identified in 81.4% (35/43) of MPM patients with no association of MC1R genotype with somatic mutations or with intrapatient concordance of somatic mutational profile. Our results support the genetic diversity of multiple melanomas and show that somatic heterogeneity is not influenced by inherited MC1R variants. Immunohistochemistry may be useful as an initial screening test. Cutaneous melanoma is one of the most aggressive human cancers and is known for its rapid progression and poor prognosis in the advanced stages.1Lo J.A. Fisher D.E. The melanoma revolution: from UV carcinogenesis to a new era in therapeutics.Science. 2014; 346: 945-949Crossref PubMed Scopus (274) Google Scholar During the past few decades its incidence has been steadily rising among Caucasian populations and is predicted to continue increasing for at least two more decades.2Nikolaou V. Stratigos A.J. Emerging trends in the epidemiology of melanoma.Br J Dermatol. 2014; 170: 11-19Crossref PubMed Scopus (246) Google Scholar Patients diagnosed with a single primary cutaneous melanoma have an approximately ninefold higher risk of developing subsequent melanomas compared with the general population.3Bradford P.T. Freedman D.M. Goldstein A.M. Tucker M.A. Increased risk of second primary cancers after a diagnosis of melanoma.Arch Dermatol. 2010; 146: 265-272Crossref PubMed Scopus (167) Google Scholar The risk is highest within the first year after initial melanoma diagnosis, whereas it decreases but remains elevated for at least 20 years.3Bradford P.T. Freedman D.M. Goldstein A.M. Tucker M.A. Increased risk of second primary cancers after a diagnosis of melanoma.Arch Dermatol. 2010; 146: 265-272Crossref PubMed Scopus (167) Google Scholar, 4Murali R. Goumas C. Kricker A. From L. Busam K.J. Begg C.B. Dwyer T. Gruber S.B. Kanetsky P.A. Orlow I. Rosso S. Thomas N.E. Berwick M. Scolyer R.A. Armstrong B.K. GEM Study GroupClinicopathologic features of incident and subsequent tumors in patients with multiple primary cutaneous melanomas.Ann Surg Oncol. 2012; 19: 1024-1033Crossref PubMed Scopus (41) Google Scholar Multiple primary melanoma (MPM) accounts for 1.2% to 8.2% of all sporadic melanoma patients in Europe and for up to 23% in populations living in high incidence countries such as Australia.3Bradford P.T. Freedman D.M. Goldstein A.M. Tucker M.A. Increased risk of second primary cancers after a diagnosis of melanoma.Arch Dermatol. 2010; 146: 265-272Crossref PubMed Scopus (167) Google Scholar, 4Murali R. Goumas C. Kricker A. From L. Busam K.J. Begg C.B. Dwyer T. Gruber S.B. Kanetsky P.A. Orlow I. Rosso S. Thomas N.E. Berwick M. Scolyer R.A. Armstrong B.K. GEM Study GroupClinicopathologic features of incident and subsequent tumors in patients with multiple primary cutaneous melanomas.Ann Surg Oncol. 2012; 19: 1024-1033Crossref PubMed Scopus (41) Google Scholar, 5Savoia P. Osella-Abate S. Deboli T. Marenco F. Stroppiana E. Novelli M. Fierro M.T. Bernengo M.G. Clinical and prognostic reports from 270 patients with multiple primary melanomas: a 34-year single-institution study.J Eur Acad Dermatol Venereol. 2012; 26: 882-888Crossref PubMed Scopus (25) Google Scholar, 6Youlden D.R. Youl P.H. Soyer H.P. Aitken J.F. Baade P.D. Distribution of subsequent primary invasive melanomas following a first primary invasive or in situ melanoma in Queensland, Australia, 1982–2010.JAMA Dermatol. 2014; 150: 526-534Crossref PubMed Scopus (55) Google Scholar MPMs are prevalent in men and in older patients4Murali R. Goumas C. Kricker A. From L. Busam K.J. Begg C.B. Dwyer T. Gruber S.B. Kanetsky P.A. Orlow I. Rosso S. Thomas N.E. Berwick M. Scolyer R.A. Armstrong B.K. GEM Study GroupClinicopathologic features of incident and subsequent tumors in patients with multiple primary cutaneous melanomas.Ann Surg Oncol. 