Congenital Melanocytic Nevi Frequently Harbor NRAS ... - Core

ORIGINAL ARTICLE

Congenital Melanocytic Nevi Frequently Harbor NRAS Mutations but no BRAF Mutations Ju¨rgen Bauer1,2, John A. Curtin3, Dan Pinkel3 and Boris C. Bastian1,3,4 Most melanocytic nevi develop on sun-exposed skin during childhood and adolescence and commonly harbor BRAF mutations or, less frequently, NRAS mutations. A small subset of nevi is present at birth, and therefore must develop independently of UV light. To assess whether these nevi have a different mutation spectrum than those that develop on sun-exposed skin, we determined the BRAF and NRAS mutation frequencies in 32 truly congenital nevi. We found no BRAF mutations, but 81% (26/32) harbored mutations in NRAS. Consistently, seven of 10 (70%) proliferating nodules that developed early in life in congenital nevi showed mutations in NRAS. A separate set of nevi that displayed histological features frequently found in nevi present at birth (‘‘congenital pattern nevi’’) but lacked a definitive history of presence at birth showed an inverse mutation pattern with common BRAF mutations (20/28 or 71%) and less frequent NRAS mutations (7/28 or 25%). Thus, nevi that develop in utero are genetically distinct from those that develop later, and histopathologic criteria alone are unable to reliably distinguish the two groups. The results are consistent with the finding in melanoma that BRAF mutations are uncommon in neoplasms that develop in the absence of sun-exposure. Journal of Investigative Dermatology (2007) 127, 179–182. doi:10.1038/sj.jid.5700490; published online 3 August 2006

INTRODUCTION Activating mutations within the kinase domain of the BRAF gene are a common somatic event in melanoma (Davies et al., 2002) as well as in benign melanocytic nevi (Pollock et al., 2003). Whereas sunlight is an undisputed etiological factor in the development of melanoma (Gandini et al., 2005) and nevi (Bauer et al., 2005), the role of UV exposure in the formation of BRAF mutations is unclear. About 90% of BRAF mutations found in melanoma and melanocytic nevi result in T-to-A transversions at nucleotide 1796 and therefore lack the typical UV radiation signature of CC-to-TT or C-to-T transitions. In melanoma, the finding of frequent BRAF mutations in tumors on intermittently sun-exposed skin, and the infrequent BRAF mutations in melanomas on relatively or completely sun-protected body sites (Maldonado et al., 2003; Cohen et al., 2004; Edwards et al., 2004; Sasaki et al., 2004; Wong et al., 2005) suggest a link, possibly indirect, between exposure to UV light and the induction of BRAF mutations. However, the lack of BRAF mutations in melanomas on skin that has received high levels of sun exposure and their frequent occurrence in histologically defined congenital nevi 1

Department of Dermatology, University of California at San Francisco, San Francisco, California, USA; 2Department of Dermatology, Eberhard Karls University, Tu¨bingen, Germany; 3Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California, USA and 4 Department of Pathology, University of California at San Francisco, San Francisco, California, USA The work was performed in San Francisco, CA, USA. Correspondence: Dr Boris C. Bastian, Comprehensive Cancer Center, University of California San Francisco, Box 0808, San Francisco, California 94143-0808, USA. E-mail: [email protected] Received 7 February 2006; revised 30 March 2006; accepted 24 May 2006; published online 3 August 2006

& 2006 The Society for Investigative Dermatology

(Pollock et al., 2003; Yazdi et al., 2003) argue against a causative link between UV light and BRAF mutations in melanocytic neoplasia. Strictly defined, congenital melanocytic nevi are nevi that are present at birth and thus have developed in utero in the absence of UV exposure. However, less stringent definitions are commonly used by the medical field, for example, nevi that arise in the first years of life are often termed ‘‘tardive’’ congenital nevi, and pathologists commonly make the diagnosis of a congenital nevus in young adults and adults based on the presence of particular histopathologic features that are found in nevi that are truly congenital. If these criteria are inadequate to recognize nevi that developed in utero, then some of the implications drawn from previous studies of BRAF mutation frequencies in nevi may be in error. In this study, we compare the mutation frequencies of BRAF and NRAS in 32 melanocytic nevi that definitively developed in utero, in 10 proliferating nodules that developed in such congenital nevi, and in 28 melanocytic nevi excised later in life and showing only histopathologic features similar to congenital nevi, the so-called ‘‘congenital pattern nevi’’. We demonstrate that these groups of nevi are very distinct in their mutation spectrum. Our results have important clinical implications, as congenital nevi are considered precursors of melanoma with estimated lifetime risks ranging from 5 to 40% (Tannous et al., 2005). RESULTS AND DISCUSSION None of the 32 melanocytic nevi that were present at birth nor any of the 10 nodular proliferations that developed in melanocytic nevi present at birth showed BRAF mutations. In contrast, 20 of 28 (71%) of the melanocytic nevi with a www.jidonline.org

