Fibroblast Growth Factor Receptor 3 Mutations in Epidermal Nevi and Associated Low Grade Bladder Tumors

ORIGINAL ARTICLE

See related commentary on pg 1572

Fibroblast Growth Factor Receptor 3 Mutations in Epidermal Nevi and Associated Low Grade Bladder Tumors Silvia Herna´ndez1, Agustı´ Toll2, Eula`lia Baselga3, Adriana Ribe´3, Javier Azua-Romeo4, Ramon M. Pujol2,5,7 and Francisco X. Real1,6 Epidermal nevi (EN) are benign lesions presenting at birth or in childhood. Based on the occurrence of fibroblast growth factor receptor 3 (FGFR3) mutations in seborrheic keratosis and urothelial carcinomas (UC), and the identification of two young patients with EN and UC, we hypothesized that mutations might occur in EN. The R248C mutation was found in 6/23 (26.1%) EN but it was absent from unaffected skin. In two patients with EN and UC, both lesions were FGFR3 wild type. Our findings indicate that: (1) FGFR3 mutations occur in mosaicism and can cause EN and (2) other genes are involved in EN. Journal of Investigative Dermatology (2007) 127, 1664–1666; doi:10.1038/sj.jid.5700705; published online 25 January 2007

INTRODUCTION Epidermal nevi (EN) are a heterogeneous group of congenital hamartomas characterized by hyperplasia of the epidermis and adnexal structures and manifested by verrucoid scaly plaques often following Blaschko’s lines. EN are distinguished by clinical, histopathological, and genetic criteria (Happle and Rogers, 2002). EN can be associated with complex developmental abnormalities (EN syndrome) and, occasionally, with cutaneous squamous and basal cell carcinomas (Sugarman, 2004). Recently, activating fibroblast growth factor receptor 3 (FGFR3) mutations were reported in 39% of seborrheic keratoses (Logie et al., 2005) and in 86% of adenoid seborrheic keratosis (Hafner et al., 2006a). The highest prevalence of somatic FGFR3 mutations occurs in urothelial carcinomas (UC) (Cappellen et al., 1999). Mutations are associated with low grade tumors (Billerey et al., 2001) with an overall good prognosis (van Rhijn et al., 2003; Hernandez et al., 2006). The same mutations have been reported in germline DNA of patients with skeletal dysplasias (Horton

1

Department de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain; 2Servei de Dermatologia, Hospital del Mar, Barcelona, Spain; 3Hospital de Sant Pau, Barcelona, Spain; 4Departamento de Anatomı´a e Histologı´a, Universidad de Zaragoza, Barcelona, Spain; 5 Universitat Auto`noma de Barcelona, Barcelona, Spain and 6Unitat de Biologia Cel.lular i Molecular, Institut Municipal d’Investigacio´ Me`dica, Barcelona, Spain 7

These authors contributed equally to this work

Correspondence: Dr Francisco X. Real, Unitat de Biologia Cel.lular i Molecular, Institut Municipal d’Investigacio´ Me`dica, Carrer del Dr Aiguader 80, E-08003 Barcelona, Spain. E-mail: [email protected] Abbreviations: EN, epidermal nevus; FGFR3, fibroblast growth factor receptor 3; UC, urothelial carcinoma Received 20 September 2006; revised 14 November 2006; accepted 16 November 2006; published online 25 January 2007

1664 Journal of Investigative Dermatology (2007), Volume 127

and Lunstrum, 2002). In UC and in skeletal dysplasias, mutations occur mainly in hotspots in exons 7, 10, and 15 (Cappellen et al., 1999; Hernandez et al., 2006). Mutations confer constitutive kinase activity and there is a good correlation between the latter and disease severity (Naski et al., 1996). We have identified a 41-year-old patient with a congenital widespread non-epidermolytic keratinocytic EN; lesions were distributed in a checkerboard pattern. The patient had had a low grade UC diagnosed at age 19. There were no skeletal abnormalities and no family history of skin or urological diseases. Three additional literature reports of EN and UC were identified (Rosenthal and Fretzin, 1986; Rongioletti and Rebora, 1991; Garcia de Jalon et al., 2004). Based on the histological similarity of seborrheic keratosis and EN, and these case reports, we hypothesized that FGFR3 mutations might cause EN. RESULTS Characteristics of cases and mutational results are shown in Table 1. In six patients (26.1%), the R248C mutation in exon 7 was found. Exons 10 or 15 were normal in all cases. Mutations were not associated with gender, age at diagnosis, location, number of EN, or histological subtype. Unaffected skin from two patients with FGFR3mut EN showed FGFR3wt sequences. FGFR3 has been shown to be overexpressed in UC (Gomez-Roman et al., 2005). Using immunohistochemistry, FGFR3 was weakly detected in the basal cell layer of 2/6 FGFR3mut and 9/13 FGFR3wt samples of EN, similar to its weak detection in normal epidermis. In the remaining samples, FGFR3 was undetectable. No sample showed evidence of increased FGFR3 expression. We analyzed the TaG1 UC of two very young patients with an associated EN. Both tumors were wild type for exons & 2007 The Society for Investigative Dermatology