2012; 19: 1024-1033Crossref PubMed Scopus (41) Google Scholar, 5Savoia P. Osella-Abate S. Deboli T. Marenco F. Stroppiana E. Novelli M. Fierro M.T. Bernengo M.G. Clinical and prognostic reports from 270 patients with multiple primary melanomas: a 34-year single-institution study.J Eur Acad Dermatol Venereol. 2012; 26: 882-888Crossref PubMed Scopus (25) Google Scholar, 6Youlden D.R. Youl P.H. Soyer H.P. Aitken J.F. Baade P.D. Distribution of subsequent primary invasive melanomas following a first primary invasive or in situ melanoma in Queensland, Australia, 1982–2010.JAMA Dermatol. 2014; 150: 526-534Crossref PubMed Scopus (55) Google Scholar, 7Ferrone C.R. Ben Porat L. Panageas K.S. Berwick M. Halpern A.C. Patel A. Coit D.G. Clinicopathological features of and risk factors for multiple primary melanomas.JAMA. 2005; 294: 1647-1654Crossref PubMed Scopus (169) Google Scholar with subsequent melanomas being more frequently thinner7Ferrone C.R. Ben Porat L. Panageas K.S. Berwick M. Halpern A.C. Patel A. Coit D.G. Clinicopathological features of and risk factors for multiple primary melanomas.JAMA. 2005; 294: 1647-1654Crossref PubMed Scopus (169) Google Scholar, 8Siskind V. Hughes M.C. Palmer J.M. Symmons J.M. Aitken J.F. Martin N.G. Hayward N.K. Whiteman D.C. Nevi, family history, and fair skin increase the risk of second primary melanoma.J Invest Dermatol. 2011; 131: 461-467Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar and contiguous to a dysplastic nevus compared with initial tumors.4Murali R. Goumas C. Kricker A. From L. Busam K.J. Begg C.B. Dwyer T. Gruber S.B. Kanetsky P.A. Orlow I. Rosso S. Thomas N.E. Berwick M. Scolyer R.A. Armstrong B.K. GEM Study GroupClinicopathologic features of incident and subsequent tumors in patients with multiple primary cutaneous melanomas.Ann Surg Oncol. 2012; 19: 1024-1033Crossref PubMed Scopus (41) Google Scholar Melanoma arises through the gradual accumulation of genetic somatic abnormalities that involve critical signaling pathways such as the mitogen-activated protein kinase signal transduction cascade.9Wangari-Talbot J. Chen S. Genetics of melanoma.Front Genet. 2013; 3: 330Crossref PubMed Scopus (23) Google Scholar Oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations occur in approximately 50% of cutaneous melanomas mainly at codon 600 in exon 15, with the most common mutation (approximately 90% of the cases) being the V600E change.9Wangari-Talbot J. Chen S. Genetics of melanoma.Front Genet. 2013; 3: 330Crossref PubMed Scopus (23) Google Scholar Activating NRAS (neuroblastoma RAS viral oncogene homolog) mutations, mostly affecting exon 2 at codon 61, have been reported in approximately 20% of cutaneous melanomas.9Wangari-Talbot J. Chen S. Genetics of melanoma.Front Genet. 2013; 3: 330Crossref PubMed Scopus (23) Google Scholar Although the somatic BRAF and NRAS mutational status is of great interest for melanoma treatment, there is no consensus so far on the best testing method.10Bruno W. Martinuzzi C. Andreotti V. Pastorino L. Spagnolo F. Dalmasso B. Cabiddu F. Gualco M. Ballestrero A. Bianchi-Scarrà G. Queirolo P. Grillo F. Mastracci L. Ghiorzo P. Italian Melanoma Intergroup (IMI)Heterogeneity and frequency of BRAF mutations in primary melanoma: comparison between molecular methods and immunohistochemistry.Oncotarget. 2017; 8: 8069-8082Crossref PubMed Scopus (27) Google Scholar Besides BRAF and NRAS, recent studies identified recurrent somatic mutations in the promoter region of the telomerase reverse transcriptase (TERT) gene occurring in early stages of melanoma development.11Heidenreich B. Nagore E. Rachakonda P.S. Garcia-Casado Z. Requena C. Traves V. Becker J. Soufir N. Hemminki K. Kumar R. Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma.Nat Commun. 