0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Case Report/Case Series |

Activating HRAS Mutation in Agminated Spitz Nevi Arising in a Nevus Spilus FREE

Kavita Y. Sarin, MD, PhD1; Bryan K. Sun, MD, PhD1; Charles D. Bangs2; Athena Cherry, MD2; Susan M. Swetter, MD1,3; Jinah Kim, MD, PhD1; Paul A. Khavari, MD, PhD1,3
[+] Author Affiliations
1Department of Dermatology, Stanford University School of Medicine, Stanford, California
2Department of Pathology, Stanford University School of Medicine, Stanford, California
3Dermatology Service, VA Palo Alto Health Care System, Palo Alto, California
JAMA Dermatol. 2013;149(9):1077-1081. doi:10.1001/jamadermatol.2013.4745.
Text Size: A A A
Published online

Importance  Spitz nevi are benign melanocytic proliferations that can sometimes be clinically and histopathologically difficult to distinguish from melanoma. Agminated Spitz nevi have been reported to arise spontaneously, in association with an underlying nevus spilus, or after radiation or chemotherapy. However, to our knowledge, the genetic mechanism for this eruption has not been described.

Observations  We report a case of agminated Spitz nevi arising in a nevus spilus and use exome sequencing to identify a clonal activating point mutation in HRAS (GenBank 3265) (c.37G→C) in the Spitz nevi and underlying nevus spilus. We also identify a secondary copy number increase involving HRAS on chromosome 11p, which occurs during the development of the Spitz nevi.

Conclusions and Relevance  Our results reveal an activating HRAS mutation in a nevus spilus that predisposes to the formation of Spitz nevi. In addition, we demonstrate a copy number increase in HRAS as a “second hit” during the formation of agminated Spitz nevi, which suggests that both multiple Spitz nevi and solitary Spitz nevi may arise through similar molecular pathways. In addition, we describe a unique investigative approach for the discovery of genetic alterations in Spitz nevi.

Figures in this Article

Spitz nevi are benign melanocytic neoplasms composed of epithelioid or spindle cell melanocytes. While Spitz nevi have distinct histologic criteria for diagnosis, a subset of Spitz nevi can be clinically and histopathologically difficult to distinguish from malignant melanoma, leading to controversy regarding the nature of these lesions.1,2 Some Spitz nevi harbor activating mutations in HRAS (GenBank 3265) and BRAF (GenBank 673), serine-threonine kinases in the mitogen-activated protein kinase pathway that play a critical role in epidermal development, homeostasis, and tumor progression.35 In addition, approximately 20% of Spitz nevi, predominantly those harboring HRAS mutations, have an increased copy number of chromosomal locus 11p, where HRAS resides.3,6 These HRAS mutations can be a favorable prognostic biomarker since HRAS is rarely mutated in melanoma.6,7

Spitz nevi usually present as solitary skin tumors but can occur in multiple patterns, having agminated, dermatomal, and disseminated forms.810 Agminated Spitz nevi occur rarely, with fewer than 50 cases reported in the literature. They have been reported to arise spontaneously, in association with an underlying nevus spilus, and after radiation or chemotherapy.1012 Despite the clinical and histopathologic resemblance to solitary Spitz nevi, the genetic alterations in these lesions remain unknown. It is unclear if these agminated lesions harbor the same mutations as solitary Spitz nevi or arise from an alternate pathway. These lesions represent a compelling approach to studying Spitz nevi since they may potentially arise from an early mutation, producing a clone of melanocytes predisposed to developing into Spitz nevi. Herein, we applied exome sequencing to identify genetic changes in agminated Spitz nevi arising in a nevus spilus and demonstrate a common mosaic mutation among them.

A 25-year-old man presented to the Stanford Pigmented Lesion and Melanoma Clinic with a 4-year history of pink papules emanating in a large congenital pigmented tan patch on his left lower back. Clinical examination revealed a more than 20-cm tan patch speckled with 1- to 2-mm hyperpigmented macules, characteristic of a nevus spilus, and containing fifteen to twenty 4- to 6-mm pink papules, characteristic of Spitz nevi (Figure 1A and B). The patient was otherwise healthy, with no personal or family history of malignant melanoma. Histopathologic specimens of 2 pink papules revealed symmetric, well-demarcated melanocytic proliferations consisting of spindle cell melanocytes with large vesicular nuclei splayed through the dermis, consistent with intradermal Spitz nevi (Figure 1C and D). To identify underlying genetic alterations, we obtained specimens from 2 additional pink papules, with histopathologic features also confirming the diagnosis of Spitz nevi. Our study complied with the Declaration of Helsinki and was approved by the institutional review board at Stanford University School of Medicine. Genomic DNA was isolated from these 2 lesional samples along with the adjacent normal skin, 1 cm outside the boundaries of the nevus spilus, and subjected to exome sequencing (eMethods and eTable in the Supplement).