S Herna´ndez et al. FGFR3 Mutations in Epidermal Nevi and Associated Bladder Cancer

Table 1. Characteristics of patients with EN included in the study in relationship to FGFR3 mutational status Single/multiple

Location

Histology

FGFR3 status

Multiple

Neck

AV-like

WT

Multiple

Upper lip

C

WT

Multiple

Upper lip

C

WT

Multiple

Neck

SK-like

WT

Multiple

Neck

SK-like

WT

Single

Cheek

SK-like

R248C

Single

Cheek/neck

C

WT

Multiple

Arm

C

R248C

Single

Frontoparietal

C

WT

Single

Chin–neck

AV-like

WT

Single

Lumbar

C

WT

Single

Forearm

SK-like

WT

Multiple

Shoulder/arm

C

R248C

Single

Shoulder

C

WT

Single

Frontal

C

WT

Single

Frontal

SK-like

WT

Single

Occipital

C

WT

Single

Axillary

C

WT

Single

NA

C

R248C

Single

NA

AV-like

R248C

Single

NA

AV-like

R248C

Multiple

Widespread

C

WT

Single

Lumbar

SK-like

WT

Single

Back

C

WT

Single

Neck

C

WT

AV, acrokeratosis verruciformis; C, common (hyperkeratosis, papillomatosis, and acanthosis with elongation of rete ridges); NA, not available; SK, seborrheic keratosis; WT, wild-type.

7 and 10–18; the corresponding patient’s EN were also FGFR3wt. DISCUSSION Our results indicate that mutations in FGFR3 occur frequently in EN. The characteristic distribution of lesions and absence of mutations in unaffected skin indicate somatic mosaicism. FGFR2 mutational somatic mosaicism in acne has also been described (Munro and Wilkie, 1998). While this paper was prepared, FGFR3 mutations were reported in 16/39 (41%) EN from 11/33 (33%) patients. In all but one case, the R248C substitution was found; in the remaining case, two mutations (G372C and G382R) were detected in exon 10. In four patients with an FGFR3mut EN, unaffected epidermis adjacent to the nevus was FGFR3wt, indicating mosaicism. In an additional case, leukocyte DNA was FGFR3wt, supporting the lack of germline mutation (Hafner et al., 2006b). Our findings provide strong support to

these data and suggest that other genes may contribute to the development of EN, and to UC as well. PIK3CA mutations are associated with low grade UC (Lopez-Knowles et al., 2006), thus making it a candidate gene. A comparison of skin and urothelium – and of benign and malignant lesions from these epithelia – may provide important clues as to the genetic mechanisms involved in tumor development and progression in these tissues. This notion is supported by the known involvement of PTCH and TSC1 alterations in both skin and bladder tumors (Aboulkassim et al., 2003; Knowles et al., 2003). The role of mosaicism in tumor development is best assessed in the skin because of its accessibility. It is tempting to speculate that somatic mutations occurring in mosaicism may also contribute to tumors in other tissues (i.e. bladder). This concept, which could account for the association of EN with UC, merits further analysis in human cancer biology. MATERIALS AND METHODS We retrieved and reviewed 23 EN showing ‘‘common’’ histopathological features, acrokeratosis verruciformis-like findings, or a seborrheic keratosis-like pattern. In addition, the UCs of the case described here and of an earlier report (Garcia de Jalon et al., 2004) were analyzed. Informed consent was obtained from patients. The study was approved by the Ethics Committee of Institut Municipal d’Investigacio´ Me`dica and was conducted according to the Declaration of Helskinki Principles. Microdissection, DNA extraction, primers, and PCR amplification of FGFR3 exons 7, 10, and 15 were performed as reported elsewhere (Hernandez et al., 2006); exons 11–18 were also analyzed in DNA from the UC. All abnormal sequences were independently confirmed. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank J Rodrı´guez and M Garrido for technical help, A Hartmann and N Malats for helpful discussions, A Ferna´ndez, E Campo, J Lloreta, E LopezKnowles, and J Ramı´rez for valuable contributions, and the Tumor Banks at Hospital Clinic, Hospital del Mar, and Hospital de Sant Pau for providing samples. This work was supported, in part, by grants C03/010 and G03/174 from Instituto de Salud Carlos III, Ministerio de Sanidad, Spain.

