The Dermoscopical and Histopathological Patterns of Nevi Correlate with the Frequency of BRAF Mutations
2010; Elsevier BV; Volume: 131; Issue: 2 Linguagem: Inglês
10.1038/jid.2010.332
ISSN1523-1747
AutoresIris Zalaudek, Christian Guelly, Giovanni Pellacani, Rainer Hofmann‐Wellenhof, Slave Trajanoski, Harald Kittler, Alon Scope, Ashfaq A. Marghoob, Caterina Longo, Bernd Leinweber, Gerardo Ferrara, Toshiaki Saida, James M. Grichnik, Giuseppe Argenziano, Jürgen C. Becker,
Tópico(s)melanin and skin pigmentation
Resumoglobular nevi mixed reticular nevi with central globules/homogeneous areas mixed reticular/homogeneous nevi with peripheral globules reticular nevi ultradeep pyrosequencing TO THE EDITOR The frequency of BRAF mutations among nevi is variable (Thomas, 2006Thomas N.E. BRAF somatic mutations in malignant melanoma and melanocytic naevi.Melanoma Res. 2006; 16: 97-103Crossref PubMed Scopus (92) Google Scholar), but little is known about the frequency among nevi, stratified by dermoscopy and by the detection method. In this study, we evaluated BRAF status in 45 nevi from 43 patients (23 women, mean age 39.9 years) using the Sanger method (Loewe et al., 2004Loewe R. Kittler H. Fischer G. et al.BRAF kinase gene V599E mutation in growing melanocytic lesions.J Invest Dermatol. 2004; 123: 733-736Crossref PubMed Scopus (48) Google Scholar); a random subset of 24 of these nevi was tested by the more sensitive ultradeep pyrosequencing (UDPS, Huse et al., 2007Huse S.M. Huber J.A. Morrison H.G. et al.Accuracy and quality of massively parallel DNA pyrosequencing.Genome Biol. 2007; 8: R143Crossref PubMed Scopus (997) Google Scholar; Tan et al., 2008Tan Y.H. Liu Y. Eu K.W. et al.Detection of BRAF V600E mutation by pyrosequencing.Pathology. 2008; 40: 295-298Crossref PubMed Scopus (77) Google Scholar; See Supplementary Data: Material and Methods S1, Supplementary Tables S1 and S2 online). Download .pdf (.14 MB) Help with pdf files Supplementary Information Patient demographics, anatomic location of nevi, dermoscopic patterns, histopathological diagnosis, and results of the genetic tests using Sanger and UDPS are summarized in Table 1. Dermoscopically, the 45 nevi consisted of 17 (37.7%) reticular nevi (R), 5 (11.1%) globular nevi (G), 12 (26.6%) mixed reticular nevi with central globules/homogeneous areas (MC), and 11 (24.4%) mixed reticular/homogeneous nevi with peripheral globules (MP).Table 1Patient data, clinical characteristics, and detection rates of BRAF mutations among 45 neviRef_BRAF coding position1798:G/A1799:T/A1800:G/A1801:A/GCodon600600600601600MutationGTG/ATGGTG/GAGGTG/GTAAAA/GAAGTG/GAGIDDXHXAge, yearsLocationSexVariant (%)Variant (%)Variant (%)Variant (%)ReadsSanger 1RComp40TrunkF0.3002.797,608GTG 2RJunct42AbdomenF000013,959GTG 3MCComp37TrunkF1.3400311,423GTG 4MCComp36TrunkM0003.387,042GTG 5MCComp46TrunkF000011,986GTG 6MCComp61AbdomenF000015,899GTG 7MCComp31TrunkM00.320012,015GTG 8MPComp42TrunkM00.4900.299,054GTG 9RJunct46TrunkM1.411.080011,370GTG10MPJunct23TrunkF01.360014,359GTG11MPComp54AbdomenF0.471.750010,017GTG12MPComp55AbdomenM02.830010,736GTG13MPComp19TrunkF0.913.7600.3210,047GTG14MPComp30TrunkF2.184.5500.414,919GTG15MPComp34TrunkF04.630010,101GTG16MPComp26AbdomenM1.584.91006,399GaG17MCComp58AbdomenM1.035.620.8407,746GTG18MPComp31TrunkM1.356.720010,000GTG19MPComp38ExtremityM0.417.34009,021GAG20MPComp26TrunkF010.540011,163GTG21MCComp27AbdomenM010.62006,525GTG22GComp52TrunkM011.120014,949GTG23RComp39AbdomenM015.380010,343GTG24GDerm27ExtremityF028.590010,118GAG25GComp24TrunkF/////GAG26MCComp35ExtremityF/////GaG27MCComp32TrunkF/////GAG28RJunct55ExtremityF/////GTG29RJunct39TrunkM/////GTG30RJunct46ExtremityM/////GTG31RJunct37AbdomenF/////GTG32RComp35TrunkF/////GTG33RComp44TrunkF/////GTG34RComp44TrunkM/////GTG35RComp25ExtremityF/////GTG36RComp38TrunkF/////GTG37RComp38AbdomenF/////GTG38RJunct39TrunkM/////GTG39RComp49TrunkF/////GTG40RComp34TrunkM/////GTG41GDerm62TrunkM/////GTG42GComp40AbdomenF/////GTG43MCJunct37AbdomenF/////GTG44MCComp33TrunkM/////GTG45MCJunct57TrunkM/////GTGAbbreviations: Comp, compound nevus; Derm, dermal nevus; F, female; G, globular; Junct, junctional nevus; M, male; MC, mixed pattern with central globules or structureless areas; MP, mixed pattern with peripheral globules; R, reticular.Columns from left to right: (1) Patient ID; (2) Dermoscopic pattern of nevi; (3) Histopathological diagnosis of nevi; (4) Age; (5) Location of nevi; (6) Sex; (7–11) Results from ultradeep pyrosequencing; (12) Results from Sanger sequencing. Sanger results with background levels indicating a minor A—variants are denoted as GaG. Data showing