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Genetic Abnormalities in Large to Giant Congenital Nevi: Beyond NRAS Mutations

  • Vanessa Martins da Silva
    Affiliations
    Melanoma Unit, Department of Dermatology, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Catalonia, Spain
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  • Estefania Martinez-Barrios
    Affiliations
    Department of Biochemical and Molecular Genetics, Hospital Clínic, IDIBAPS, University of Barcelona, Catalonia, Spain
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  • Gemma Tell-Martí
    Affiliations
    Melanoma Unit, Department of Dermatology, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Catalonia, Spain

    Centro de Investigación Biomédica en Red en Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Catalonia, Spain
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  • Marc Dabad
    Affiliations
    CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Catalonia, Spain

    Universitat Pompeu Fabra, Barcelona, Catalonia, Spain
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  • Cristina Carrera
    Affiliations
    Melanoma Unit, Department of Dermatology, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Catalonia, Spain

    Centro de Investigación Biomédica en Red en Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Catalonia, Spain
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  • Paula Aguilera
    Affiliations
    Melanoma Unit, Department of Dermatology, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Catalonia, Spain

    Centro de Investigación Biomédica en Red en Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Catalonia, Spain
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  • Daniel Brualla
    Affiliations
    Department of Pediatric Dermatology, Hospital San Joan de Déu, Barcelona, Spain
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  • Anna Esteve-Codina
    Affiliations
    CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Catalonia, Spain

    Universitat Pompeu Fabra, Barcelona, Catalonia, Spain
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  • Asunción Vicente
    Affiliations
    Department of Pediatric Dermatology, Hospital San Joan de Déu, Barcelona, Spain
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  • Susana Puig
    Affiliations
    Melanoma Unit, Department of Dermatology, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Catalonia, Spain

    Centro de Investigación Biomédica en Red en Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Catalonia, Spain
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  • Author Footnotes
    8 These authors equally contributed to this work.
    Joan Anton Puig-Butillé
    Correspondence
    Correspondence: Joan Anton Puig-Butille, Hospital Clínic de Barcelona, Molecular Biology CORE, Biochemistry and Molecular Genetics Department, C/Villarroel, 170, Esc 7-5, 08036 Barcelona, Spain.
    Footnotes
    8 These authors equally contributed to this work.
    Affiliations
    Department of Biochemical and Molecular Genetics, Hospital Clínic, IDIBAPS, University of Barcelona, Catalonia, Spain

    Centro de Investigación Biomédica en Red en Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Catalonia, Spain

    Molecular Biology CORE, Hospital Clínic, IDIBAPS, University of Barcelona, Catalonia, Spain
    Search for articles by this author
  • Author Footnotes
    8 These authors equally contributed to this work.
    Josep Malvehy
    Footnotes
    8 These authors equally contributed to this work.
    Affiliations
    Melanoma Unit, Department of Dermatology, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Catalonia, Spain

    Department of Biochemical and Molecular Genetics, Hospital Clínic, IDIBAPS, University of Barcelona, Catalonia, Spain
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  • Author Footnotes
    8 These authors equally contributed to this work.
Open ArchivePublished:October 22, 2018DOI:https://doi.org/10.1016/j.jid.2018.07.045
      Large and giant congenital melanocytic nevi (CMN) are rare melanocytic lesions mostly caused by postzygotic NRAS alteration. Molecular characterization is usually focused on NRAS and BRAF genes in a unique biopsy sample of the CMN. However, large/giant CMN may exhibit phenotypic differences among distinct areas, and patients differ in features such as presence of multiple CMN or spilus-like lesions. Herein, we have characterized a series of 21 large/giant CMN including patients with spilus-type nevi (9/21 patients, 42.8%). Overall, 53 fresh frozen biopsy samples corresponding to 40 phenotypically characterized areas of large/giant CMNs and 13 satellite lesions were analyzed with a multigene panel and RNA sequencing. Mutational screening showed mutations in 76.2% (16/21) of large/giant CMNs. A NRAS mutation was found in 57.1% (12/21) of patients, and mutations in other genes such as BRAF, KRAS, APC, and MET were detected in 14.3% (3/21) of patients. RNA sequencing showed the fusion transcript ZEB2-ALK and SOX5-RAF1 in large/giant CMN from two patients without missense mutations. Both alterations were not detected in unaffected skin and were detected in different areas of affected skin. These findings suggest that large/giant CMN may result from distinct molecular events in addition to NRAS mutations, including point mutations and fusion transcripts.

      Abbreviation:

      CMN (congenital melanocytic nevus)