2014; 5: 3401Crossref PubMed Scopus (139) Google Scholar, 12Kanetsky P.A. Rebbeck T.R. Hummer A.J. Panossian S. Armstrong B.K. Kricker A. Marrett L.D. Millikan R.C. Gruber S.B. Culver H.A. Zanetti R. Gallagher R.P. Dwyer T. Busam K. From L. Mujumdar U. Wilcox H. Begg C.B. Berwick M. Population-based study of natural variation in the melanocortin-1 receptor gene and melanoma.Cancer Res. 2006; 66: 9330-9337Crossref PubMed Scopus (104) Google Scholar The MC1R (melanocortin-1 receptor) gene is a key determinant of human pigmentation and specific germline allelic variants, defined R variants, show the strongest effect on melanoma susceptibility.12Kanetsky P.A. Rebbeck T.R. Hummer A.J. Panossian S. Armstrong B.K. Kricker A. Marrett L.D. Millikan R.C. Gruber S.B. Culver H.A. Zanetti R. Gallagher R.P. Dwyer T. Busam K. From L. Mujumdar U. Wilcox H. Begg C.B. Berwick M. Population-based study of natural variation in the melanocortin-1 receptor gene and melanoma.Cancer Res. 2006; 66: 9330-9337Crossref PubMed Scopus (104) Google Scholar MPM patients have an increased probability to harbor two or more MC1R variants compared with patients with a single primary melanoma.13Goldstein A.M. Landi M.T. Tsang S. Fraser M.C. Munroe D.J. Tucker M.A. Association of MC1R variants and risk of melanoma in melanoma-prone families with CDKN2A mutations.Cancer Epidemiol Biomarkers Prev. 2005; 14: 2208-2212Crossref PubMed Google Scholar, 14Pastorino L. Bonelli L. Ghiorzo P. Queirolo P. Battistuzzi L. Balleari E. Nasti S. Gargiulo S. Gliori S. Savoia P. Abate Osella S. Bernengo M.G. Bianchi Scarrà G. CDKN2A mutations and MC1R variants in Italian patients with single or multiple primary melanoma.Pigment Cell Melanoma Res. 2008; 21: 700-709Crossref PubMed Scopus (43) Google Scholar, 15Puntervoll H.E. Yang X.R. Vetti H.H. Bachmann I.M. Avril M.F. Benfodda M. Catricalà C. Dalle S. Duval-Modeste A.B. Ghiorzo P. Grammatico P. Harland M. Hayward N.K. Hu H.H. Jouary T. Martin-Denavit T. Ozola A. Palmer J.M. Pastorino L. Pjanova D. Soufir N. Steine S.J. Stratigos A.J. Thomas L. Tinat J. Tsao H. Veinalde R. Tucker M.A. Bressac-de Paillerets B. Newton-Bishop J.A. Goldstein A.M. Akslen L.A. Molven A. Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants.J Med Genet. 2013; 50: 264-270Crossref PubMed Scopus (35) Google Scholar A synergistic association between somatic BRAF mutations and germline MC1R variants, with MC1R genotype conferring an increased risk of developing BRAF-mutated melanomas, was observed in Italian16Fargnoli M.C. Pike K. Pfeiffer R.M. Tsang S. Rozenblum E. Munroe D.J. Golubeva Y. Calista D. Seidenari S. Massi D. Carli P. Bauer J. Elder D.E. Bastian B.C. Peris K. Landi M.T. MC1R variants increase risk of melanomas harboring BRAF mutations.J Invest Dermatol. 2008; 128: 2485-2490Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar and US patients17Landi M.T. Bauer J. Pfeiffer R.M. Elder D.E. Hulley B. Minghetti P. Calista D. Kanetsky P.A. Pinkel D. Bastian B.C. MC1R germline variants confer risk for BRAF-mutant melanoma.Science. 2006; 313: 521-522Crossref PubMed Scopus (269) Google Scholar but not confirmed in other populations.18Thomas N.E. Kanetsky P.A. Edmiston S.N. Alexander A. Begg C.B. Groben P.A. Hao H. Busam K. Ollila D.W. Berwick M. Conway K. Relationship between germline MC1R variants and BRAF-mutant melanoma in a North Carolina population-based study.J Invest Dermatol. 2010; 130: 1463-1465Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Hacker E. Nagore E. Cerroni L. Woods S.L. Hayward N.K. Chapman B. Montgomery G.W. Soyer H.P. Whiteman D.C. NRAS and BRAF mutations in cutaneous melanoma and the association with MC1R genotype: findings from Spanish and Austrian populations.J Invest Dermatol. 