Place holder to copy figure label and caption
Figure 1.
Clinical and Histopathologic Features of the Agminated Spitz Nevi Arising in a Nevus Spilus

A and B, Photograph of a large tan patch on the left lower back with 1- to 2-mm hyperpigmented macules and 4- to 6-mm pink papules. C, Pink papule showing plump melanocytes splayed through desmoplastic collagen, consistent with Spitz nevi (hematoxylin-eosin, original magnification ×10). D, Melanocytes with amphophilic cytoplasm in the dermis (hematoxylin-eosin, original magnification ×20).

Graphic Jump Location

Comparison of recurrent variants from the exome sequencing identified an HRAS point mutation (c.37G→C, p.Gly13Arg) in both Spitz nevi that was absent in the adjacent normal skin (Figure 2A). No other recurrent somatic mutations were detected (eMethods in the Supplement). Sanger sequencing confirmed the presence of the HRAS mutation in both Spitz lesions. We performed Sanger sequencing on DNA derived from 2 additional formalin-fixed, paraffin-embedded Spitz nevi obtained from the same patient that also demonstrated the HRAS point mutation (Figure 2B). Therefore, all 4 Spitz nevi obtained from our patient harbored the same single-nucleotide variation.

Place holder to copy figure label and caption
Figure 2.
Activating HRAS Mutations and Amplification of Chromosome 11p in Agminated Spitz Nevi

A, Sanger sequencing of a representative Spitz nevus and adjacent unaffected skin demonstrates a c.37G→C, p.Gly13Arg mutation specific to the lesional tissue. B, Table of HRAS mutations showing the HRAS mutation is present in all 4 Spitz nevi but undetectable in the adjacent normal skin. C, Chromosomal amplifications predicted by SeqGene CNV and displayed with an Integrative Genomics Viewer (http://www.broadinstitute.org/igv/). Both Spitz nevi have a predicted amplification (red bars) over chromosome 11p. D, Dual-color fluorescent in situ hybridization (FISH) with HRAS probe (red signals) and a reference centromeric probe for chromosome 11 (green signals) showing a focus of melanocytes with increased red signal significantly above reference green signals, indicating HRAS amplification (arrows). E, Dual-colored FISH showing a focus of melanocytes with polysomy demonstrated by increased HRAS (red) and centromeric (green) signals in the nucleus (arrows). F, Dual-color FISH showing epidermal keratinocytes and papillary dermal fibroblasts with equivalent red and green signals. G, Sanger sequencing of AciI1-digested DNA from a Spitz nevus, nevus spilus, and the adjacent normal skin demonstrating the HRAS mutation in the nevus spilus and Spitz nevus but not in the normal skin. H, Diagram of 2-hit model of a nevus spilus, with the first hit leading to the macular portion of the nevus spilus and the second hit leading to the formation of Spitz nevi. WT indicates wild type.

Graphic Jump Location

To evaluate for copy number changes, we used SeqGene-CNV on the exome sequencing data.13 This algorithm detects regions with abnormal copy number changes using circular binary segmentation. This revealed a copy number increase in chromosome 11p in both Spitz nevi compared with the normal skin control (Figure 2C). We then performed fluorescent in situ hybridization using an HRAS probe that confirmed amplification of HRAS in the melanocytes from 2 Spitz nevi (Figure 2D) and polysomy in the melanocytes from a third Spitz nevus (Figure 2E). No HRAS amplification was detected in adjacent fibroblasts or epidermal keratinocytes (Figure 2F).