      Introduction

      Congenital melanocytic nevi (CMNs) are benign melanocytic tumors developed in utero as result of postzygotic somatic mutations and are clinically classified according to the maximal diameter predicted to attain in adulthood. Large and giant CMN are lesions with diameters larger than 20 cm or 40 cm in adult life, respectively (
      • Krengel S.
      • Scope A.
      • Dusza S.W.
      • Vonthein R.
      • Marghoob A.A.
      New recommendations for the categorization of cutaneous features of congenital melanocytic nevi.
      ). CMNs larger than 20 cm are rare and have an estimated incidence of 1 in 20,000 (
      • Castilla E.E.
      • da Graça Dutra M.
      • Orioli-Parreiras I.M.
      Epidemiology of congenital pigmented naevi: I. incidence rates and relative frequencies.
      ). The clinical importance of CMNs is the increased risk of melanomas and other malignancies observed in these patients. The size of the lesion is one of the major predictors for melanoma development, alongside other factors such as number of satellites and abnormal magnetic resonance image of the central nervous system in the first year of life (
      • Kinsler V.A.
      • O’Hare P.
      • Bulstrode N.
      • Calonje J.E.
      • Chong W.K.
      • Hargrave D.
      • et al.
      Melanoma in congenital melanocytic naevi.
      ,
      • Krengel S.
      • Hauschild A.
      • Schäfer T.
      Melanoma risk in congenital melanocytic naevi: a systematic review.
      ). The term satellite describes any smaller melanocytic lesion around a large one; however, a satellite can be of similar size or be far from the largest.
      • Kinsler V.
      Satellite lesions in congenital melanocytic nevi—time for a change of name.
      proposed categorizing patients as single or multiple CMN cases. Additional categorization has been established based on phenotypical features such as the Krengel classification (
      • Krengel S.
      • Scope A.
      • Dusza S.W.
      • Vonthein R.
      • Marghoob A.A.
      New recommendations for the categorization of cutaneous features of congenital melanocytic nevi.
      ) or based on body distribution such as the 6B classification (
      • Martins da Silva V.P.
      • Marghoob A.
      • Pigem R.
      • Carrera C.
      • Aguilera P.
      • Puig-Butillé J.A.
      • et al.
      Patterns of distribution of giant congenital melanocytic nevi (GCMN): the 6B rule.
      ).
      Most CMNs result from somatic alterations in NRAS or BRAF genes. NRAS alterations (p.Q61K, p.Q61R) are detected in approximately 80% of patients with large/giant CMN or multiple CMN (
      • Bauer J.
      • Curtin J.A.
      • Pinkel D.
      • Bastian B.C.
      Congenital melanocytic nevi frequently harbor NRAS mutations but no BRAF mutations.
      ,
      • Charbel C.
      • Fontaine R.H.
      • Malouf G.G.
      • Picard A.
      • Kadlub N.
      • El-Murr N.
      • et al.
      NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi.
      ,
      • Dessars B.
      • De Raeve L.E.
      • Morandini R.
      • Lefort A.
      • El Housni H.
      • Ghanem G.E.
      • et al.
      Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.
      ,
      • Kinsler V.A.
      • Thomas A.C.
      • Ishida M.
      • Bulstrode N.W.
      • Loughlin S.
      • Hing S.
      • et al.
      Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS.
      ,
      • Salgado C.M.
      • Basu D.
      • Nikiforova M.
      • Bauer B.S.
      • Johnson D.
      • Rundell V.
      • et al.
      BRAF mutations are also associated with neurocutaneous melanocytosis and large/giant congenital melanocytic nevi.
      ), and BRAF mutations are frequently detected in small lesions (
      • Ichii-Nakato N.
      • Takata M.
      • Takayanagi S.
      • Takashima S.
      • Lin J.
      • Murata H.
      • et al.
      High frequency of BRAFV600E mutation in acquired nevi and small congenital nevi, but low frequency of mutation in medium-sized congenital nevi.
      ).
      • Kinsler V.A.
      • Thomas A.C.
      • Ishida M.
      • Bulstrode N.W.
      • Loughlin S.
      • Hing S.
      • et al.
      Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS.
      analyzed NRAS status in multiple CMN biopsy samples from each patient and found that lesions from the same patient present the same mutational status.
      There is a subset of CMN, termed spilus-type CMN, that present as large cafe-au-lait macules with superimposed macular or papular speckled lesions. These speckled spots can exhibit a wide variety of colors and sizes (
      • Kinsler V.A.
      • Krengel S.
      • Riviere J.-B.
      • Waelchli R.
      • Chapusot C.
      • Al-Olabi L.
      • et al.
      Next-generation sequencing of nevus spilus-type congenital melanocytic nevus: exquisite genotype-phenotype correlation in mosaic RASopathies.
      ,
      • Schaffer J.V.
      • Orlow S.J.
      • Lazova R.
      • Bolognia J.L.
      Speckled lentiginous nevus: within the spectrum of congenital melanocytic nevi.
      ). Less recurrent NRAS mutations have been observed in this subtype of lesions (
      • Dessars B.
      • De Raeve L.E.
      • Morandini R.
      • Lefort A.
      • El Housni H.
      • Ghanem G.E.
      • et al.
      Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.
      ,
      • Kinsler V.A.
      • Krengel S.
      • Riviere J.-B.
      • Waelchli R.
      • Chapusot C.
      • Al-Olabi L.
      • et al.
      Next-generation sequencing of nevus spilus-type congenital melanocytic nevus: exquisite genotype-phenotype correlation in mosaic RASopathies.
      ,
      • Krengel S.
      • Widmer D.S.
      • Kerl K.
      • Levesque M.P.
      • Schiestl C.
      • Weibel L.
      Naevus spilus-type congenital melanocytic naevus associated with a novel NRAS codon 61 mutation.
      ,
      • Shih F.
      • Yip S.
      • McDonald P.J.
      • Chudley A.E.
      • Del Bigio M.R.
      Oncogenic codon 13 NRAS mutation in a primary mesenchymal brain neoplasm and nevus of a child with neurocutaneous melanosis.
      ).
      The aim of this study was to analyze clinical and molecular characteristics of a cohort of patients with large/giant CMNs or spilus–type CMNs. We conducted a molecular characterization of different areas of CMNs or satellites using next-generation sequencing approaches such a multigene panel and RNA sequencing.