2013; 133: 1027-1033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 20Scherer D. Rachakonda P.S. Angelini S. Mehnert F. Sucker A. Egberts F. Hauschild A. Hemminki K. Schadendorf D. Kumar R. Association between the germline MC1R variants and somatic BRAF/NRAS mutations in melanoma tumors.J Invest Dermatol. 2010; 130: 2844-2848Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 21Hacker E. Hayward N.K. Dumenil T. James M.R. Whiteman D.C. The association between MC1R genotype and BRAF mutation status in cutaneous melanoma: findings from an Australian population.J Invest Dermatol. 2010; 130: 241-248Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Recently, the entire somatic mutational load in melanoma has been shown to be influenced by germline MC1R variants with a higher somatic mutational burden in melanomas of patients carrying R variants compared with those of noncarriers, thus supporting a germline-somatic interaction.22Robles-Espinoza C.D. Roberts N.D. Chen S. Leacy F.P. Alexandrov L.B. Pornputtapong N. Halaban R. Krauthammer M. Cui R. Timothy Bishop D. Adams D.J. Germline MC1R status influences somatic mutation burden in melanoma.Nat Commun. 2016; 7: 12064Crossref PubMed Scopus (83) Google Scholar To date, only two studies investigated the somatic heterogeneity of multiple melanomas within the same patient, evaluating alterations in main candidate genes by molecular methods.23Colombino M. Sini M. Lissia A. De Giorgi V. Stanganelli I. Ayala F. Massi D. Rubino C. Manca A. Paliogiannis P. Rossari S. Magi S. Mazzoni L. Botti G. Capone M. Palla M. Ascierto P.A. Cossu A. Palmieri G. Italian Melanoma Intergroup (IMI)Discrepant alterations in main candidate genes among multiple primary melanomas.J Transl Med. 2014; 12: 117Crossref PubMed Scopus (25) Google Scholar, 24Egberts F. Bohne A.S. Krüger S. Hedderich J. Rompel R. Haag J. Röcken C. Hauschild A. Varying mutational alterations in multiple primary melanomas.J Mol Diagn. 2016; 18: 75-83Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar However, neither of the two analyzed the possible influence of germline mutations on the somatic diversity of MPM. In this study, we investigated the mutational status of BRAF, NRAS, and TERT promoter genes in MPM patients to evaluate the intrapatient genetic heterogeneity of multiple melanomas and to compare the consistency of mutational findings obtained by molecular analysis and by immunohistochemistry. In addition, we explored the potential influence of germline MC1R genotype on the occurrence of somatic mutations. Patients with histologically proven diagnosis of cutaneous MPM followed at the Department of Dermatology, University of L'Aquila (in 2010 to 2015), and at the Institute of Dermatology, Catholic University of the Sacred Heart, Rome, Italy (in 2014 to 2015), were included in the study. For each patient, synchronous (diagnosis of a second melanoma within 6 months from the first diagnosis) and metachronous multiple primary melanomas (including in situ) were retrieved. Clinical information such as sex, age at diagnosis, anatomic site, family history of melanoma, personal and family history of other cutaneous or visceral neoplasms, and phenotypic characteristics such as hair color, eye color, number of melanocytic nevi, and presence or absence of clinically atypical nevi were collected for each patient. Hematoxylin and eosin–stained sections of all melanomas were reviewed by two pathologists (G.C. and M.D.P.) to confirm the histopathologic diagnosis. Features of tumors, including histopathologic variant [superficial spreading melanoma (SSM), nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and others], Breslow thickness, and presence of melanoma-associated nevus were recorded. Representative sections from each melanoma were selected for immunohistochemistry (IHC), and tumor-rich areas were marked for molecular analysis. Approval for this study was obtained from the local ethics committees (ASL L'Aquila-Teramo, protocol