Spitz nevi are heterogeneous melanocytic tumors, with less than 20% of these lesions harboring HRAS activating mutations and even fewer containing the HRAS point mutation.3 Thus, it would be highly improbable for all Spitz nevi obtained from our patient to develop identical mutations if they represented independent lesions. We hypothesized that these Spitz nevi arose in an agminated fashion from a common postzygotic clone of melanocytes, likely demarcated by the nevus spilus. To improve our sensitivity to detect this mutation in the nevus spilus, we performed polymerase chain reaction amplification of the genomic DNA followed by enzymatic digestion with Aci1, which digests the wild-type sequence but not the mutant sequence (eMethods and eFigures 1 and 2 in the Supplement). Subsequent Sanger sequencing reproducibly detected the HRAS point mutation in the nevus spilus and Spitz nevi but not in the adjacent normal skin (Figure 2G). This implicates the HRAS point mutation as the initiating mutation predisposing melanocytes to develop into Spitz nevi. In this model, a “second hit” may be required for the formation of Spitz nevi (Figure 2H). Our data support HRAS amplification as a secondary change because its mechanism was not identical in all Spitz nevi, with 1 nevus demonstrating polysomy of chromosome 11.

Multiple Spitz nevi can occur rarely in agminated and disseminated forms, but the genetic alterations that lead to these occurrences are unknown. To our knowledge, this is the first report demonstrating mosaicism in agminated Spitz nevi and identifying an activating HRAS mutation in agminated Spitz nevi. Mosaic HRAS mutations were recently recognized in the nevi sebacei and nevi spili in patients with phacomatosis pigmentokeratotica.14 This report extends this finding by demonstrating an HRAS mutation in a sporadic nevus spilus. Interestingly, the HRAS point mutation, in particular, has been detected in a variety of benign skin neoplasms, including epidermal and sebaceous nevi, Spitz nevi, and nevi spili, providing a unique example of genetic pleiotropy within the ectodermal lineage.15

Our data indicate that multiple Spitz nevi may have a similar pathogenesis to that of solitary Spitz nevi since a subset of solitary Spitz nevi also harbors activating mutations in HRAS and copy number increases in chromosome 11p.3 However, similar to solitary Spitz nevi, other genetic alterations also may play a role in the pathogenesis of multiple Spitz nevi. Gantner et al9 recently demonstrated the absence of HRAS-activating mutations in a patient with eruptive Spitz nevi, suggesting that alternate genetic alterations may be responsible for the lesions in this patient. It is tempting to speculate that many cases of multiple Spitz nevi may result from an early clonal mutation, as demonstrated in our patient.

Recently, significant progress has been made in understanding the genetic alterations in cutaneous tumors, in part due to the advances in sequencing technology. Many of these technologies rely on a large number of samples to determine recurrent mutations. This approach may be difficult in solitary Spitz nevi since the lesions are uncommon and possess heterogeneous mutations. Identifying clonal mutations in patients with multiple Spitz nevi presents a promising approach to distinguish genetic alterations in all Spitz nevi. Insight into these genetic changes is critical to improve our ability to diagnose and manage these controversial lesions.

Accepted for Publication: April 2, 2013.

Corresponding Author: Kavita Y. Sarin, MD, PhD, Department of Dermatology, Stanford University School of Medicine, 450 Broadway St, Pavilion B, Fourth Floor, MC 5338, Redwood City, CA 94063 (ksarin@stanford.edu)

Published Online: July 24, 2013. doi:10.1001/jamadermatol.2013.4745.

Author Contributions: Drs Sarin, Swetter, Kim, and Khavari had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Sarin, Kim.

Acquisition of data: Sarin, Sun, Bangs, Cherry, Swetter, Kim.

Analysis and interpretation of data: Sarin, Sun, Bangs, Cherry, Kim, Khavari.

Drafting of the manuscript: Sarin, Bangs, Swetter, Kim.

Critical revision of the manuscript for important intellectual content: Sarin, Sun, Cherry, Swetter, Kim, Khavari.

Statistical analysis: Sarin.

Administrative, technical, and material support: Sun, Bangs, Cherry, Kim, Khavari.

Study supervision: Kim, Khavari.

Conflict of Interest Disclosures: None reported.