mutations in BRAF are given in bold. Open table in a new tab Abbreviations: Comp, compound nevus; Derm, dermal nevus; F, female; G, globular; Junct, junctional nevus; M, male; MC, mixed pattern with central globules or structureless areas; MP, mixed pattern with peripheral globules; R, reticular. Columns from left to right: (1) Patient ID; (2) Dermoscopic pattern of nevi; (3) Histopathological diagnosis of nevi; (4) Age; (5) Location of nevi; (6) Sex; (7–11) Results from ultradeep pyrosequencing; (12) Results from Sanger sequencing. Sanger results with background levels indicating a minor A—variants are denoted as GaG. Data showing mutations in BRAF are given in bold. Using Sanger method, 6 of the 45 nevi (13.3%) contained BRAFV600E; their dermoscopic patterns were G (n=2), MC (n=2), and MP (n=2), and all (n=6) were compound nevi on histopathology. None of the nevi classified as R on dermoscopy or as junctional nevi on histopathology showed BRAF mutations. Patients with nevi harboring BRAFV600E were younger than those with wild-type BRAF (30 versus 41 years, respectively; P=0.03). Using UDPS, 19 (79.2%) of the 24 randomly selected nevi were BRAFV600E positive, including G nevi (100%), MP nevi (90.9%), R nevi (75%), and MC nevi (57%). Six nevi showed heterogeneity of mutations (patients 3, 9, 14 and 16–18) and two nevi (patients 1 and 3), which were negative for BRAFV600E, revealed p.K601E variant. Representative electropherogramms are depicted in Figure 1 (lanes 3 and 4). There was no significant age difference between patients with BRAFV600E nevi and patient whose nevi showed wild-type BRAF (37 versus 44 years, respectively; P=0.20). The frequency of BRAF mutations among nevi was highly dependent on the sensitivity of the employed method; only 13.3% of nevi showed BRAF mutation using Sanger method, whereas 79.2% showed mutation with UDPS. The finding of BRAF mutation by UDPS and not by Sanger method probably attests to clonal heterogeneity within the nevus, whereby clones of BRAF mutations and clones with wild-type BRAF coexist (Lin et al., 2009Lin J. Takata M. Murata H. et al.Polyclonality of BRAF mutations in acquired melanocytic nevi.J Natl Cancer Inst. 2009; 101: 1423-1427Crossref PubMed Scopus (55) Google Scholar). The age difference seen between patients with BRAFV600E nevi and patients with wild-type nevi was only found using Sanger method, but not with UDPS. Although marked differences between the methods were found in BRAF mutation frequency among R nevi (0% by Sanger and 75% by UDPS), both methods found BRAF mutations to be more frequent among nevi classified as G and MP by dermoscopy and as dermal and compound nevi by histopathology. These findings suggest that BRAF-mutant clones predominate in younger age, and possibly in G nevi (Table 1), which mostly correlate with compound or dermal nevi on histopathology. In contrast, among R nevi, which usually are predominantly junctional nevi, BRAF-mutated clones are the minority. This challenges the theory of nevogenesis, which states that intermittent UV exposure induces BRAF mutations, which in turn stimulate melanocytic proliferation (Wu et al., 2007Wu J. Rosenbaum E. Begum S. et al.Distribution of BRAF T1799A(V600E) mutations across various types of benign nevi: implications for melanocytic tumorigenesis.Am J Dermatopathol. 2007; 29: 534-537Crossref PubMed Scopus (59) Google Scholar). One would expect, by this theory, a higher frequency of BRAF mutations among nevi that are predominantly junctional (for example, R nevi) than in nevi that are predominantly dermal (for example, G nevi); junctional melanocytes are likely to be exposed to higher levels of UV than dermal melanocytes. The reports of high rates of BRAF mutations among nevi from sun-protected body areas (Wu et al., 2007Wu J. Rosenbaum E. Begum S. et al.Distribution of BRAF T1799A(V600E) mutations across various types of benign nevi: implications for melanocytic tumorigenesis.Am J Dermatopathol. 2007; 29: 534-537Crossref PubMed Scopus (59) Google Scholar; Takata et al., 2010Takata M. Murata H. Saida T. Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma.Pigment Cell Melanoma Res. 2010; 23: 64-71Crossref PubMed Scopus (48) Google Scholar) further doubt the notion that UV exposure has an important role in acquisition of BRAF mutations. The finding that BRAF mutation among nevi is age related is interesting, as the dermoscopic pattern of nevi was also found to be age dependent (Zalaudek et al., 2006Zalaudek I. Grinschgl S. Argenziano G. et al.Age-related prevalence of dermoscopy patterns in acquired melanocytic naevi.Br J Dermatol. 