no. 30/CE15; Fondazione Policlinico Universitario Gemelli, protocol no. 25779/14). A written informed consent was signed by all patients. The study was performed according to the Helsinki Declaration. Somatic DNA was extracted from five formalin-fixed, paraffin-embedded (FFPE) tissue sections (each of 10-μ thickness) by microdissection of marked melanoma tissue with the use of a QIAmp Micro tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The coding sequence and splice junctions of exon 15 of BRAF (NG_007873.2), exon 3 of NRAS (NG_007572.1), and core promoter region of TERT gene (NC_000005.9: 1,295,149 to 1,295,267, hg19 GRCh37) were screened by Sanger sequencing, and the BRAFV600E, BRAFV600K, and NRASQ61R mutations were also analyzed by competitive allele-specific TaqMan PCR (castPCR Technology) to overcome the limit of detection of Sanger sequencing. For germline analysis, the DNA was extracted from whole blood with the use of a QIAamp DNA-blood midi kit (Qiagen). Mutational screening of the entire open reading frame of MC1R (NG_012026.1) was performed by direct sequencing as above. For Sanger sequencing analysis, PCR amplification of the regions of interest was performed in a Simply-Amp PCR-System (Thermo Fisher, Foster City, CA), using previously reported primers.25Massi D. Simi L. Sensi E. Baroni G. Xue G. Scatena C. Caldarella A. Pinzani P. Fontanini G. Carobbio A. Urso C. Mandalà M. 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, 26Chen Y.L. Jeng Y.M. Chang C.N. Lee H.J. Hsu H.C. Lai P.L. Yuan R.H. TERT promoter mutation in resectable hepatocellular carcinomas: a strong association with hepatitis C infection and absence of hepatitis B infection.Int J Surg. 2014; 12: 659-665Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 27Pellegrini C. Maturo M.G. Martorelli C. Suppa M. Antonini A. Kostaki D. Verna L. Landi M.T. Peris K. Fargnoli M.C. Characterization of melanoma susceptibility genes in high-risk patients from Central Italy.Melanoma Res. 2017; 27: 258-267Crossref PubMed Scopus (23) Google Scholar PCR experiments were performed with 1.25 U of AmpliTaq Gold-360 (Thermo Fisher) in a 50-μL volume, containing the 1X reaction buffer provided by the manufacturer, 1.6 mmol/L of MgCl2, 200 μmol/L of each deoxynucleoside triphosphate, 0.2 μmol/L of each primer, and 30 ng of genomic DNA template. Five percent dimethyl sulfoxide or 5% glycerol (for TERT promoter amplification) were added to the reaction solution. PCR amplification was performed as previously described.27Pellegrini C. Maturo M.G. Martorelli C. Suppa M. Antonini A. Kostaki D. Verna L. Landi M.T. Peris K. Fargnoli M.C. Characterization of melanoma susceptibility genes in high-risk patients from Central Italy.Melanoma Res. 2017; 27: 258-267Crossref PubMed Scopus (23) Google Scholar Water controls and positive controls were run in parallel with DNA of samples. Sequencing of amplicons was performed with the 3500 Genetic Analyzer (Thermo Fisher). A nucleotide sequence was considered valid when the quality value was >20 (<1/100 error probability). The variants were detected using the Applied Biosystems Minor Variant Finder software version 1.0 (Thermo Fisher), specific to calling low-frequency somatic variants. For competitive allele-specific TaqMan assay, PCRs containing 20 ng of DNA, 1X TaqMan Mutation Detection Assays (assay Hs00000111_mu for BRAFV600E, Hs000000002_rm for BRAFV600K, Hs000000083_rm for NRASQ61R), 1X TaqMan Genotyping Master Mix (Thermo Fisher), and water to reach the final volume of 20 μL were performed in 96-well plates with the use of the standard TaqMan protocol on 7500 Fast Real Time-PCR System (Thermo Fisher). Five DNA samples extracted from FFPE sections of melanomas positive for BRAFV600E or BRAFV600K and three extracted from FFPE sections of normal skin of