REFERENCES Aboulkassim TO, LaRue H, Lemieux P, Rousseau F, Fradet Y (2003) Alteration of the PATCHED locus in superficial bladder cancer. Oncogene 22:2967–71 Billerey C, Chopin D, Aubriot-Lorton MH, Ricol D, Gil Diez de Medina S, Van Rhijn B et al. (2001) Frequent FGFR3 mutations in papillary noninvasive bladder (pTa) tumors. Am J Pathol 158:1955–9 Cappellen D, De Oliveira C, Ricol D, de Medina S, Bourdin J, Sastre-Garau X (1999) Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat Genet 23:18–20 Garcia de Jalon A, Azua-Romeo J, Trivez MA, Pascual D, Blas M, Rioja LA (2004) Epidermal naevus syndrome (Solomon’s syndrome) associated with bladder cancer in a 20-year-old female. Scand J Urol Nephrol 38:85–7 Gomez-Roman JJ, Saenz P, Molina M, Cuevas Gonzalez J, Escuredo K, Santa Cruz S et al. (2005) Fibroblast growth factor receptor 3 is overexpressed in urinary tract carcinomas and modulates the neoplastic cell growth. Clin Cancer Res 11:459–65

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S Herna´ndez et al. FGFR3 Mutations in Epidermal Nevi and Associated Bladder Cancer

Hafner C, van Oers JM, Hartmann A, Landthaler M, Stoehr R, Blaszyk H et al. (2006a) High frequency of FGFR3 mutations in adenoid seborrheic keratoses. J Invest Dermatol 126:2404–7 Hafner C, van Oers JM, Vogt T, Landthaler M, Stoehr R, Blaszyk H et al. (2006b) Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi. J Clin Invest 116:2201–7 Happle R, Rogers M (2002) Epidermal nevi. Adv Dermatol 18:175–201

Lopez-Knowles E, Hernandez S, Malats N, Kogevinas M, Lloreta J, Carrato A et al. (2006) PIK3CA mutations are an early genetic alteration associated with FGFR3 mutations in superficial papillary bladder tumors. Cancer Res 66:7401–4 Munro CS, Wilkie AO (1998) Epidermal mosaicism producing localised acne: somatic mutation in FGFR2. Lancet 352:704–5 Naski MC, Wang Q, Xu J, Ornitz DM (1996) Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nat Genet 13:233–7

Hernandez S, Lopez-Knowles E, Lloreta J, Kogevinas M, Amoros A, Tardon A et al. (2006) Prospective study of FGFR3 mutations as a prognostic factor in nonmuscle invasive urothelial bladder carcinomas. J Clin Oncol 24:3664–71

Rongioletti F, Rebora A (1991) Epidermal nevus with transitional cell carcinomas of the urinary tract. J Am Acad Dermatol 25:856–8

Horton WA, Lunstrum GP (2002) Fibroblast growth factor receptor 3 mutations in achondroplasia and related forms of dwarfism. Rev Endocr Metab Disord 3:381–5

Rosenthal D, Fretzin DF (1986) Epidermal nevus syndrome: report of association with transitional cell carcinoma of the bladder. Pediatr Dermatol 3:455–8

Knowles MA, Habuchi T, Kennedy W, Cuthbert-Heavens D (2003) Mutation spectrum of the 9q34 tuberous sclerosis gene TSC1 in transitional cell carcinoma of the bladder. Cancer Res 63:7652–6

Sugarman JL (2004) Epidermal nevus syndromes. Semin Cutan Med Surg 23:145–57

Logie A, Dunois-Larde C, Rosty C, Levrel O, Blanche M, Ribeiro A et al. (2005) Activating mutations of the tyrosine kinase receptor FGFR3 are associated with benign skin tumors in mice and humans. Hum Mol Genet 14:1153–60

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van Rhijn BW, Vis AN, van der Kwast TH, Kirkels WJ, Radvanyi F, Ooms EC et al. (2003) Molecular grading of urothelial cell carcinoma with fibroblast growth factor receptor 3 and MIB-1 is superior to pathologic grade for the prediction of clinical outcome. J Clin Oncol 21: 1912–21