      Results

      Clinical characteristics of patients with large/giant CMNs

      Twenty-one patients with large/giant CMNs were included in this study. The clinical characteristics are indicated in Table 1. The median age of the patients was 27 years (range = 0.12–64 years). The male/female sex ratio was 0.50 (7 males/14 females). The phenotype of patients were classified according to the Krengel classification (
      • Krengel S.
      • Widmer D.S.
      • Kerl K.
      • Levesque M.P.
      • Schiestl C.
      • Weibel L.
      Naevus spilus-type congenital melanocytic naevus associated with a novel NRAS codon 61 mutation.
      ) and 6B pattern of distribution (
      • Martins da Silva V.P.
      • Marghoob A.
      • Pigem R.
      • Carrera C.
      • Aguilera P.
      • Puig-Butillé J.A.
      • et al.
      Patterns of distribution of giant congenital melanocytic nevi (GCMN): the 6B rule.
      ) (see Supplementary Table S1 online for definitions). According to the Krengel classification, 76.2% of patients had a giant CMN (projected adult size > 40 cm), and 57.1% of them had more than 50 satellites. The most frequently found characteristics among these patients were moderate color heterogeneity (47.6% of patients), no surface rugosity (47.6% of patients), no nodularity (47.6% of patients), and no hypertrichosis (61.9% of patients).
      Table 1Clinical Characteristics Patients with Large or Giant CMNs
      Patient NumberSexAge at Biopsy DateCMN Subgroup6B Pattern of DistributionKrengel ClassificationMelanoma HistoryNCM HistoryNumber of CMN Biopsy SamplesNumber of Satellite Biopsies
      1F43ClassicBoleroG2 C1 R1 N0 H2 S3NoNo11
      2F58ClassicBoleroG1 C1 R2 N2 H1 S3NoNo11
      3M19ClassicBoleroG1 C2 R1 N2 H0 S3NoNo11
      4M15ClassicBathing trunkG2 C2 R1 N0 H2 S3NoNo21
      5F14ClassicBathing trunkG2 C2 R2 N2 H0 S2NoNo11
      6F47ClassicBoleroG2 C0 R2 N2 H0 S3NoNo11
      7F15ClassicBodyG2 C0 R0 N2 H0 S3NoNo21
      8F0.12ClassicExtremityG1 C2 R1 N1 H1 S3NoNo11
      9M0.33ClassicBodyG2 C2 R1 N1 H0 S3NoNo31
      10F4ClassicBodyG2 C2 R1 N1 H1 S3NoYes30
      11F30ClassicNAL1 C1 R0 N2 H1 S3NoNo10
      12F2.6ClassicNAL1 C1 R1 N0 H2 S0NoNo10
      13M20Spilus typeBathing trunkG2 C0 R0 N0 H0 S2NoNo31
      14M3Spilus typeBathing trunkG1 C1 R0 N0 H0 S1YesNo11
      15F53Spilus typeBackG1 C2 R0 N0 H0 S3NoNo20
      16F20Spilus typeNAL1 C1 R1 N1 H0 S0NoNo70
      17M56Spilus typeNAL1 C1 R0 N0 H0 S1Yes
      The patient developed three primary melanomas during adulthood within the CMN lesion.
      No31
      18M11Spilus typeBathing trunkG2 C1 R0 N0 H0 S1NoNo10
      19F56Spilus typeBathing trunkG2 C1 R0 N0 H0 S0Yes
      Melanoma did not develop within the CMN lesion. The patient developed a leiomyosarcoma within the area of the CMN in adulthood.
      No20
      20F36Spilus typeBreast/bellyG1 C2 R0 N1 H1 S3NoNo21
      21F64Spilus typeNAL1 C1 R0 N0 H0 S0YesNo10
      Abbreviations: CMN, congenital melanocytic nevi; F, female; M, male; NA, not applicable (i.e., non-giant CMN).
      1 The patient developed three primary melanomas during adulthood within the CMN lesion.
      2 Melanoma did not develop within the CMN lesion. The patient developed a leiomyosarcoma within the area of the CMN in adulthood.
      Patients were classified as having classic CMN phenotype or spilus-like CMNs based on clinical characteristics of the lesion. CMNs with a lighter background with superimposed spots within the lesion were classified as spilus nevi (
      • Schaffer J.V.
      • Orlow S.J.
      • Lazova R.
      • Bolognia J.L.
      Speckled lentiginous nevus: within the spectrum of congenital melanocytic nevi.
      ). Those CMNs that did not exhibit these features were classified as classic CMNs. A spilus-like CMN was observed in 42.8% of patients. Four spilus-type CMN patients developed melanoma during adulthood, and a tumor arose on the CMN in three patients. Presence of neurocutaneous melanosis was confirmed in one patient.

      Oncogenic mutations in large/giant CMN lesions detected by next-generation sequencing

      Molecular characterization was conducted on 53 fresh skin biopsy samples by TruSight Tumor 26 Panel or TruSight Tumor 15 Panel (Illumina, San Diego, CA), which allow deep sequencing of key genes involved in the development of melanocytic lesions. We characterized a unique biopsy sample of the largest CMN from 11 patients and different areas of the largest CMN from 10 patients. An additional satellite lesion was collected from 13 patients (Table 1).
      The molecular screening detected 14 somatic mutations in the APC, BRAF, EGFR, GNAQ, KRAS, MET, NRAS, and PIK3CA genes. We detected an NRAS mutation in 57.1% of lesions (12 patients) with four c.181C>A, p.Q61K mutations; four c.182A>G, p.Q61R mutations; two c.182T>A, p.Q61L mutations; and two c.37G>C, p.G13R mutations. The percentage of mutant NRAS alleles within a nevus varied from 3% to 59%. In addition to NRAS mutations, we found other somatic variants with a mutant allele fraction ranging from 3.5% to 40% (Table 2). The failure to identify somatic mutations in CMNs could result from a low nevus/non-nevus cell ratio within the biopsy or from next-generation sequencing methodological issues such as low read depth. In all nonmutant NRAS/BRAF biopsy samples, we evaluated the reads mapped at codons 12, 13, and 61 of NRAS and codon 600 of BRAF by visual inspection. Reads evaluation did not identify any mutated tag in the NRAS and BRAF region, which reached a median coverage of ×18,927 (range = ×13.507–×25,436) (see Supplementary Table S2 online). Moreover, we analyzed more than one biopsy sample in most wild-type BRAF/NRAS lesions. These data indicate that cells from these lesions do not harbor recurrent NRAS or BRAF mutations.
      Table 2Molecular Status of the CMN
      Patient NumberSexCMN SubgroupKrengel ClassificationNRAS StatusBRAF StatusOther Alterations
      1FClassicG2 C1 R1 N0 H2 S3NRASQ61RWTPIK3CAR524K(
      Passenger alteration, not oncogenic.
      )
      2FClassicG1 C1 R2 N2 H1 S3WTWT
      3MClassicG1 C2 R1 N2 H0 S3WTWTZEB2-ALK
      Fusion transcript alteration.
      4MClassicG2 C2 R1 N0 H2 S3WTWTSOX5-RAF1
      Fusion transcript alteration.
      5FClassicG2 C2 R2 N2 H0 S2NRASQ61KWT
      6FClassicG2 C0 R2 N2 H0 S3WTWT
      7FClassicG2 C0 R0 N2 H0 S3NRASQ61RWTGGNBP2-MYO19
      Passenger alteration, not oncogenic.
      8FClassicG1 C2 R1 N1 H1 S3NRASQ61KWT
      9MClassicG2 C2 R1 N1 H0 S3NRASQ61KWT
      10FClassicG2 C2 R1 N1 H1 S3NRASQ61RWT
      11FClassicL1 C1 R0 N2 H1 S3WTWT
      12FClassicL1 C1 R1 N0 H2 S0NRASQ61KWT
      13MSpilus likeG2 C0 R0 N0 H0 S2WTWTKRASG174S
      14MSpilus likeG1 C1 R0 N0 H0 S1NRASQ61LWT
      15FSpilus likeG1 C2 R0 N0 H0 S3NRASG13RWT
      16
      In this patient, each BRAF alteration was found in a different biopsy sample from the same CMN. No mutations were found in other biopsy samples.
      FSpilus likeL1 C1 R1 N1 H0 S0WTBRAFG464E