unaffected individuals were used to set the ΔCt cutoff for experiments of mutation detection. Immunohistochemical analysis was performed on FFPE sections of 4-μm thickness, obtained from the same tissue block used for molecular analysis. BRAFV600E and NRASQ61R mutants were evaluated with the following monoclonal antibodies: BRAFV600E VE1 clone (Spring Bioscience, Pleasanton, CA) and NRASQ61R SP174 clone (Spring Bioscience) at a dilution of 1:30 and 1:80, respectively. For both BRAFV600E and NRASQ61R IHC, sections were freshly cut, dried at 60°C for 30 minutes, deparaffinized, and rehydrated. Immunoreactions were performed on Ventana BenchMark XT immune stainer (Ventana Medical Systems Inc., Tucson, AZ) with the use of the ultraView Universal RED Detection Kit, as previously reported.25Massi D. Simi L. Sensi E. Baroni G. Xue G. Scatena C. Caldarella A. Pinzani P. Fontanini G. Carobbio A. Urso C. Mandalà M. 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 No chromogen was detected when primary antibody was omitted. Positive and negative controls were mounted on each section subjected to immunostaining. Glass slides for immunohistochemistry were gifted from USC Diagnostic (Rome, Italy). The evaluation of IHC status was performed independently by two observers (G.C. and M.D.P.) blinded to the mutational molecular status; disagreement was resolved by consensus. Cytoplasmic staining of BRAFV600E VE1 or NRASQ61R SP174 antibodies in melanoma cells was interpreted as positive or negative. In detail, negative staining was defined as absence of any cytoplasmic labeling either in single interspersed melanoma cells (<10%) or in cells of histiocytic/macrophage lineage; positive staining was classified as homogeneous (staining in >95% of cells) or heterogeneous (staining in <95% of cells) according to the percentage of cytoplasmic staining in melanoma cells, as previous described.10Bruno W. Martinuzzi C. Andreotti V. Pastorino L. Spagnolo F. Dalmasso B. Cabiddu F. Gualco M. Ballestrero A. Bianchi-Scarrà G. Queirolo P. Grillo F. Mastracci L. Ghiorzo P. Italian Melanoma Intergroup (IMI)Heterogeneity and frequency of BRAF mutations in primary melanoma: comparison between molecular methods and immunohistochemistry.Oncotarget. 2017; 8: 8069-8082Crossref PubMed Scopus (27) Google Scholar, 28Tetzlaff M.T. Pattanaprichakul P. Wargo J. Fox P.S. Patel K.P. Estrella J.S. Broaddus R.R. Williams M.D. Davies M.A. Routbort M.J. Lazar A.J. Woodman S.E. Hwu W.J. Gershenwald J.E. Prieto V.G. Torres-Cabala C.A. Curry J.L. Utility of BRAF V600E immunohistochemistry expression pattern as a surrogate of BRAF mutation status in 154 patients with advanced melanoma.Hum Pathol. 2015; 46: 1101-1110Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Intensity of the staining was graded as weak, moderate, or strong.10Bruno W. Martinuzzi C. Andreotti V. Pastorino L. Spagnolo F. Dalmasso B. Cabiddu F. Gualco M. Ballestrero A. Bianchi-Scarrà G. Queirolo P. Grillo F. Mastracci L. Ghiorzo P. Italian Melanoma Intergroup (IMI)Heterogeneity and frequency of BRAF mutations in primary melanoma: comparison between molecular methods and immunohistochemistry.Oncotarget. 2017; 8: 8069-8082Crossref PubMed Scopus (27) Google Scholar Descriptive statistics and molecular and IHC data are given as means, medians, or proportions, as appropriate. Variables were categorized as follows: median age at melanoma diagnosis (≤40 years, >40 years), number of common melanocytic nevi (≤50, >50), number of primary melanomas (2, >2), occurrence of synchronous melanomas, histopathologic subtype (SSM or other subtypes), melanoma thickness (in situ, invasive; Breslow thickness ≤1 mm, >1 mm), and melanoma anatomic site (head/neck, trunk, extremities). For MC1R analysis, we evaluate the association of germline variants with a patient's phenotypical characteristics as skin type (I/II, III/IV), hair color (red/blond, light brown, dark brown/black), and eye color (blue/green, light brown, dark brown). The R142H, R151C, R160W, D294H, and I155T MC1R variants were considered as R variants, whereas all of the others were termed as r.18Thomas N.E. Kanetsky P.A. Edmiston S.N. Alexander A. Begg C.B. Groben P.A. Hao H. Busam K. Ollila D.W. Berwick M. Conway K. Relationship between germline MC1R variants and BRAF-mutant melanoma in a North Carolina population-based study.J Invest Dermatol. 