Da Forno  PD, Fletcher  A, Pringle  JH, Saldanha  GS.  Understanding spitzoid tumours: new insights from molecular pathology. Br J Dermatol. 2008;158(1):4-14.
PubMed
Mones  JM, Ackerman  AB.  “Atypical” Spitz’s nevus, “malignant” Spitz’s nevus, and “metastasizing” Spitz’s nevus: a critique in historical perspective of three concepts flawed fatally. Am J Dermatopathol. 2004;26(4):310-333.
PubMed   |  Link to Article
Bastian  BC, LeBoit  PE, Pinkel  D.  Mutations and copy number increase of HRAS in Spitz nevi with distinctive histopathological features. Am J Pathol. 2000;157(3):967-972.
PubMed   |  Link to Article
Fullen  DR, Poynter  JN, Lowe  L,  et al.  BRAF and NRAS mutations in spitzoid melanocytic lesions. Mod Pathol. 2006;19(10):1324-1332.
PubMed   |  Link to Article
Wiesner  T, Murali  R, Fried  I,  et al.  A distinct subset of atypical Spitz tumors is characterized by BRAF mutation and loss of BAP1 expression. Am J Surg Pathol. 2012;36(6):818-830.
PubMed   |  Link to Article
Bastian  BC, Wesselmann  U, Pinkel  D, Leboit  PE.  Molecular cytogenetic analysis of Spitz nevi shows clear differences to melanoma. J Invest Dermatol. 1999;113(6):1065-1069.
PubMed   |  Link to Article
van Dijk  MC, Bernsen  MR, Ruiter  DJ.  Analysis of mutations in B-RAF, N-RAS, and H-RAS genes in the differential diagnosis of Spitz nevus and spitzoid melanoma. Am J Surg Pathol. 2005;29(9):1145-1151.
PubMed   |  Link to Article
Harris  K, Florell  SR, Papenfuss  J,  et al.  Melanoma mimic: a case of multiple pagetoid Spitz nevi. Arch Dermatol. 2012;148(3):370-374.
PubMed   |  Link to Article
Gantner  S, Wiesner  T, Cerroni  L,  et al.  Absence of BRAF and HRAS mutations in eruptive Spitz naevi. Br J Dermatol. 2011;164(4):873-877.
PubMed   |  Link to Article
Boone  SL, Busam  KJ, Marghoob  AA,  et al.  Two cases of multiple Spitz nevi: correlating clinical, histologic, and fluorescence in situ hybridization findings. Arch Dermatol. 2011;147(2):227-231.
PubMed   |  Link to Article
Aloi  F, Tomasini  C, Pippione  M.  Agminated Spitz nevi occurring within a congenital speckled lentiginous nevus. Am J Dermatopathol. 1995;17(6):594-598.
PubMed   |  Link to Article
Berk  DR, Lane  AT.  Acquired bilateral agminated Spitz nevi in a child with Langerhans cell histiocytosis. Pediatr Dermatol. 2010;27(3):282-284.
PubMed   |  Link to Article
Deng  X.  SeqGene: a comprehensive software solution for mining exome- and transcriptome-sequencing data. BMC Bioinformatics. 2011;12:267. doi:10.1186/1471-2105-12-267.
PubMed   |  Link to Article
Groesser  L, Herschberger  E, Sagrera  A,  et al.  Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell [published online January 21, 2013]. J Invest Dermatol. doi:10.1038/jid.2013.24.
PubMed
Hafner  C, Groesser  L.  Mosaic RASopathies. Cell Cycle. 2013;12(1):43-50.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.
Clinical and Histopathologic Features of the Agminated Spitz Nevi Arising in a Nevus Spilus

A and B, Photograph of a large tan patch on the left lower back with 1- to 2-mm hyperpigmented macules and 4- to 6-mm pink papules. C, Pink papule showing plump melanocytes splayed through desmoplastic collagen, consistent with Spitz nevi (hematoxylin-eosin, original magnification ×10). D, Melanocytes with amphophilic cytoplasm in the dermis (hematoxylin-eosin, original magnification ×20).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Activating HRAS Mutations and Amplification of Chromosome 11p in Agminated Spitz Nevi

A, Sanger sequencing of a representative Spitz nevus and adjacent unaffected skin demonstrates a c.37G→C, p.Gly13Arg mutation specific to the lesional tissue. B, Table of HRAS mutations showing the HRAS mutation is present in all 4 Spitz nevi but undetectable in the adjacent normal skin. C, Chromosomal amplifications predicted by SeqGene CNV and displayed with an Integrative Genomics Viewer (http://www.broadinstitute.org/igv/). Both Spitz nevi have a predicted amplification (red bars) over chromosome 11p. D, Dual-color fluorescent in situ hybridization (FISH) with HRAS probe (red signals) and a reference centromeric probe for chromosome 11 (green signals) showing a focus of melanocytes with increased red signal significantly above reference green signals, indicating HRAS amplification (arrows). E, Dual-colored FISH showing a focus of melanocytes with polysomy demonstrated by increased HRAS (red) and centromeric (green) signals in the nucleus (arrows). F, Dual-color FISH showing epidermal keratinocytes and papillary dermal fibroblasts with equivalent red and green signals. G, Sanger sequencing of AciI1-digested DNA from a Spitz nevus, nevus spilus, and the adjacent normal skin demonstrating the HRAS mutation in the nevus spilus and Spitz nevus but not in the normal skin. H, Diagram of 2-hit model of a nevus spilus, with the first hit leading to the macular portion of the nevus spilus and the second hit leading to the formation of Spitz nevi. WT indicates wild type.