      BRAFL584F
      17
      In this patient, all mutations were detected in the same skin biopsy sample, and only the APC p.P1268L and MET p.E1232K mutations are oncogenic. No mutations were found in the other biopsy samples.
      MSpilus likeL1 C1 R0 N0 H0 S1WTWTGNAQI190I/APCP1268L/METS290F METE1232K/EGFRL760L
      18MSpilus likeG2 C1 R0 N0 H0 S1NRAS Q61RWT
      19FSpilus likeG2 C1 R0 N0 H0 S0WTWTPIK3CAR524K(
      Passenger alteration, not oncogenic.
      )
      20FSpilus likeG1 C2 R0 N1 H1 S3NRASG13RWTGNAQQ209P(
      The alteration was additionally detected in an affected NRASG13R skin biopsy sample showing the blue nevi phenotype.
      )
      21FSpilus likeL1 C1 R0 N0 H0 S0NRASQ61LWT
      Abbreviations: CMN, congenital melanocytic nevi; F, female; M, male; WT, wild type.
      1 In this patient, each BRAF alteration was found in a different biopsy sample from the same CMN. No mutations were found in other biopsy samples.
      2 In this patient, all mutations were detected in the same skin biopsy sample, and only the APC p.P1268L and MET p.E1232K mutations are oncogenic. No mutations were found in the other biopsy samples.
      3 Passenger alteration, not oncogenic.
      4 Fusion transcript alteration.
      5 The alteration was additionally detected in an affected NRASG13R skin biopsy sample showing the blue nevi phenotype.
      In silico evaluation indicated that BRAFG464E (c.1391G>A), BRAFL584F (c.1750C>T), KRASG174S (c.520G>A), APCP1268L (c.3803C>T), and METE1232K (c.3694G>A) variants have oncogenic properties. In contrast, PIK3CAR524K (c.1571G>A), EGFRL760L (c.2280C>T), METS290F (c.869C>T), and GNAQI190I (c.570G>A) variants were classified as passenger alterations. Altogether, molecular screening found an oncogenic alteration in NRAS or other genes in 76.2% (16/21) of large/giant CMNs.

      Molecular characterization of multiple areas of the main lesions and satellite lesions from patients with large/giant CMNs

      We analyzed distinct areas of the largest CMN in 10 patients. We observed the same genetic background in each CMN from the same individual except for three spilus-type CMN patients. Patient 16 harbored the BRAFG464E and BRAFL584F mutations in two distinct biopsy samples, and no mutations were found in five additional biopsy samples. Patient 17, who developed melanoma within the CMN, presented both METE1232K and APCP1268L mutations in the same area, and no mutations were found in three additional biopsy samples. The larger CMN from patient 20 showed a hyperpigmented brown area and a blue area (Figure 1). Both areas harbored the NRASG13R mutation but, additionally, we detected the GNAQQ209P (c.626A>C) mutation within the blue area.
      Figure thumbnail gr1
      Figure 1Patient with giant breast/belly spilus-type CMN. (a) Phenotypically distinct biopsied areas marked in blue ink. (b) Dermoscopy of hypopigmented brown background area. (c) Dermoscopy of hyperpigmented brown area. (d) Dermoscopy of homogeneous blue area. Patient consented for the publication of the images.
      The variant allele frequency of GNAQQ209P was lower than that of NRASG13R (8% vs. 15%), suggesting that GNAQQ209P most likely occurred after the NRASG13R acquisition during nevus formation. The CMNs from patients 9 and 15 also had an area showing phenotypically blue nevus features. In both cases, GNAQ mutations were not present within the blue nevus area. Recently, it was described that wild-type GNAQ–acquired blue nevi can present the CYSLTR2L129Q mutation (
      • Möller I.
      • Murali R.
      • Müller H.
      • Wiesner T.
      • Jackett L.A.
      • Scholz S.L.
      • et al.
      Activating cysteinyl leukotriene receptor 2 (CYSLTR2) mutations in blue nevi.
      ). We assessed the presence of this mutation with a less sensitive technique such as Sanger sequencing in the wild-type GNAQ blue lesions, and no mutations were found.
      We analyzed satellite lesions from 61% of patients (13 patients), and we observed the same molecular background (mutations or transcript fusions) among satellite and largest CMNs in 92.3% of patients (12 patients). Patient 17 presented a discordant molecular background among biopsy samples (from the largest CMN and satellite).

      Distribution of NRAS alterations in CMNs and spilus-type CMNs

      Oncogenic alterations were detected in 58.8% (7/12) of patients with classic CMN and 66.7% (6/9) of patients with spilus-like CMN. Although in classic CMNs all mutations were located at codon 61 of NRAS (p.Q61R and p.Q61K), spilus-like CMNs showed a broad spectrum of mutations, including all NRASG13R lesions (Table 2). Patients with an NRASG13R lesion presented a peculiar spilus phenotype, with multiple speckled spots of different sizes and colors and often with large spots (Figure 2a).
      Figure thumbnail gr2
      Figure 2Heterogeneous phenotypes of large to giant congenital melanocytic nevi (CMNs). (a) Giant spilus-type CMNs with breast/belly pattern of distribution and NRASG13R mutation. Multiple speckled lesions in a large lighter background can be seen. Note the variety in size and color of the spots. (b) Patient with giant classic-type CMN with bolero pattern of distribution. Patients consented for publication of their images.
      We assessed whether the presence of NRAS mutations was associated with clinical and phenotypical characteristics. No differences were observed in terms of patterns of anatomical distribution, rugosity, hypertricosis, and number of satellites among NRASmut lesions versus NRASwildtype lesions.