2010; 130: 1463-1465Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Synonymous variants were considered as wild type. Molecular findings by allele-specific TaqMan assays were used as the gold standard for statistical analysis. Semiquantitative data (age at diagnosis, Breslow thickness) were analyzed by means of t-test or by medians with U-test. Univariate analysis by χ2 test or by Fisher exact test was used to test the significance of mutation frequency according to clinical characteristics of melanoma patients and clinicopathologic features of melanoma lesions. Odds ratio was calculated to determine the magnitude of differences among mutated and wild-type melanoma groups. Cohen κ coefficient test was used to measure the agreement between molecular and IHC methods in determining BRAF mutational status. In general, P < 0.05 was considered statistically significant. Statistical analysis was performed with the statistical package SPSS software version 17.0 (SPSS Incorporated, Chicago, IL). Demographic and clinical characteristics of patients and related tumors are summarized in Table 1.Table 1Demographic and Clinical Characteristics of Melanoma Patients (n = 44) and Histopathologic Features of Tumors (n = 97)CharacteristicValuePatients SexMale24 (54.6)Female20 (45.4) Age, yearsMedian (range)49 (15–80)≤4017 (38.6)>4027 (61.4) Nevus count∗Numbers do not add up to the total because of missing data.≤5012 (27.3)>5031 (70.4) Clinically atypical nevi∗Numbers do not add up to the total because of missing data.No28 (63.6)Yes11 (25.0) Skin typeI5 (11.4)II22 (50.0)III14 (31.8)IV3 (6.8) No. of primary melanomas235 (79.5)39 (20.5) Family history of melanomaYes9 (20.4)No35 (79.5) Synchronous/metachronous†One patient with three melanomas developed two synchronous tumors and the third tumor 2 years later was excluded.Synchronous10 (22.2)Metachronous33 (75.0)Tumors Breslow thicknessIn situ58 (59.8)Invasive39 (40.2)≤1 mm34 (35.1)>1 mm5 (5.1)Median (range), mm0.5 (0.2–2.0) Histopathologic subtypeSSM94 (97.0)NM1 (1.0)LM/LMM1 (1.0)Others (spitzoid)1 (1.0) Anatomic siteHead/neck1 (1.0)Trunk57 (58.8)Extremities39 (40.2) Nevus associationYes39 (40.2)No58 (59.8)Values are n (%) unless otherwise specified.LM/LMM, lentigo maligna/lentigo maligna melanoma; NM, nodular melanoma; SSM, superficial spreading melanoma.∗ Numbers do not add up to the total because of missing data.† One patient with three melanomas developed two synchronous tumors and the third tumor 2 years later was excluded. Open table in a new tab Values are n (%) unless otherwise specified. LM/LMM, lentigo maligna/lentigo maligna melanoma; NM, nodular melanoma; SSM, superficial spreading melanoma. Samples of primary and subsequent melanomas were obtained from 44 MPM patients with stage I to II (24 men and 20 women; median age at first diagnosis, 49.0 years; range, 15 to 80 years). We analyzed a total of 97 melanoma tissues (35 patients [79.5%] with two paired tumor tissues, and nine [20.5%] with three tumor tissues; mean, 2.2 melanomas per patient). Synchronous melanomas occurred in 10 MPM patients (22.2%): nine patients with two melanomas and one patient with three melanomas. Metachronous lesions were diagnosed in 33 MPM patients (75.0%); one patient with three melanomas developed two synchr