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‘‘congenital pattern’’ showed the common BRAF V600E mutation. Mutation analysis of NRAS in the cases that were wild type for BRAF revealed an inverse distribution: 26 of the 32 nevi present at birth (81%) and seven of the 10 (70%) proliferating nodules showed mutations in NRAS (Tables 1 and 2). Previous studies on truly congenital nevi also found frequent NRAS mutations in codon 61 (Carr and Mackie, 1994) and no BRAF mutations (Papp et al., 1999; De Raeve et al., 2006). Only seven of 28 (25%) nevi with a ‘‘congenital pattern’’ showed NRAS mutations. These nevi may thus represent ‘‘true’’ congenital nevi in which NRAS mutations developed in utero, or acquired nevi in which the mutations developed later in life. These results are in striking contrast to earlier studies that have reported BRAF mutations in six out of seven (85.7%) (Pollock et al., 2003) or in six out of 13 (46.2 %) melanocytic nevi that were classified as congenital (Yazdi et al., 2003). Several explanations for this discrepancy are possible. We could have missed BRAF mutations in nevi present at birth because of normal cell contamination or because our patients were young, and mutations could still develop later in life in such nevi. Both possibilities are highly unlikely because (i) nevi present at birth showed contiguous areas of melanocytes that could be easily dissected away from normal tissue, and (ii) we found frequent activating mutations in NRAS, which are virtually never found when the BRAF V600E mutation is present (Davies et al., 2002), making it very unlikely that BRAF mutations were missed or arose later in progression of these nevi. We believe that the difference between our mutation analysis and previous results is based on the fact that the classification of nevi as congenital in the previous studies was based on histological criteria, not actual presence at birth. Nevi present at birth frequently show distinctive microscopic features including splaying of nevus cells between collagen bundles in the lower two-thirds of the reticular dermis, within hair follicles, eccrine ducts, or blood vessel walls (Mark et al., 1973). These criteria are commonly used to classify nevi whose age of appearance is unknown as ‘‘congenital pattern nevi’’. These are sometimes assumed to be truly congenital. However, several studies have shown that these histopathologic features are not specific to nevi present at birth. In one study, nevi that were not present at examination at birth but occurred in the first 3 years of life showed a congenital pattern in 47% of the cases (Clemmensen and Kroon, 1988). Two other studies compared melanocytic nevi in children based on clinical files or parents’ information as to whether a

nevus was acquired or congenital. These studies concluded that the histopathologic features had limited specificity as predictor of presence at birth (Rhodes et al., 1985; Cribier et al., 1999). The nevi present at birth in our study were at least several centimeters in diameter, qualifying as medium to large size congenital nevi by conventional criteria (Kopf et al., 1979), whereas the nevi with a ‘‘congenital pattern’’ were significantly smaller with a maximum diameter of 13 mm. Reliable histories regarding presence at birth of small nevi are difficult to obtain and were not available for the group of ‘‘congenital pattern’’ nevi in our study. This group is thus expected to contain a mixture of nevi acquired after birth and nevi present at birth. Because a subset of nevi in the ‘‘congenital pattern’’ group also contained NRAS mutations that were common in the group of nevi present at birth, it is possible that these also developed before birth. Alternatively, it is conceivable that the marked differences in the mutation frequencies of BRAF and NRAS do not apply for small nevi that developed before birth, indicating genetic differences between truly congenital nevi that are small and those that are large. Such differences would thus be independent of environmental factors such as UV light and depend more likely on the developmental stage of the organism or melanocyte at which the mutations occur. Some studies have found a relatively high risk of progression to melanoma for small histologically defined congenital nevi (Rhodes and Melski, 1982; Rhodes et al., 1982). Therefore, an improved understanding of the mutation spectrum in nevi of all sizes that are present at birth, ‘‘tardive’’ congenital nevi that develop during the first year of life, and histologically defined congenital nevi may allow more sophisticated partitioning of progression risk among the nevus subtypes and substantially improve patient management. The high frequency of NRAS mutations in nevi that developed in utero demonstrates that UV light is not required

Table 2. Frequencies of BRAF and NRAS mutations Congenital Proliferating melanocytic nodules in congenital Congenital nevi melanocytic nevi pattern nevi BRAF mutations

0 (0%)

NRAS mutations No mutations

0 (0%)

20 (71.4%)