      Fusion transcripts detected in wild-type NRAS large/giant CMNs

      We asked whether other alterations, such as chromosomal rearrangements, may drive the CMN pathogenesis in those CMN lesions without mutations. We analyzed the transcriptome of 49 CMN biopsy samples from 19 patients by RNA sequencing to identify fusion transcripts. We detected candidate fusion transcripts in three patients with classic CMN, corresponding to two nevi without mutations and one NRAS mutant nevus (Table 2). All fusion transcripts detected were validated by reverse transcriptase-PCR and Sanger sequencing. Patient 3 harbored a ZEB2-ALK transcript fusion in the large CMN and in a satellite (Figure 3a). Patient 4 harbored a SOX5-RAF1 transcript fusion (Figure 3b) in two distinct areas of the largest CMN (a hyperpigmented hairy area and one area of the CMN in spontaneous involution) and in a satellite. In both patients, fusion transcript expression was restricted to the melanocytic lesions, and no expression was detected in unaffected skin, supporting the oncogenic potential of both gene transcripts.
      Figure thumbnail gr3
      Figure 3Gene fusion detected in large/giant congenital melanocytic lesions. RNA sequencing data were used to identify gene fusions. Gene Expression Omnibus accession number GSE120597. (a) ALK gene (transcript ENST00000389048.7) fusion transcript was detected with ZEB2 transcript (ENST00000627532.2). The fusion transcript is expected to produce a protein that retains the ALK kinase domain. (b) RAF1 gene (transcript ENST00000251849.8) fusion transcript was detected for the SOX5 transcript (ENST00000451604.6). The fusion transcript is expected to produce a protein that retains the RAF1 kinase domain. (c) The GGNBP2-MYO19 fusion transcript (ENST00000613102.4 and ENST00000610930.4), which is expected to produce a transcript with a stop codon and without active domains, respective domains. All fusion transcripts were validated by reverse transcriptase-PCR and Sanger sequencing in large/giant CMN biopsy samples (CMN), satellites (SAT), or nonlesional skin (NS). Image of agarose gel and Sanger sequencing are indicated for all gene fusions. The GGNBP2-MYO19 transcript was detected in affected skin and nonlesional skin.
      Patient 7, who harbored an NRASQ61R CMN lesion, presented a GGNBP2-MYO19 fusion transcript, which created a transcript with no active domains (Figure 3c). The transcript fusion was detected in CMN biopsies, a satellite, and unaffected skin. Altogether, this suggests that the GGNBP2-MYO19 fusion has no functional impact and was considered to be a passenger alteration.