2006; 154: 299-304Crossref PubMed Scopus (122) Google Scholar; Scope et al., 2008Scope A. Marghoob A.A. Dusza S.W. et al.Dermoscopic patterns of naevi in fifth grade children of the Framingham school system.Br J Dermatol. 2008; 158: 1041-1049Crossref PubMed Scopus (57) Google Scholar). Most nevi before puberty are G nevi, whereas MP nevi are most commonly seen during puberty and adolescence (Kittler et al., 2000Kittler H. Seltenheim M. Dawid M. et al.Frequency and characteristics of enlarging common melanocytic nevi.Arch Dermatol. 2000; 136: 316-320Crossref PubMed Scopus (141) Google Scholar). Using digital dermoscopic follow-up, MP pattern was shown to indicate nevus growth; MP nevi enlarge symmetrically, while progressively developing central network or structureless brown area, until finally, the peripheral globules disappear indicating that active growth has ceased. At this stage, nevi reveal either R or MC pattern. Indeed, in adults, nevi mostly show the R pattern and, to a lesser extent, the MC pattern. The observation that BRAF mutations are frequent among nevi whose onset is during early childhood (G nevi) and among growing nevi (MP nevi) during adolescence, supports the hypothesis that BRAF mutations are acquired early in nevogenesis; it also stands to reason that BRAF-mutant cells affect the nevus’ phenotype (for example, dermoscopic pattern). Furthermore, our findings are in line with a previous study reporting a 13-fold higher frequency of BRAFV600E in growing compared with stable melanocytic neoplasms (Loewe et al., 2004Loewe R. Kittler H. Fischer G. et al.BRAF kinase gene V599E mutation in growing melanocytic lesions.J Invest Dermatol. 2004; 123: 733-736Crossref PubMed Scopus (48) Google Scholar); in their series, 5 of 20 changing nevi (25%) harbored BRAFV600E, as detected by Sanger sequencing, whereas only 2 of 35 stable nevi (6%) were BRAFV600E positive. If MC- and R nevi represent a later phase in the life of a nevus, while MP pattern represents an early stage, it seems reasonable that BRAF-mutant clones predominate in nevi during their growth phase. Thus, BRAF-mutant cells differentially contribute to the life of nevi. Indeed, previous studies showed that, initially, abnormal BRAF expression leads to moderate melanocyte proliferation, whereas sustained expression leads to oncogene-induced senescence, attributed to the induction of p16INK4a and acidic β-galactosidase activity, and to cell-cycle arrest (Michaloglou et al., 2005Michaloglou C. Vredeveld L.C. Soengas M.S. et al.BRAFE600-associated senescence-like cell cycle arrest of human naevi.Nature. 2005; 436: 720-724Crossref PubMed Scopus (1681) Google Scholar; Gray-Schopfer et al., 2006Gray-Schopfer V.C. Cheong S.C. Chong H. et al.Cellular senescence in naevi and immortalisation in melanoma: a role for p16?.Br J Cancer. 2006; 95: 496-505Crossref PubMed Scopus (311) Google Scholar; Houben et al., 2009Houben R. Ortmann S. Drasche A. et al.Proliferation arrest in B-Raf mutant melanoma cell lines upon MAPK pathway activation.J Invest Dermatol. 2009; 129: 406-414Crossref PubMed Scopus (16) Google Scholar; Scurr et al., 2010Scurr L.L. Pupo G.M. Becker T.M. et al.IGFBP7 is not required for B-RAF-induced melanocyte senescence.Cell. 2010; 141: 717-727Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Our study has several limitations: first, the sample number is small, particularly because nevi were subcategorized by dermoscopic pattern. Second, we were able to perform UDPS in only a subset of nevi; however, we attempted to mitigate sampling bias by randomly selecting nevi for UDPS testing. Third, the discordant results of BRAF mutations among R nevi obtained by Sanger and UDPS methods, precludes definitive conclusions for this nevus subtype. In summary, this study demonstrates that the frequency and predominance of BRAF mutations in nevi depends on dermoscopic pattern, the histopathological subtype, and the stage of nevus development. Moreover, the method employed to test BRAF mutations strongly impacts the observed mutation frequency; therefore, comparison between studies should take into account the detection methods. Future studies analyzing genetic alterations in nevi should consider stratifying nevi by dermoscopic pattern and by growth phase. This study was supported by the Elise Richter Program (V9-B05) of the Austrian Science Fund (http://www.fwf.ac.at). The sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data; or in the preparation, review, or approval of the manuscript. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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