26 (81.3%)

7 (70.0%)

7 (25.0%)

6 (18.8%)

3 (30.0%)

1 (3.6%)

Table 1. Clinical characteristics of tumors and patients Congenital melanocytic nevi

Proliferating nodules in congenital melanocytic nevi

Congenital pattern nevi

n (samples)

32

10

28

Male/female

14/18

2/7

9/13

1 day to 30 years (1.3 years)

2–135 days (40 days)

15–67 years (40 years)

Age range (median)

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for NRAS mutations. In contrast, the complete absence of BRAF mutations in nevi present at birth, but their frequent occurrence in acquired nevi, parallels the finding in melanoma in which BRAF mutations are found in melanomas that occur on intermittently sun-exposed body sites but are rare to absent in melanomas developing in completely sunprotected mucosa-lined body cavities (Cohen et al., 2004; Edwards et al., 2004; Sasaki et al., 2004; Wong et al., 2005). These striking patterns suggest a possible link between UV exposure and BRAF mutations in melanocytic neoplasia. However, any connection is likely to be complex as the mutations also occur in other cancers and do not show typical signatures of UV induction (Davies et al., 2002). Any link between BRAF mutations and UV light is further complicated by the fact that melanomas arising on skin areas with the highest UV exposure rarely have BRAF mutations (Maldonado et al., 2003). We therefore proposed that UV exposure might be an important causative factor for the formation of BRAF mutations in melanocytic tumors, but is not sufficient. Recent molecular (Curtin et al., 2005) and epidemiologic (Whiteman et al., 2003) studies have led us to suggest that a hypothesis that integrates these results. We have proposed that there are genetically distinct types of melanomas with regard to their mutation spectrum, and that individuals have a genetically determined susceptibility to UV exposure that strongly influences the genetic characteristics of a melanoma that they may develop. The hypothesized susceptibility operates either through a higher probability of melanocytes to acquire BRAF mutations if exposed to UV or to proliferate if such a mutation occurs. The present study extends these observations and the hypothesis to the development of nevi. Thus, we propose that truly congenital nevi, none of which have BRAF mutations, may be found on both (or either) susceptible and resistant individuals. Acquired nevi, most of which have BRAF mutations (Pollock et al., 2003; Yazdi et al., 2003), and melanomas with BRAF mutations occur in the more susceptible individuals and require modest UV exposure for their formation. Resistant individuals do not develop melanomas until they have had high total UV exposures, and these melanomas predominantly do not have BRAF mutations. Further study is required to determine the validity of this susceptibility hypothesis, and to define its genetic basis. However, until a formal proof of this hypothesis can be rendered, other mechanisms explaining the higher prevalence of BRAF mutations in sun-exposed sites have to be considered. In summary, our data show that nevi that develop in utero, in the absence of UV exposure, are genetically distinct from nevi developing after birth, even those that have histological similarities with truly congenital nevi. Our findings are consistent with the observation in melanoma that BRAF mutations are rare in neoplasms developing in UV-protected sites suggesting a connection, whether direct or indirect, between BRAF mutations and UV exposure. Better understanding of the genetic differences among histologically similar nevi with overlapping histological appearances may substantially improve the ability to make clinical decisions concerning the risk of progression to melanoma.

MATERIALS AND METHODS We selected 32 melanocytic nevi from the archives of the Department of Pathology of the University of California, San Francisco, in which there was a documented history that the nevus was present at birth. The study was approved by the Institutional Review Board of the University of California, San Francisco. The patient ages ranged from 1 day to 30 years (median 16 months, Table 1 and Table S1). The DNA was extracted from micro-dissected paraffin sections and BRAF exon 15 was amplified as published previously (Maldonado et al., 2003). For all tumors in which no mutations were found in exon 15, we also sequenced NRAS codon 61. PCR products were purified using ExoSAP-IT (USB Corporation, Cleveland, OH) and sequenced directly using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). In addition, we sequenced 10 atypical nodular cellular proliferations of a separate set of patients with melanocytic nevi present at birth. Atypical nodular proliferations typically present as rapidly growing tumors that develop shortly after birth and can simulate melanoma clinically and histopathologically. For comparison, we retrieved 28 melanocytic nevi from 22 patients that showed histopathologic features similar to congenital nevi (‘‘congenital pattern nevi’’) as defined by the presence of melanocytes clustered in the vicinity of adnexal structures, vessels or nerves, or splayed between the collagen bundles of the reticular dermis (Mark et al., 1973). No information was available as to whether these nevi were present at birth. The median age of this group of patients was 40 and the age ranged from 15 to 67 years. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS This work was supported by National Cancer Institute Grant R33 CA95300 to BCB and Deutsche Forschungsgemeinschaft stipend BA 2852/1-1 to JB.

SUPPLEMENTARY MATERIAL Table S1. Demographic data and mutation status of studied cases.

REFERENCES Bauer J, Buttner P, Wiecker TS, Luther H, Garbe C (2005) Risk factors of incident melanocytic nevi: a longitudinal study in a cohort of 1,232 young German children. Int J Cancer 115:121–6 Carr J, Mackie RM (1994) Point mutations in the N-ras oncogene in malignant melanoma and congenital naevi. Br J Dermatol 131:72–7 Clemmensen OJ, Kroon S (1988) The histology of ‘‘congenital features’’ in early acquired melanocytic nevi. J Am Acad Dermatol 19:742–6 Cohen Y, Rosenbaum E, Begum S, Goldenberg D, Esche C, Lavie O et al. (2004) Exon 15 BRAF mutations are uncommon in melanomas arising in nonsun-exposed sites. Clin Cancer Res 10:3444–7 Cribier BJ, Santinelli F, Grosshans E (1999) Lack of clinical–pathological correlation in the diagnosis of congenital naevi. Br J Dermatol 141:1004–9 Curtin JA, Fridlyand J, Kageshita T, Patel H, Busam K, Kutzner H et al. (2005) Distinct sets of genetic alterations in melanoma. N Engl J Med 353:2135–47 Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. (2002) Mutations of the BRAF gene in human cancer. Nature 417:949–54 De Raeve LE, Claes A, Ruiter DJ, van Muijen GN, Roseeuw D, van Kempen LC (2006) Distinct phenotypic changes between the superficial and deep component of giant congenital melanocytic nevi: a rationale for curettage. Br J Dermatol 154:485–92

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Edwards RH, Ward MR, Wu H, Medina CA, Brose MS, Volpe P et al. (2004) Absence of BRAF mutations in UV-protected mucosal melanomas. J Med Genet 41:270–2

Rhodes AR, Silverman RA, Harrist TJ, Melski JW (1985) A histologic comparison of congenital and acquired nevomelanocytic nevi. Arch Dermatol 121:1266–73

Gandini S, Sera F, Cattaruzza MS, Pasquini P, Picconi O, Boyle P et al. (2005) Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure. Eur J Cancer 41:45–60

Rhodes AR, Sober AJ, Day CL, Melski JW, Harrist TJ, Mihm MC et al. (1982) The malignant potential of small congenital nevocellular nevi. An estimate of association based on a histologic study of 234 primary cutaneous melanomas. J Am Acad Dermatol 6:230–41

Kopf AW, Bart RS, Hennessey P (1979) Congenital nevocytic nevi and malignant melanomas. J Am Acad Dermatol 1:123–30 Maldonado JL, Fridlyand J, Patel H, Jain AN, Busam K, Kageshita T et al. (2003) Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst 95:1878–90 Mark GJ, Mihm MC, Liteplo MG, Reed RJ, Clark WH (1973) Congenital melanocytic nevi of the small and garment type. Clinical, histologic, and ultrastructural studies. Hum Pathol 4:395–418 Papp T, Pemsel H, Zimmermann R, Bastrop R, Weiss DG, Schiffmann D (1999) Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi. J Med Genet 36:610–4 Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM et al. (2003) High frequency of BRAF mutations in nevi. Nat Genet 33:19–20 Rhodes AR, Melski JW (1982) Small congenital nevocellular nevi and the risk of cutaneous melanoma. J Pediatr 100:219–24

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Sasaki Y, Niu C, Makino R, Kudo C, Sun C, Watanabe H et al. (2004) BRAF point mutations in primary melanoma show different prevalences by subtype. J Invest Dermatol 123:177–83 Tannous ZS, Mihm MC Jr, Sober AJ, Duncan LM (2005) Congenital melanocytic nevi: clinical and histopathologic features, risk of melanoma, and clinical management. J Am Acad Dermatol 52:197–203 Whiteman DC, Watt P, Purdie DM, Hughes MC, Hayward NK, Green AC (2003) Melanocytic nevi, solar keratoses, and divergent pathways to cutaneous melanoma. J Natl Cancer Inst 95:806–12 Wong CW, Fan YS, Chan TL, Chan AS, Ho LC, Ma TK et al. (2005) BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs. J Clin Pathol 58:640–4 Yazdi AS, Palmedo G, Flaig MJ, Puchta U, Reckwerth A, Rutten A et al. (2003) Mutations of the BRAF gene in benign and malignant melanocytic lesions. J Invest Dermatol 121:1160–2