      Discussion

      Melanocytic nevi are a heterogeneous group of lesions in terms of clinical-histological characteristics (
      • Rogers T.
      • Marino M.L.
      • Raciti P.
      • Jain M.
      • Busam K.J.
      • Marchetti M.A.
      • et al.
      Biologically distinct subsets of nevi.
      ) and molecular background (
      • Roh M.R.
      • Eliades P.
      • Gupta S.
      • Tsao H.
      Genetics of melanocytic nevi.
      ). In this study, we report the molecular characterization of a cohort of large/giant CMN patients by next-generation sequencing and RNA sequencing methods. Using these highly sensitive techniques, we found NRAS mutations in 57.1% of patients, and no BRAFV600E mutation was detected. Previous studies including similar numbers of patients found NRAS mutations and the BRAFV600E mutation in 70–94.7% and 0%–15% of patients with large/giant CMN, respectively (
      • Charbel C.
      • Fontaine R.H.
      • Malouf G.G.
      • Picard A.
      • Kadlub N.
      • El-Murr N.
      • et al.
      NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi.
      ,
      • Dessars B.
      • De Raeve L.E.
      • Morandini R.
      • Lefort A.
      • El Housni H.
      • Ghanem G.E.
      • et al.
      Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.
      ,
      • Kinsler V.A.
      • Thomas A.C.
      • Ishida M.
      • Bulstrode N.W.
      • Loughlin S.
      • Hing S.
      • et al.
      Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS.
      ,
      • Phadke P.A.
      • Rakheja D.
      • Le L.P.
      • Selim M.A.
      • Kapur P.
      • Davis A.
      • et al.
      Proliferative nodules arising within congenital melanocytic nevi: a histologic, immunohistochemical, and molecular analyses of 43 cases.
      ,
      • Wu D.
      • Wang M.
      • Wang X.
      • Yin N.
      • Song T.
      • Li H.
      • et al.
      Lack of BRAFV600E mutations in giant congenital melanocytic nevi in a Chinese population.
      ). Although we found a lower prevalence of NRAS mutations than previous studies, our study also supports the idea that BRAF mutations are less associated with CMNs than NRAS mutations. Discrepancies in the prevalence of NRAS mutations among studies may reflect differences in study methodology or biological heterogeneity of nevi. Comparison among studies is difficult because in most of them, the lesions were categorized exclusively by the projected adult size information (
      • Ruiz-Maldonado R.
      Measuring congenital melanocytic nevi.
      ). It may be possible that distinct molecular results are due to phenotypic differences among samples rather than lesion size. CMNs usually present areas with very distinct characteristics in terms of rugosity, color, or hairiness, as shown in the spilus-type CMN, which represents almost 50% of our patients. Moreover, even classic CMNs can present phenotypically distinct areas, as in patient 9, who presented a nodular blue area inside the main nevus, or the patient shown in Figure 2b, who has a quite homogenous color throughout the lesion with distinct areas in terms of rugosity, nodularity, and hairiness. Therefore, the description of the lesions via current proposed classifications (
      • Krengel S.
      • Scope A.
      • Dusza S.W.
      • Vonthein R.
      • Marghoob A.A.
      New recommendations for the categorization of cutaneous features of congenital melanocytic nevi.
      ,
      • Martins da Silva V.P.
      • Marghoob A.
      • Pigem R.
      • Carrera C.
      • Aguilera P.
      • Puig-Butillé J.A.
      • et al.
      Patterns of distribution of giant congenital melanocytic nevi (GCMN): the 6B rule.
      ) is crucial in molecular studies carried out in large to giant CMNs.
      A spilus-type CMN is composed of a large cafe-au-lait macule extending across the midline and superimposed hypertrichotic medium to large CMNs, mostly present at birth (
      • Lee S.B.
      • Hummel H.-M.
      • Krengel S.
      • Enk A.
      • Haenssle H.A.
      Mosaic RASopathies: phenotypic and genotypic differentiation of naevus spilus-type congenital melanocytic naevus and segmental naevus spilus.
      ). We have characterized many patients with spilus-type CMN and confirm that NRAS alterations are found in a subset of lesions. However, NRAS mutations detected in this subgroup of lesions included less frequently reported alterations such as p.Q61L, p.Q61H, or p.G13R, which have been previously described in three CMN patients (
      • Dessars B.
      • De Raeve L.E.
      • Morandini R.
      • Lefort A.
      • El Housni H.
      • Ghanem G.E.
      • et al.
      Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.
      • Kinsler V.A.
      • Krengel S.
      • Riviere J.-B.
      • Waelchli R.
      • Chapusot C.
      • Al-Olabi L.
      • et al.
      Next-generation sequencing of nevus spilus-type congenital melanocytic nevus: exquisite genotype-phenotype correlation in mosaic RASopathies.
      • Shih F.
      • Yip S.
      • McDonald P.J.
      • Chudley A.E.
      • Del Bigio M.R.
      Oncogenic codon 13 NRAS mutation in a primary mesenchymal brain neoplasm and nevus of a child with neurocutaneous melanosis.
      ). Among previously reported NRASG13R CMNs, two patients also presented a spilus-type phenotype (
      • Shih F.
      • Yip S.
      • McDonald P.J.
      • Chudley A.E.
      • Del Bigio M.R.
      Oncogenic codon 13 NRAS mutation in a primary mesenchymal brain neoplasm and nevus of a child with neurocutaneous melanosis.
      ). Unfortunately, a detailed description of the lesion was not presented in the third case (
      • Dessars B.
      • De Raeve L.E.
      • Morandini R.
      • Lefort A.
      • El Housni H.
      • Ghanem G.E.
      • et al.
      Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.
      ).
      A previous NRASG13R spilus-type CMN patient presented neurocutaneous melanosis and mesenchymal brain tumor (
      • Shih F.
      • Yip S.
      • McDonald P.J.
      • Chudley A.E.
      • Del Bigio M.R.
      Oncogenic codon 13 NRAS mutation in a primary mesenchymal brain neoplasm and nevus of a child with neurocutaneous melanosis.
      ). Although in our series none of the NRASG13R spilus-type CMN patients presented neurocutaneous melanosis or developed melanoma, all patients who developed melanoma had a spilus-type CMN. Inconsistent data exist regarding melanoma risk in spilus nevus patients.
      • Gathings R.M.
      • Reddy R.
      • Bhatia A.C.
      • Brodell R.T.
      Nevus spilus: is the presence of hair associated with an increased risk for melanoma?.
      suggested that nevus spilus have a notably lower risk for malignant transformation than other CMNs of the same size, but
      • Boot-Bloemen M.C.T.
      • de Kort W.J.A.
      • van der Spek-Keijser L.M.T.
      • Kukutsch N.A.
      Melanoma in segmental naevus spilus: a case series and literature review.
      reported a series of five patients with large/giant spilus-type CMNs in which three patients developed melanoma (
      • Boot-Bloemen M.C.T.
      • de Kort W.J.A.
      • van der Spek-Keijser L.M.T.
      • Kukutsch N.A.
      Melanoma in segmental naevus spilus: a case series and literature review.
      ). In our series, all patients with spilus-type CMNs developed a cutaneous malignancy during adulthood, but no central nervous system melanoma has developed so far. These melanomas were of the superficial spreading type and presented with a reticulated pattern by dermoscopy. In our experience, these melanomas tend to manifest as hyperpigmented lesions, with changes detected on digital follow-up. These pigmented areas develop inside a hyperpigmented spot of the spilus-type nevus and not on the light café-au-lait background.
      Few studies in large/giant CMNs have been focused on genes other than BRAF and NRAS. We observed that wild-type NRAS large/giant CMNs can harbor a diversity of oncogenic alterations, including point mutations and fusion transcripts involving oncogenes, some representing new alternative therapeutic targeting opportunities. Mostly point mutations have been observed in tumors; either melanomas such as BRAFG464E (
      • Dahl C.
      • Christensen C.
      • Jönsson G.
      • Lorentzen A.
      • Skjødt M.L.
      • Borg Å.
      • et al.
      Mutual exclusivity analysis of genetic and epigenetic drivers in melanoma identifies a link between p14 ARF and RARβ signaling.
      ), BRAFL584F (
      • Nikolaev S.I.
      • Rimoldi D.
      • Iseli C.
      • Valsesia A.
      • Robyr D.
      • Gehrig C.
      • et al.
      Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma.
      ,
      • Siroy A.E.
      • Boland G.M.
      • Milton D.R.
      • Roszik J.
      • Frankian S.
      • Malke J.
      • et al.
      Beyond BRAFV600: clinical mutation panel testing by next-generation sequencing in advanced melanoma.
      ), KRASG174S (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ), and GNAQQ209P (
      • Roh M.R.
      • Eliades P.
      • Gupta S.
      • Tsao H.
      Genetics of melanocytic nevi.
      ) or lymphoid neoplasm such as METE1232K (
      • McGirt L.Y.
      • Jia P.
      • Baerenwald D.A.
      • Duszynski R.J.
      • Dahlman K.B.
      • Zic J.A.
      • et al.
      Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides.
      ). We detected gene fusions involving the ALK gene, which is a membrane-bound tyrosine kinase receptor, or the RAF1 gene encoding serine threonine kinase. ALK fusion has been found in Spitz nevi, atypical Spitz tumors, and spitzoid melanomas (
      • Wiesner T.
      • He J.
      • Yelensky R.
      • Esteve-Puig R.
      • Botton T.
      • Yeh I.
      • et al.
      Kinase fusions are frequent in Spitz tumors and spitzoid melanomas.
      ), and RAF1 fusions are recurrent alterations observed across solid cancers, including skin cancers (
      • Kumar-Sinha C.
      • Kalyana-Sundaram S.
      • Chinnaiyan A.M.
      Landscape of gene fusions in epithelial cancers: seq and ye shall find.
      ).
      In all cases except patients 7, 16, 17, we detected the same alteration in all affected skin (CMN and satellites) but not in unaffected skin, as previously suggested (
      • Kinsler V.A.
      • Thomas A.C.
      • Ishida M.
      • Bulstrode N.W.
      • Loughlin S.
      • Hing S.
      • et al.
      Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS.
      ), supporting the idea that satellites and larger CMNs result from the same cell origin. Based on this, we hypothesize that mutations found in patients 7, 16, 17 may not be the molecular event shared by all melanocytic cells of the lesions in these patients. Because plausible oncogenic mutations can be detected in a particular area of a lesion, we suggest that molecular characterization of a large/giant CMN should be conducted in different areas of the lesions.
      The analysis of distinct areas of a lesion resulted in the identification of the coexistence of GNAQQ209P and NRASG13R mutations in a blue area of a CMN. The GNAQQ209P mutation is observed in blue nevi and related dermal melanocytic lesions (
      • Roh M.R.
      • Eliades P.
      • Gupta S.
      • Tsao H.
      Genetics of melanocytic nevi.
      ). Unfortunately, additional studies to assess whether both alterations are present in same melanocytic cells were not possible. However, because the NRAS mutation was detected in all affected areas, including a satellite, we hypothesize that the GNAQQ209P mutation was an additional molecular event that occurred in a mutant NRAS cell located in a part of the larger CMN. This uncommon finding illustrates that certain mutations may be associated with lesions showing particular clinical-pathological features.
      • Salgado C.M.
      • Basu D.
      • Nikiforova M.
      • Bauer B.S.
      • Johnson D.
      • Rundell V.
      • et al.
      BRAF mutations are also associated with neurocutaneous melanocytosis and large/giant congenital melanocytic nevi.
      suggest that BRAFV600E mutation is associated with increased dermal/subcutaneous nodules within the CMN. Our series had no patient with BRAF mutations, so a direct comparison with these results is not possible. We compared patients with mutated versus wild-type NRAS. We failed to identify a genotype-phenotype correlation associated with NRAS mutation. It may be because our series includes only patients with large/giant CMNs and more spilus-like CMNs than other series. Moreover, in most cases, the same mutation was present in all phenotypically distinct areas of the same CMN. Altogether, the data suggests that phenotypic characteristics such as nodules, hypertricosis, or rugosity are determined by factors other than the BRAF/NRAS mutations.
      In conclusion, this study supports the finding that large/giant CMNs and satellite lesions in patients with multiple CMNs originate from the same molecular event, which in most large/giant CMNs, including spilus-type lesions, is the acquisition of an NRAS alteration. However, these melanocytic proliferations can be associated with genetic abnormalities such as point mutations or gene fusions involving other genes. In addition, many spilus-type CMN patients had melanoma, indicating that these patients should be followed up routinely as are patients with large/giant CMNs of the classic type.

      Materials and Methods

      Participants and tissue samples

      We included patients diagnosed with large or giant CMNs who attended the Hospital Clinic of Barcelona between 2013 and 2015. Skin biopsy samples were taken from different phenotypic areas of the largest nevus and satellites. At least one biopsy sample of the largest CMN and clinical information (sex, age at time of biopsy, previous history of melanoma or neurocutaneous melanosis) was collected in all patients. Clinical and dermoscopic photos were taken from all areas biopsied. Patients were classified according to Krengel classification (
      • Krengel S.
      • Scope A.
      • Dusza S.W.
      • Vonthein R.
      • Marghoob A.A.
      New recommendations for the categorization of cutaneous features of congenital melanocytic nevi.
      ) and patients with giant CMNs by the B6 classification (
      • Martins da Silva V.P.
      • Marghoob A.
      • Pigem R.
      • Carrera C.
      • Aguilera P.
      • Puig-Butillé J.A.
      • et al.
      Patterns of distribution of giant congenital melanocytic nevi (GCMN): the 6B rule.
      ). CMNs were classified clinically as spilus or classic CMNs.
      All patients, or their parents where appropriate, gave written informed consent before participation. The study was approved by the medical ethics committee of the Hospital Clinic of Barcelona and complies with the Declaration of Helsinki Principles. Patients consented for the publication of their images presented in this study.

      DNA and RNA extraction

      DNA was extracted from fresh tissue using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. DNA was quantified using KAPA SYBR FAST qPCR Kit Master Mix (2X) universal (Kapa Biosystems, Wilmington, MA) and Qubit dsDNA BR Assay Kit (Invitrogen, Waltham, MA). Total RNA was isolated from fresh tissue with TRIzol (Invitrogen), followed by purification with the RNeasy mini kit (Qiagen). RNA quantification was performed using Qubit RNA BR Assay Kit, and integrity was measured via Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA).

      Somatic mutation detection by next-generation sequencing

      Molecular characterization was conducted using the TruSight Tumor 26 Panel (TST26, Illumina) in 46 lesions. An additional eight lesions were analyzed by TruSight Tumor 15 Panel (TST15; Illumina). The TST26 is focused on 174 exons of 26 genes (AKT1, EGFR, GNAS, ALK, ERBB2, KIT, APC, FBXW7, KRAS, NRAS, STK11, PDGFRA, TP53, PIK3CA, BRAF, FGFR2, MAP2K1, PTEN, CDH1, FOXL2, MET, CTNNB1, GNAQ, MSH6, SRC, and SMAD4). AKT1, EGFR, ERBB2, KIT, KRAS, NRAS, PDGFRA, TP53, PIK3CA, BRAF, FOXL2, MET, and GNAQ were included in both panels.
      Libraries were generated using TruSight Rapid Capture along with the TST26/TST15 sequencing panel (Illumina) following the manufacturer’s instructions. Approximately 400–500 ng (TST26) or 20 ng (TST15) of DNA was used to prepare the libraries. The enriched libraries were quantified and evaluated using a Bioanalyzer 2100 and DNA-1000 Kit (Agilent Technologies). Each DNA library was pooled using up to 24 different barcodes and diluted to obtain a 4-nmol/L final concentration. Libraries were sequenced in a MiSeq sequencer platform (Illumina).

      CYSLTR2 mutational screening

      All blue nevus areas were screened for the CYSLTR2L129Q mutation by Sanger sequencing. The PCRs were carried out using the PCR Master Mix (Promega, Madison, WI). PCR conditions were as follows: initial denaturizing step at 95 °C for 5 minutes, followed by 35 cycles (95 °C for 1 minute, touchdown 65–55 °C for 1 minute, 72 °C for 1 minute), and a final extension at 72 °C for 10 minutes. Mutations were screened by direct sequencing of the amplified PCR products, using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) in an ABI3100 automatic sequencer (Applied Biosystems).
      Primers for CYSLTR2 were forward, 5′-CCCTTCAGGGCTGACTATTA and reverse, 5′-CCAGGAGCATTATTGAGGAA.

      Bioinformatics analysis

      A custom pipeline was developed for analyzing data from TST26/TST15 panels. Initial quality check of raw reads (fastq files) and sequencing reads statistics were conducted by using FastQC 0.11.5 (Babraham Bioinformatics, Cambridge, United Kingdom). Reads were aligned to the Hg19 reference genome using the Burrows-Wheeler Aligner (BWA 0.7.5a) software package with the bwa-mem option (
      • Li H.
      • Durbin R.
      Fast and accurate short read alignment with Burrows-Wheeler transform.
      ) and standard sequence alignment map (i.e., SAM) files were obtained. Samtools 0.1.19 (
      • Li H.
      • Handsaker B.
      • Wysoker A.
      • Fennell T.
      • Ruan J.
      • Homer N.
      • et al.
      The Sequence Alignment/Map format and SAMtools.
      ) was used to pass the SAM format into a BAM format and to sort mappings. Statistical coverage was calculated from the BAM file using Bedtools 2.17.0 (
      • Quinlan A.R.
      • Hall I.M.
      BEDTools: a flexible suite of utilities for comparing genomic features.
      ) and Picard 1.97 (http://broadinstitute.github.io/picard/). Both tools give useful information for interpretation of the variants. Detection of variants was conducted by FreeBayes 9.9.13 (
      • Garrison E.
      • Marth G.
      Haplotype-based variant detection from short-read sequencing.
      ) by using the standard filters for FreeBayes and keeping only reads with mapping quality greater than 30 and base quality greater than 20. In addition to the standard filters, variant caller showed all variants present in more than 1% of the reads. Obtained variants were normalized by Bcftools 0.2.0-rc7. Finally, we cross-referenced variants against dbSNP 137 with GATK-lite 2.3-9 VariantAnnotator (https://www.broadinstitute.org/), and we annotated variants using ENSEMBL Variant Effect Predictor 72 (www.ensembl.org). Only variants (single-nucleotide variants or small insertions/deletions) in the coding region were evaluated. We filtered out all known single-nucleotide variants and insertions/deletions in the Exome Aggregation Consortium (ExAC) and dbSNP database (National Institutes of Health, Bethesda, MD). We evaluated the oncogenic potential of variants detected by the Cancer Genome Interpreter tool (available at www.cancergenomeinterpreter.org) and the Catalogue of Somatic Mutations in Cancer (i.e., COSMIC) database (available at cancer.sanger.ac.uk).
      Sequencing resulted in a mean read depth of ×18,978 ± ×2,665 per sample for the captured region. The coverage of BRAF codon 600 and NRAS codons 13 and 61 for all the samples are shown in Supplementary Table S2.

      RNA sequencing and transcript fusion analysis

      Stranded mRNA library preparation and sequencing, bioinformatics fusion transcript analysis, and validation of fusion transcripts by reverse transcriptase-PCR and Sanger sequencing are detailed in Supplementary Materials and Methods.

      Statistical analyses

      Descriptive data were reported as median ± standard deviation and/or as percentages. Association between molecular status and categorical variables was calculated by cross-tabulations and Fisher exact test by classifying the CMNs in patients with mutated NRAS versus other CMNs (lesions with other oncogenic alterations and those with no alteration detected). P-values less than 0.05 were considered as statistically significant. All conventional analyses were performed using SPSS statistics software, version 22.0 (IBM, Armonk, NY).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We are grateful to our patients and relatives and to nurses Daniel Gabriel, Pablo Iglesias, Asuncio Arnaiz, and M. Eugenia Moliner and technicians Abel Caño, Mireia Domınguez, and Beatriz Alejo for their help. This work was mainly supported by the Spanish Federation of Neuromuscular Disease (FEDASEM), the Spanish Federation of Rare Diseases (FEDER), and Isabel Gemio Research Foundation of muscular dystrophy and other rare diseases (FIG) through the Call for Research Projects on Rare Diseases–2014 through the initiative “We Are Rare, All Are Unique.” The research at the Melanoma Unit in Barcelona is partially funded by grant numbers PI15/00716 and PI15/00956 from Fondo de Investigaciones Sanitarias, Spain; by the CIBER de Enfermedades Raras of the Instituto de Salud Carlos III, Spain; by the AGAUR 2014_SGR_603 of the Catalan Government, Spain; by the European Commission under the 6th Framework Programme, contract no. LSHC-CT-2006-018702 (GenoMEL), under the 7th Framework Programme (Diagnoptics); by the MARATÓ de TV3 Foundation; and by the Leo Messi Foundation.

      Supplementary Material

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      Linked Article

      • Large-Giant Congenital Melanocytic Nevi: Moving Beyond NRAS Mutations
        Journal of Investigative DermatologyVol. 139Issue 4
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          Large-giant congenital melanocytic nevi have been well characterized clinically, yet questions remain about the heterogenous phenotypes observed. Martins da Silva et al. (2018) highlight the genotypic diversity between “classic” and “spilus-like” congenital melanocytic nevi by analyzing multiple biopsy sites and matching satellite nevi. This study provides evidence for alternative modes of development beyond the well-established NRAS mutation paradigm.
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