Graphic Jump Location

Tables

References

Da Forno  PD, Fletcher  A, Pringle  JH, Saldanha  GS.  Understanding spitzoid tumours: new insights from molecular pathology. Br J Dermatol. 2008;158(1):4-14.
PubMed
Mones  JM, Ackerman  AB.  “Atypical” Spitz’s nevus, “malignant” Spitz’s nevus, and “metastasizing” Spitz’s nevus: a critique in historical perspective of three concepts flawed fatally. Am J Dermatopathol. 2004;26(4):310-333.
PubMed   |  Link to Article
Bastian  BC, LeBoit  PE, Pinkel  D.  Mutations and copy number increase of HRAS in Spitz nevi with distinctive histopathological features. Am J Pathol. 2000;157(3):967-972.
PubMed   |  Link to Article
Fullen  DR, Poynter  JN, Lowe  L,  et al.  BRAF and NRAS mutations in spitzoid melanocytic lesions. Mod Pathol. 2006;19(10):1324-1332.
PubMed   |  Link to Article
Wiesner  T, Murali  R, Fried  I,  et al.  A distinct subset of atypical Spitz tumors is characterized by BRAF mutation and loss of BAP1 expression. Am J Surg Pathol. 2012;36(6):818-830.
PubMed   |  Link to Article
Bastian  BC, Wesselmann  U, Pinkel  D, Leboit  PE.  Molecular cytogenetic analysis of Spitz nevi shows clear differences to melanoma. J Invest Dermatol. 1999;113(6):1065-1069.
PubMed   |  Link to Article
van Dijk  MC, Bernsen  MR, Ruiter  DJ.  Analysis of mutations in B-RAF, N-RAS, and H-RAS genes in the differential diagnosis of Spitz nevus and spitzoid melanoma. Am J Surg Pathol. 2005;29(9):1145-1151.
PubMed   |  Link to Article
Harris  K, Florell  SR, Papenfuss  J,  et al.  Melanoma mimic: a case of multiple pagetoid Spitz nevi. Arch Dermatol. 2012;148(3):370-374.
PubMed   |  Link to Article
Gantner  S, Wiesner  T, Cerroni  L,  et al.  Absence of BRAF and HRAS mutations in eruptive Spitz naevi. Br J Dermatol. 2011;164(4):873-877.
PubMed   |  Link to Article
Boone  SL, Busam  KJ, Marghoob  AA,  et al.  Two cases of multiple Spitz nevi: correlating clinical, histologic, and fluorescence in situ hybridization findings. Arch Dermatol. 2011;147(2):227-231.
PubMed   |  Link to Article
Aloi  F, Tomasini  C, Pippione  M.  Agminated Spitz nevi occurring within a congenital speckled lentiginous nevus. Am J Dermatopathol. 1995;17(6):594-598.
PubMed   |  Link to Article
Berk  DR, Lane  AT.  Acquired bilateral agminated Spitz nevi in a child with Langerhans cell histiocytosis. Pediatr Dermatol. 2010;27(3):282-284.
PubMed   |  Link to Article
Deng  X.  SeqGene: a comprehensive software solution for mining exome- and transcriptome-sequencing data. BMC Bioinformatics. 2011;12:267. doi:10.1186/1471-2105-12-267.
PubMed   |  Link to Article
Groesser  L, Herschberger  E, Sagrera  A,  et al.  Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell [published online January 21, 2013]. J Invest Dermatol. doi:10.1038/jid.2013.24.
PubMed
Hafner  C, Groesser  L.  Mosaic RASopathies. Cell Cycle. 2013;12(1):43-50.
PubMed   |  Link to Article

Correspondence

CME
Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Supplement.

eMethods.

eTable. List of Common Non-synonymous Single-Nucleotide Variants in Spitz1 and Spitz2 Called by DNAnexus Prior to Our Filters

eFigure 1. HRAS Point Mutation (c.37G?C) Disrupts the Aci1 Digestion Site

eFigure 2. Enrichment of HRAS Point Mutation (c.37G?C) After Aci1 Digestion

Supplemental Content

Some tools below are only available to our subscribers or users with an online account.

Web of Science® Times Cited: 1

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections