mitogen-activated protein kinase signaling pathway in sporadic trichoblastoma and syringocystadenoma papilliferum

Human Pathology (2015) 46, 272–276

www.elsevier.com/locate/humpath

Original contribution

Activating mutations in the RAS/mitogen-activated protein kinase signaling pathway in sporadic trichoblastoma and syringocystadenoma papilliferum☆ Anne-Sophie Shen a , Eva Peterhof a , Peter Kind MD b , Arno Rütten MD c , Bernhard Zelger MD d , Michael Landthaler MD a , Mark Berneburg MD a , Christian Hafner MD a,e , Leopold Groesser MD a,⁎ a

Department of Dermatology, University of Regensburg, 93053 Regensburg, Germany Dermatohistological Laboratory Prof. Kind, 63065 Offenbach, Germany c Dermatopathology Friedrichshafen, 88048 Friedrichshafen, Germany d Department of Dermatology and Venerology, Medical University Innsbruck, 6020 Innsbruck, Austria e Dermatohistological Laboratory, Sonnenstr. 7, 80331 Munich, Germany b

Received 2 August 2014; revised 24 October 2014; accepted 1 November 2014

Keywords: HRAS; KRAS; BRAF; Trichoblastoma; Syringocystadenoma papilliferum

Summary Trichoblastoma (TB) and syringocystadenoma papilliferum (SCAP) are both rare adnexal skin lesions occurring either sporadically or as secondary neoplasms in sebaceous nevi. TB and SCAP associated with sebaceous nevi have been shown to carry the same HRAS mutation as the underlying nevus. However, the genetic background of sporadic TB and SCAP has remained unknown. Therefore, we screened 18 sporadic TBs and 23 sporadic syringocystadenoma papillifera from 41 patients for the presence of activating mutations in RAS genes and other oncogenes. Using a RAS SNaPshot assay, HRAS mutations were detected in 2 (11%) of 18 sporadic TB and 6 (26%) of 23 sporadic syringocystadenoma papillifera. A KRAS mutation was identified in 1 sporadic SCAP. High-throughput oncogene mutation profiling furthermore identified BRAF V600E mutations in sporadic syringocystadenoma papillifera, which could be validated in 12 (52%) of 23 lesions using a BRAF SNaPshot assay. BRAF and RAS mutations were mutually exclusive in sporadic syringocystadenoma papillifera. No BRAF mutation could be detected in 3 syringocystadenoma papillifera secondarily arisen from a sebaceous nevus as well as in sporadic TB. In 14 lesions carrying an oncogenic mutation, nonlesional control tissue from the epidermal margin revealed a wild-type sequence, thus proving the somatic character of the mutation. Our results indicate that activation of the RAS-mitogen-activated protein kinase pathway by BRAF and RAS mutations contributes significantly to the tumorigenesis of sporadic SCAP and, less frequently, of sporadic TB. © 2015 Elsevier Inc. All rights reserved.

1. Introduction ☆

Disclosures: The authors declare no conflict of interest. ⁎ Corresponding author. Department of Dermatology, University of Regensburg, Franz-Josef Strauss-Allee 11, 93053 Regensburg, Germany. E-mail address: [email protected] (L. Groesser). http://dx.doi.org/10.1016/j.humpath.2014.11.002 0046-8177/© 2015 Elsevier Inc. All rights reserved.

Trichoblastoma (TB) and syringocystadenoma papilliferum (SCAP) are rare benign adnexal skin lesions, which may occur sporadically or in association with sebaceous nevi [1,2].

RAS/BRAF mutations in benign adnexal skin lesions TB are clinically not distinctive adnexal lesions, that present histopathologically as symmetric, well-circumscribed nests and lobules of small monomorphous basaloid cells with peripheral palisade formation of the nuclei [3]. These nests are found in close association with a loose spindle cell– containing stroma, which is characteristically separated by clefts from the surrounding normal dermis (Figure). Although TB are generally considered benign, trichoblastic carcinomas

273 arising within TB have been reported [4]. Despite the identification of CTNNB1 mutations in a very small percentage of sporadic TB [5], the genetic basis of this rare adnexal lesion remains to be elucidated. SCAP is a benign neoplasia mainly affecting the skin of the head and neck region and arising from pluripotent cells [2]. Clinically, this lesion presents as a hairless plaque or nodule measuring 1 to 4 cm in diameter, which is often first noted at birth or during early childhood [2]. Histopathologically, SCAP is localized in a semicystic depression in the dermis, which is in continuity with the epidermis and is lined by a papillary proliferation (Figure). Papillary projections are lined by cuboidal and in the luminar layer by low columnar cells with decapitation secretion. The stroma of the papillary structures is rich in plasma cells and other chronic inflammatory cells [6]. The pathogenesis of this mainly sporadic lesion remains enigmatic, although it has been found that loss of p16 may be relevant in the development of SCAP [7]. HRAS mutations have recently been identified in sebaceous nevi as well as in TB and SCAP associated with this nevus [8]. As these findings made it tempting to speculate on a pathogenic role of the Ras-Rafmitogen-activated protein kinase (MAPK) pathway in sporadic TB and SCAP, we analyzed these lesions using a RAS multiplex SNaPshot assay and the high-throughput oncogene mutation profiling assays OncoCarta Panel versions 1.0 and 2.0 (Sequenom, San Diego, CA).

2. Materials and methods 2.1. Samples Samples of 44 benign adnexal skin lesions were collected from the histologic archives of the Department of Dermatology, University of Regensburg, Regensburg, Germany; the Dermatohistological Laboratory Prof. Kind, Offenbach, Germany; the Dermatopathology Friedrichshafen, Friedrichshafen, Germany; and the Department of Dermatology, University of Innsbruck, Innsbruck, Austria. The Ethics Committee of the University of Regensburg approved this study, which was performed according to the Declaration of Helsinki.

2.2. Mutation analysis Figure Histologic and genetic characteristics of sporadic TB (A) and SCAP (B). A, TB presents as well-circumscribed nests of basaloid monorphous cells. SNaPshot analysis revealed a HRAS c.37GNC point mutation in the tumor nests, whereas the adjacent epidermis showed a HRAS wild type at codon 13. B, Prominent epithelial papillary invaginations are a typical feature of SCAP. Whereas the lesional tissue showed the BRAF c.1799 TNA mutation, the adjacent epidermis showed a wild-type sequence at codon 600 of BRAF, thus indicating that the mutation is somatic.

HRAS, KRAS, and NRAS mutations were analyzed as described previously [9]. To screen for mutations in other oncogenes, the OncoCarta Panels versions 1.0 and 2.0 (Sequenom) were used for a subset of 2 sporadic SCAP and 2 sporadic TB being wild type for RAS mutations [10]. In addition, all samples were screened for the BRAF c.1799 TNA mutation as previously described [11]. A second independent polymerase chain reaction was used to confirm all detected mutations.

274 Table

A. -S. Shen et al. RAS and BRAF mutations in sporadic TB and SCAP

Sample

Sex

TB 1 F 2 F 3 F 4 F 5 F 6 F 7 M 8 M 9 F 10 F 11 F 12 F 13 F 14 M 15 F 16 F 17 F 18 F SCAP 1 F 2 M 3 M 4 F 5 F 6 F 7 M 8 F 9 F 10 M 11 M 12 F 13 na 14 F 15 M 16 M 17 M 18 F 19 M 20 F 21 M 22 M 23 F Secondary SCAP 1 M 2 F 3 na

Age

Localization

Diagnosis

HRAS

KRAS

NRAS

BRAF

59 53 37 62 62 78 27 55 81 72 69 69 51 82 66 58 40 59

Occiput Cheek Chin Nose Occiput Occiput Gluteal Forehead na Knee Nose Ear Scalp Thigh Upper arm Nose Nose Temple

TB TB TB TB TB TB TB TB TB TB TB TB TB TB TB TB TB TB

G13R a G13R a wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt

wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt

wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt

wt wt wt wt wt wt wt wt wt wt wt wt wt na wt wt wt wt

42 82 78 48 44 69 43 53 26 34 34 35 na 57 62 72 67 70 49 69 60 22 61

Forehead Occiput Thigh Calf Scalp Auricle Neck Scapula Occiput Cheek Scalp Chin na na Abdomen Thorax Temple Heel Temple Occiput Occiput Thorax Gluteal

SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP SCAP

wt wt wt wt wt G13R a wt G13R a G13R a wt G13R wt G13R a wt wt wt wt wt G13R a wt wt wt wt

wt wt wt G12D a wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt

wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt

V600E V600E V600E wt na wt V600E a wt wt V600E wt V600E a wt V600E V600E a wt V600E a wt wt V600E a V600E wt V600E a

29 57 33

Forehead Forehead Occiput

SCAP on nevus sebaceus SCAP on nevus sebaceus SCAP on nevus sebaceus

G13R G13R G13R

wt wt wt

wt wt wt

wt wt wt

Abbreviations: F, female; M, male; na, not available, age at the time of the excision in years; wt, wild type; HRAS G13R, c37G>C; KRAS G12D, c.35G>A; BRAF V600E, c1799T>A. a Control tissue from adjacent skin was analyzed wild type.

3. Results Tissues from 41 patients with sporadic TB and SCAP were available for genetic analysis. The HRAS c.37GNC

mutation resulting in a p.Gly13Arg substitution was detected in 2 (11%) of 18 sporadic TB and in 6 (26%) of 23 sporadic SCAP (Table). In 1 sporadic SCAP, a KRAS c.35GNA leading to a p.Gly12Asp substitution was identified. No

RAS/BRAF mutations in benign adnexal skin lesions NRAS mutation could be detected in TB and SCAP. After the HRAS c.37GNC mutation has previously been shown in 100% of TB and SCAP associated with a sebaceous nevus, this hotspot mutation appears to be the most common RAS mutation in sporadic TB and SCAP as well [8]. To identify additional oncogenes involved in the pathogenesis of TB and SCAP, we analyzed 2 sporadic TB and 2 sporadic SCAP being wild type for HRAS and KRAS using the OncoCarta Panels versions 1.0 and 2.0. These panels cover 238 mutations in 19 oncogenes and 152 mutations in 18 oncogenes, respectively, and detected a BRAF c.1799 TNA mutation, resulting in a p.Val600Glu substitution, in 1 sporadic SCAP. Therefore, we screened all sporadic SCAP for the BRAF c.1799 TNA mutation, which was detected in 12 (52%) of 23 lesions (Table). Thereby, BRAF and RAS mutations proved to be mutually exclusive. This observation was further substantiated by a BRAF wild-type status in 3 additionally investigated SCAP, which had developed within a nevus sebaceus and showed a HRAS c.37GNC mutation. To the best of our knowledge, neither BRAF nor HRAS/KRAS mutations have been reported in sporadic SCAP so far. In contrast to the sporadic SCAP, all sporadic TB investigated showed a BRAF wild-type sequence at codon 600. To prove the somatic nature of the RAS and BRAF mutations identified, we analyzed perilesional epidermis of 2 sporadic TB and 12 sporadic SCAP. None of these samples showed the mutation found in the lesional tissue, thus confirming the somatic character of the mutation and indicating a strong genotype phenotype correlation (Table and Figure). In summary, 2 (11%) of 18 sporadic TB and 19 (83%) of 23 sporadic SCAP harbored an oncogenic mutation in the Ras-MAPK pathway.

4. Discussion Recent data on postzygotic HRAS mutations in TB and SCAP arising in sebaceous nevi made it tempting to speculate on a pathogenic role of the Ras-MAPK pathway in sporadic TB and SCAP [8,12]. In this study, HRAS mutations were present in 11% of sporadic TB and 26% of sporadic SCAP. Interestingly, the HRAS c.37GNC hotspot mutation was the predominant RAS mutation in sporadic TB and SCAP. This mutation has previously been shown to be the most frequent somatic mutation in nevus sebaceous [8], epidermal nevi [13], phacomatosis pigmentokeratotica [14], nevus marginatus [15], and sporadic nevi spili [16]. In 1 sporadic SCAP, a KRAS c.35GNA mutation was identified, which is the most frequent somatic KRAS substitution found in human cancer according to the Catalogue of Somatic Mutations in Cancer database and has also been found in a small percentage of sebaceous nevi. The mutational profile in sporadic SCAP and TB with respect to RAS is thus in accord with the mutational spectrum found in sebaceous nevi [8]. As the cells of secondary TB and SCAP have been shown to derive directly from the sebaceous nevus cells [8], the HRAS

275 c.37GNC mutation as well as the KRAS c.35GNA mutation furthermore appears to be involved into the development of TB and SCAP. This may not least be suggested by data obtained from a Cre-inducible mouse model for constitutive activation of KRas (KRAS c.35GNA), where wounding has been shown to precipitate tumor formation after KRas activation [17]. Beside the concerted RAS mutation profiles in sporadic and secondary TB and SCAP, the identification of the somatic BRAF c.1799 TNA mutation in approximately 50% of sporadic SCAP is the most intriguing finding of this study. This mutation is found to be mutually exclusive with the aforementioned HRAS and KRAS mutations of sporadic and secondary SCAP, which may be viewed as an indicator of tumor homogeneity. BRAF mutations have been reported in a wide variety of benign and malignant human tumors such as benign colorectal lesions, colorectal carcinoma, papillary thyroid carcinomas, and hairy cell leukemia [18]. Among cutaneous neoplasms, the BRAF c.1799 TNA mutation is primarily associated with melanocytic lesions; however, it has also been reported in sebaceous hyperplasias of patients belonging to MYH-associated polyposis pedigrees [19]. These data thus expand the spectrum of benign nonmelanocytic skin lesions carrying BRAF mutations and could furthermore hint at potential therapeutic targets in syringocystadenocarcinoma papilliferum, which is a rare malignant counterpart of SCAP with the ability to develop locoregional metastases [20]. Taken together, our study for the first time demonstrates somatic HRAS, KRAS, and BRAF mutations in the vast majority of sporadic SCAP. In sporadic TB, RAS mutations occur far less frequently. These data shed light on the hitherto undetermined genetic basis of these rare adnexal lesions.

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