KY Sarin et al. HRAS Mutation in Nevus Spilus
V600E-driven melanoma by upregulating VEGF-A. Cancer Discov 2:344–55 Miller AJ, Mihm MC Jr (2006) Melanoma. N Engl J Med 355:51–65 Pryor JG, Brown-Kipphut BA, Iqbal A et al. (2011) Microarray comparative genomic hybridization detection of copy number changes in desmoplastic melanoma and malignant peripheral nerve sheath tumor. Am J Dermatopathol 33:780–5 Remmele W, Stegner HE (1987) Recommendation for uniform definition of an
immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue. Pathologe 8:138–40
Yuan P, Ito K, Perez-Lorenzo R et al. (2013) Phenformin enhances the therapeutic benefit of BRAFV600E inhibition in melanoma. Proc Natl Acad Sci USA 110:18226–31
Shen CH, Yuan P, Perez-Lorenzo R et al. (2013) Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation. Mol Cell 52:161–72
Zheng B, Jeong JH, Asara JM et al. (2009) Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation. Mol Cell 33:237–47
William WN, Kim JS, Liu DD et al. (2012) The impact of phosphorylated AMP-activated protein kinase expression on lung cancer survival. Ann Oncol 23:78–85
Zheng L, Yang W, Wu F et al. (2013) Prognostic significance of AMPK activation and therapeutic effects of metformin in hepatocellular carcinoma. Clin Cancer Res 19:5372–80
Activating HRAS Mutation in Nevus Spilus Journal of Investigative Dermatology (2014) 134, 1766–1768; doi:10.1038/jid.2014.6; published online 30 January 2014
TO THE EDITOR Nevus spilus, also known as speckled lentiginous nevus, is a congenital hyperpigmented patch, which progressively evolves, developing dark macules and papules during childhood and adolescence (Vidaurri-de la Cruz and Happle, 2006) (Figure 1a, b). Over time, nevus spilus has been reported to give rise to common lentigines, melanocytic nevi, Spitz nevi, and melanoma (Bolognia, 1991; Aloi et al., 1995; Betti et al., 1997; Vente et al., 2004). These congenital lesions are postulated to result from a postzygotic genetic alteration of the melanocytic lineage, which gives rise to a clone of melanocytes predisposed to developing neoplasms (Happle, 1993). Using exome sequencing, we recently identified an activating HRAS point mutation (c.37G-4C, p.Gly13Arg) in a nevus spilus giving rise to agminated Spitz nevi that was absent from adjacent normal skin (Sarin et al., 2013). However, it remained unclear whether this mutation is unique to this atypical case or represented a common mutation in nevi spili. In this report, we identify the presence of the HRAS point mutation (c.37G-4C, p.Gly13Arg) in eight additional sporadic nevi spili, thus implicating the HRAS mutation (c.37G-
4C, p.Gly13Arg) as the predominant causative mutation for nevi spili. Genomic DNA was isolated from paraffin tissue biopsies of eight samples that were clinically diagnosed as nevi spili by board-certified dermatologists and confirmed as being consistent with nevi spili by board-certified dermatopathologists. These tissue biopsies were obtained from the lentiginous areas as well as the nevi within the lesion. Phenotypic and histopathologic data on each nevus spilus sample are detailed in Supplementary Table S1 online. Initial attempts at Sanger sequencing of whole paraffin sections for the presence of the HRAS point mutation (c.37G-4C, p.Gly13Arg) were negative. We hypothesized that the causative mutation may be undetectable by traditional Sanger sequencing as nevi spili melanocytes comprise a minute fraction of the total cells in the section. One method for enrichment of this small population is through laser capture microdissection. However, this technique is time and labor intensive and thus not easily adaptable into a screening assay. To enrich for a potential HRAS mutation, we used a rapid screening assay exploiting the fact that the HRAS point mutation (c.37G-4C, p.Gly13Arg) obliterates an Aci1 enzymatic digestion site (Supple-
Abbreviation: HRAS, v-Ha-ras Harvey rat sarcoma viral oncogene homolog Accepted article preview online 3 January 2014; published online 30 January 2014
1766 Journal of Investigative Dermatology (2014), Volume 134
mentary Figures S1, S2 and Supplementary Methods online). Briefly, DNA isolated from whole paraffin sections of nevi spili was amplified with HRAS primers 50 -CCTATCCTGGCTGTGTCCT G-30 and 50 -CAGGAGACCCTGTAGGAG GA-30 spanning exon 2. The HRAS exon 2 amplicon was then subjected to digestion for 16 hours at 37 degrees with Aci1, which preferentially digests wildtype HRAS sequences in the sample, thereby enriching for DNA sequences containing the HRAS mutation (c.37G4C, p.Gly13Arg). Following digestion, the DNA was subjected to a second round of amplification with nested HRAS primers 50 -CTCACCTCTATAGTGGGGT CGT-30 and 50 -TGAGGAGCGATGACG GAATA-30 spanning the digestion site. Sanger sequencing using the internal primers in both directions now detected this point mutation in all eight samples but not in normal control skin (Figure 2a and b). The mutation was also confirmed using an independent set of HRAS primers 50 -ATGACGGAATATAAGCTG-30 and 50 -CTCTATAGTGGGGTCGTA-30 for the second round amplification and Sanger sequencing. Although the digestion using Aci1 was incomplete as evidenced by the persistent detection of the wild-type allele, it was sufficient to enrich the proportion of DNA containing the HRAS mutation to detectable levels. Recently, activating HRAS mutations were identified in the keratinocytes and
KY Sarin et al. HRAS Mutation in Nevus Spilus
melanocytes of patients with phakomatosis pigmentokeratotica, suggesting a postzygotic mutation in an early common progenitor cell. We hypothesized that sporadic nevus spilus is likely to occur from a postzygotic mutation affecting specifically the melanocytic lineage. To assess this, we performed laser capture microdissection to isolate
DNA from the lentiginous melanocytes and junctional melanocytic nests, the superficial epidermis, and the surrounding dermis on two nevus spilus samples. In both cases, the HRAS mutation (c.37G-4C, p.Gly13Arg) was specifically detected in the melanocyte isolate but not in the keratinocytes or dermal fibroblasts (Figure 2c, Supplementary
Figure 1. Clinical and histopathological features of a nevus spilus. (a) Photograph of a tan patch on the left abdomen speckled with 1–2 mm hyperpigmented macules. (b) Hematoxylin and eosin staining of a nevus spilus biopsy showing scattered melanocytes and melanocytic nests along the dermo–epidermal junction. Bar ¼ 50 mm.
G C G G C C G T G T Nevus spilus G C G G C C G TGT
Normal skin
Fibroblasts
Lesion HRAS Frequency Spilus c.37G->C, p.Gly13Arg 8/8 (100%) Normal skin wt 2/2 (100%)
pERK
Melanocytes Keratinocytes
MITF
Figure 2. Activating HRAS mutation (c.37G-4C, p.Gly13Arg) in nevus spilus. (a) Sanger sequencing of Aci1-digested DNA from nevus spilus and control normal skin demonstrating the HRAS mutation in the nevus spilus but not in normal skin. (b) Table of HRAS mutations showing that the HRAS mutation (c.37G-4C, p.Gly13Arg) is present in all eight nevi spili but is undetectable in the control normal skin. wt, wild type. (c) Sanger sequencing from microdissection of two nevi spili samples demonstrates the HRAS mutation (c.37G-4C, p.Gly13Arg) in the melanocytic isolate and not in the keratinocyte isolate or dermal fibroblasts. Bar ¼ 50 mm. (d) Serial immunohistochemical staining for phosphorylated ERK (pERK), a downstream effector of the RAS/MAPK pathway (left panel), and microphthalmia-associated transcription factor, a marker of melanocytes (right panel), reveals selective activation of the RAS/MAPK pathway in the melanocytes (arrow), but not in the surrounding keratinocytes or dermal fibroblasts. Bar ¼ 50 mm.
Figure S3 online). These data suggest that sporadic nevi spili result from a postzygotic mutation in the melanocytic lineage. Of note, the percent of DNA containing the HRAS mutation (c.37G-4C, p.Gly13Arg) was less than the fifty percent that was expected of a heterozygous activating mutation. One possible explanation for this finding is the neighboring keratinocyte contamination in the laser capture microdissection of the melanocytes. RAS promotes cell growth predominantly through activation of the mitogen-activated protein kinase (MAPK) signal-transduction pathway. To evaluate downstream MAPK signaling, we performed phosphorylated ERK (pERK) immunostaining on a set of five nevi spili with confirmed HRAS mutations. Serial immunohistochemistry for pERK and microphthalmia-associated transcription factor (MITF), a marker of melanocytes, revealed acquired pERK staining in the melanocytes not detected in the neighboring keratinocytes or stromal fibroblasts, indicating a selective activation of the MAPK pathway in the melanocytic lineage (Figure 2d). The secondary antibody–only control was negative (Supplementary Figure S4 online). Our results implicate the activating HRAS mutation (c.37G-4C, p.Gly13Arg) as the predominant mutation in nevus spilus and demonstrate that this mutation is sufficient to cause activation of the MAPK signaling pathway. Nevi spili can manifest heterogeneously with macular and/or papular lesions and can be seen in different settings, with macular lesions seen in phacomatosis spilorosea and papular lesions seen in phacomatosis pigmentokeratotica (Happle, 2010). Our study is unable to fully assess whether HRAS mutations are represented differentially in these two phenotypes due to limited clinical phenotypic detail and sample size. Furthermore, it is likely that other mutations may also underlie nevi spili as, in addition to the HRAS mutation p.Gly13Arg, the activating HRAS mutation p. Gln61Arg has been identified in two nevi spili in phacomatosis pigmentokeratotica patients (Groesser et al., 2013). Future studies are needed to investigate the mechanisms underlying www.jidonline.org 1767
ZA Qadeer et al. ATRX Loss in Melanoma
the phenotypic heterogeneity of nevi spili. HRAS mutations have recently been demonstrated in a number of hamartomatous cutaneous neoplasms including epidermal nevi and nevus sebaceous (Groesser et al., 2012; Hafner et al., 2012; Levinsohn et al., 2012; Sun et al., 2013). When these alterations occur early in development or affect multiple cell lineages, they can result in developmental syndromes called ‘RASopathies,’ such as Schimmelpenning, epidermal nevus syndrome, and phacomatosis pigmentokeratotica (Hafner and Groesser, 2013). The HRAS point mutation (c.37G-4C, p.Gly13Arg), in particular, appears to be overrepresented among HRAS mutations in these cutaneous congenital conditions. This mutation allows constitutive activation of RAS pathway likely facilitating the development of secondary neoplasms through accrual of additional genetic alterations. Our finding adds sporadic nevus spilus to the spectrum of congenital cutaneous lesions that harbor activating mutations in HRAS. In nevus spilus, we speculate that the melanocytic neoplasms that arise from these tan patches likely acquire additional genetic alterations that enable progression. Indeed, we have recently demonstrated such a secondary change through amplification of HRAS in Spitz nevi arising out of a nevus spilus (Sarin et al., 2013). However, the secondary mutations that give rise to the diverse spectrum of melano-
cytic neoplasms have not yet been identified. The knowledge of the genetic basis of nevus spilus represents a further step towards understanding the genetic etiology underlying melanocytic neoplasms. In addition, these lesions represent a unique opportunity to study the genetic alterations that allow progression from a nevus spilus to a nevus or melanoma in the setting of constitutive RAS/MAPK activation. CONFLICT OF INTEREST The authors state no conflict of interest.
Kavita Y. Sarin1, Jennifer M. McNiff2, Shirley Kwok3, Jinah Kim1,3 and Paul A. Khavari1,4 1
Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA; 2Department of Dermatology, Yale Medical School, New Haven, Connecticut, USA; 3Department of Pathology, Stanford University School of Medicine, Stanford, California, USA and 4Dermatology Service, VA Palo Alto Health Care System, Palo Alto, California, USA E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Aloi F, Tomasini C, Pippione M (1995) Agminated Spitz nevi occurring within a congenital speckled lentiginous nevus. Am J Dermatopathol 17:594–8 Betti R, Inselvini E, Palvarini M et al. (1997) Agminated intradermal Spitz nevi arising on an unusual speckled lentiginous nevus with
localized lentiginosis: a continuum? Am J Dermatopathol 19:524–7 Bolognia JL (1991) Fatal melanoma arising in a zosteriform speckled lentiginous nevus. Arch Dermatol 127:1240–1 Groesser L, Herschberger E, Ruetten A et al. (2012) Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome. Nat Genet 44:783–7 Groesser L, Herschberger E, Sagrera A et al. (2013) Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell. J Invest Dermatol 133:1998–2003 Hafner C, Groesser L (2013) Mosaic RASopathies. Cell Cycle 12:43–50 Hafner C, Toll A, Gantner S et al. (2012) Keratinocytic epidermal nevi are associated with mosaic RAS mutations. J Med Genet 49:249–53 Happle R (1993) Mosaicism in human skin. Understanding the patterns and mechanisms. Arch Dermatol 129:1460–70 Happle R (2010) Speckled lentiginous nevi: no longer one single disorder. Arch Dermatol 146:204 Levinsohn JL, Tian LC, Boyden LM et al. (2012) Whole-exome sequencing reveals somatic mutations in HRAS and KRAS, which cause nevus sebaceus. J Invest Dermatol 133:827–30 Sarin KY, Sun BK, Bangs CD et al. (2013) Activating HRAS mutation in agminated spitz nevi arising in a nevus spilus. JAMA Dermatol 149:1077–81 Sun BK, Saggini A, Sarin KY et al. (2013) Mosaic activating RAS mutations in nevus sebaceus and nevus sebaceus syndrome. J Invest Dermatol 133:824–7 Vente C, Neumann C, Bertsch H et al. (2004) Speckled lentiginous nevus syndrome: report of a further case. Dermatology 209:228–9 Vidaurri-de la Cruz H, Happle R (2006) Two distinct types of speckled lentiginous nevi characterized by macular versus papular speckles. Dermatology 212:53–8
Decreased Expression of the Chromatin Remodeler ATRX Associates with Melanoma Progression Journal of Investigative Dermatology (2014) 134, 1768–1772; doi:10.1038/jid.2014.45; published online 27 February 2014
TO THE EDITOR ATRX is a member of the SWI/SNF family of chromatin remodelers, originally identified as mutated in patients with Alpha Thalassemia/Mental Retar-
dation, X-linked syndrome. The protein product contains several highly conserved domains, including an ADD (ATRX-DNMT3-DNMT3L) domain that binds methylated histone H3 at lysine 9
Abbreviations: ATRX, alpha thalassemia/mental retardation, X-linked; IHC, immunohistochemistry Accepted article preview online 27 January 2014; published online 27 February 2014
1768 Journal of Investigative Dermatology (2014), Volume 134
and an ATPase domain responsible for its remodeling activities (Ratnakumar and Bernstein, 2013). Recently, wholegenome sequencing studies identified ATRX mutations in multiple tumors, including those of neural crest cell origin: neuroblastoma, low-grade glioma, and glioblastoma (Heaphy et al., 2011a; Jiao et al., 2011; Cheung et al., 2012;
V600E-driven melanoma by upregulating VEGF-A. Cancer Discov 2:344–55 Miller AJ, Mihm MC Jr (2006) Melanoma. N Engl J Med 355:51–65 Pryor JG, Brown-Kipphut BA, Iqbal A et al. (2011) Microarray comparative genomic hybridization detection of copy number changes in desmoplastic melanoma and malignant peripheral nerve sheath tumor. Am J Dermatopathol 33:780–5 Remmele W, Stegner HE (1987) Recommendation for uniform definition of an
immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue. Pathologe 8:138–40
Yuan P, Ito K, Perez-Lorenzo R et al. (2013) Phenformin enhances the therapeutic benefit of BRAFV600E inhibition in melanoma. Proc Natl Acad Sci USA 110:18226–31
Shen CH, Yuan P, Perez-Lorenzo R et al. (2013) Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation. Mol Cell 52:161–72
Zheng B, Jeong JH, Asara JM et al. (2009) Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation. Mol Cell 33:237–47
William WN, Kim JS, Liu DD et al. (2012) The impact of phosphorylated AMP-activated protein kinase expression on lung cancer survival. Ann Oncol 23:78–85
Zheng L, Yang W, Wu F et al. (2013) Prognostic significance of AMPK activation and therapeutic effects of metformin in hepatocellular carcinoma. Clin Cancer Res 19:5372–80
Activating HRAS Mutation in Nevus Spilus Journal of Investigative Dermatology (2014) 134, 1766–1768; doi:10.1038/jid.2014.6; published online 30 January 2014
TO THE EDITOR Nevus spilus, also known as speckled lentiginous nevus, is a congenital hyperpigmented patch, which progressively evolves, developing dark macules and papules during childhood and adolescence (Vidaurri-de la Cruz and Happle, 2006) (Figure 1a, b). Over time, nevus spilus has been reported to give rise to common lentigines, melanocytic nevi, Spitz nevi, and melanoma (Bolognia, 1991; Aloi et al., 1995; Betti et al., 1997; Vente et al., 2004). These congenital lesions are postulated to result from a postzygotic genetic alteration of the melanocytic lineage, which gives rise to a clone of melanocytes predisposed to developing neoplasms (Happle, 1993). Using exome sequencing, we recently identified an activating HRAS point mutation (c.37G-4C, p.Gly13Arg) in a nevus spilus giving rise to agminated Spitz nevi that was absent from adjacent normal skin (Sarin et al., 2013). However, it remained unclear whether this mutation is unique to this atypical case or represented a common mutation in nevi spili. In this report, we identify the presence of the HRAS point mutation (c.37G-4C, p.Gly13Arg) in eight additional sporadic nevi spili, thus implicating the HRAS mutation (c.37G-
4C, p.Gly13Arg) as the predominant causative mutation for nevi spili. Genomic DNA was isolated from paraffin tissue biopsies of eight samples that were clinically diagnosed as nevi spili by board-certified dermatologists and confirmed as being consistent with nevi spili by board-certified dermatopathologists. These tissue biopsies were obtained from the lentiginous areas as well as the nevi within the lesion. Phenotypic and histopathologic data on each nevus spilus sample are detailed in Supplementary Table S1 online. Initial attempts at Sanger sequencing of whole paraffin sections for the presence of the HRAS point mutation (c.37G-4C, p.Gly13Arg) were negative. We hypothesized that the causative mutation may be undetectable by traditional Sanger sequencing as nevi spili melanocytes comprise a minute fraction of the total cells in the section. One method for enrichment of this small population is through laser capture microdissection. However, this technique is time and labor intensive and thus not easily adaptable into a screening assay. To enrich for a potential HRAS mutation, we used a rapid screening assay exploiting the fact that the HRAS point mutation (c.37G-4C, p.Gly13Arg) obliterates an Aci1 enzymatic digestion site (Supple-
Abbreviation: HRAS, v-Ha-ras Harvey rat sarcoma viral oncogene homolog Accepted article preview online 3 January 2014; published online 30 January 2014
1766 Journal of Investigative Dermatology (2014), Volume 134
mentary Figures S1, S2 and Supplementary Methods online). Briefly, DNA isolated from whole paraffin sections of nevi spili was amplified with HRAS primers 50 -CCTATCCTGGCTGTGTCCT G-30 and 50 -CAGGAGACCCTGTAGGAG GA-30 spanning exon 2. The HRAS exon 2 amplicon was then subjected to digestion for 16 hours at 37 degrees with Aci1, which preferentially digests wildtype HRAS sequences in the sample, thereby enriching for DNA sequences containing the HRAS mutation (c.37G4C, p.Gly13Arg). Following digestion, the DNA was subjected to a second round of amplification with nested HRAS primers 50 -CTCACCTCTATAGTGGGGT CGT-30 and 50 -TGAGGAGCGATGACG GAATA-30 spanning the digestion site. Sanger sequencing using the internal primers in both directions now detected this point mutation in all eight samples but not in normal control skin (Figure 2a and b). The mutation was also confirmed using an independent set of HRAS primers 50 -ATGACGGAATATAAGCTG-30 and 50 -CTCTATAGTGGGGTCGTA-30 for the second round amplification and Sanger sequencing. Although the digestion using Aci1 was incomplete as evidenced by the persistent detection of the wild-type allele, it was sufficient to enrich the proportion of DNA containing the HRAS mutation to detectable levels. Recently, activating HRAS mutations were identified in the keratinocytes and
KY Sarin et al. HRAS Mutation in Nevus Spilus
melanocytes of patients with phakomatosis pigmentokeratotica, suggesting a postzygotic mutation in an early common progenitor cell. We hypothesized that sporadic nevus spilus is likely to occur from a postzygotic mutation affecting specifically the melanocytic lineage. To assess this, we performed laser capture microdissection to isolate
DNA from the lentiginous melanocytes and junctional melanocytic nests, the superficial epidermis, and the surrounding dermis on two nevus spilus samples. In both cases, the HRAS mutation (c.37G-4C, p.Gly13Arg) was specifically detected in the melanocyte isolate but not in the keratinocytes or dermal fibroblasts (Figure 2c, Supplementary
Figure 1. Clinical and histopathological features of a nevus spilus. (a) Photograph of a tan patch on the left abdomen speckled with 1–2 mm hyperpigmented macules. (b) Hematoxylin and eosin staining of a nevus spilus biopsy showing scattered melanocytes and melanocytic nests along the dermo–epidermal junction. Bar ¼ 50 mm.
G C G G C C G T G T Nevus spilus G C G G C C G TGT
Normal skin
Fibroblasts
Lesion HRAS Frequency Spilus c.37G->C, p.Gly13Arg 8/8 (100%) Normal skin wt 2/2 (100%)
pERK
Melanocytes Keratinocytes
MITF
Figure 2. Activating HRAS mutation (c.37G-4C, p.Gly13Arg) in nevus spilus. (a) Sanger sequencing of Aci1-digested DNA from nevus spilus and control normal skin demonstrating the HRAS mutation in the nevus spilus but not in normal skin. (b) Table of HRAS mutations showing that the HRAS mutation (c.37G-4C, p.Gly13Arg) is present in all eight nevi spili but is undetectable in the control normal skin. wt, wild type. (c) Sanger sequencing from microdissection of two nevi spili samples demonstrates the HRAS mutation (c.37G-4C, p.Gly13Arg) in the melanocytic isolate and not in the keratinocyte isolate or dermal fibroblasts. Bar ¼ 50 mm. (d) Serial immunohistochemical staining for phosphorylated ERK (pERK), a downstream effector of the RAS/MAPK pathway (left panel), and microphthalmia-associated transcription factor, a marker of melanocytes (right panel), reveals selective activation of the RAS/MAPK pathway in the melanocytes (arrow), but not in the surrounding keratinocytes or dermal fibroblasts. Bar ¼ 50 mm.
Figure S3 online). These data suggest that sporadic nevi spili result from a postzygotic mutation in the melanocytic lineage. Of note, the percent of DNA containing the HRAS mutation (c.37G-4C, p.Gly13Arg) was less than the fifty percent that was expected of a heterozygous activating mutation. One possible explanation for this finding is the neighboring keratinocyte contamination in the laser capture microdissection of the melanocytes. RAS promotes cell growth predominantly through activation of the mitogen-activated protein kinase (MAPK) signal-transduction pathway. To evaluate downstream MAPK signaling, we performed phosphorylated ERK (pERK) immunostaining on a set of five nevi spili with confirmed HRAS mutations. Serial immunohistochemistry for pERK and microphthalmia-associated transcription factor (MITF), a marker of melanocytes, revealed acquired pERK staining in the melanocytes not detected in the neighboring keratinocytes or stromal fibroblasts, indicating a selective activation of the MAPK pathway in the melanocytic lineage (Figure 2d). The secondary antibody–only control was negative (Supplementary Figure S4 online). Our results implicate the activating HRAS mutation (c.37G-4C, p.Gly13Arg) as the predominant mutation in nevus spilus and demonstrate that this mutation is sufficient to cause activation of the MAPK signaling pathway. Nevi spili can manifest heterogeneously with macular and/or papular lesions and can be seen in different settings, with macular lesions seen in phacomatosis spilorosea and papular lesions seen in phacomatosis pigmentokeratotica (Happle, 2010). Our study is unable to fully assess whether HRAS mutations are represented differentially in these two phenotypes due to limited clinical phenotypic detail and sample size. Furthermore, it is likely that other mutations may also underlie nevi spili as, in addition to the HRAS mutation p.Gly13Arg, the activating HRAS mutation p. Gln61Arg has been identified in two nevi spili in phacomatosis pigmentokeratotica patients (Groesser et al., 2013). Future studies are needed to investigate the mechanisms underlying www.jidonline.org 1767
ZA Qadeer et al. ATRX Loss in Melanoma
the phenotypic heterogeneity of nevi spili. HRAS mutations have recently been demonstrated in a number of hamartomatous cutaneous neoplasms including epidermal nevi and nevus sebaceous (Groesser et al., 2012; Hafner et al., 2012; Levinsohn et al., 2012; Sun et al., 2013). When these alterations occur early in development or affect multiple cell lineages, they can result in developmental syndromes called ‘RASopathies,’ such as Schimmelpenning, epidermal nevus syndrome, and phacomatosis pigmentokeratotica (Hafner and Groesser, 2013). The HRAS point mutation (c.37G-4C, p.Gly13Arg), in particular, appears to be overrepresented among HRAS mutations in these cutaneous congenital conditions. This mutation allows constitutive activation of RAS pathway likely facilitating the development of secondary neoplasms through accrual of additional genetic alterations. Our finding adds sporadic nevus spilus to the spectrum of congenital cutaneous lesions that harbor activating mutations in HRAS. In nevus spilus, we speculate that the melanocytic neoplasms that arise from these tan patches likely acquire additional genetic alterations that enable progression. Indeed, we have recently demonstrated such a secondary change through amplification of HRAS in Spitz nevi arising out of a nevus spilus (Sarin et al., 2013). However, the secondary mutations that give rise to the diverse spectrum of melano-
cytic neoplasms have not yet been identified. The knowledge of the genetic basis of nevus spilus represents a further step towards understanding the genetic etiology underlying melanocytic neoplasms. In addition, these lesions represent a unique opportunity to study the genetic alterations that allow progression from a nevus spilus to a nevus or melanoma in the setting of constitutive RAS/MAPK activation. CONFLICT OF INTEREST The authors state no conflict of interest.
Kavita Y. Sarin1, Jennifer M. McNiff2, Shirley Kwok3, Jinah Kim1,3 and Paul A. Khavari1,4 1
Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA; 2Department of Dermatology, Yale Medical School, New Haven, Connecticut, USA; 3Department of Pathology, Stanford University School of Medicine, Stanford, California, USA and 4Dermatology Service, VA Palo Alto Health Care System, Palo Alto, California, USA E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Aloi F, Tomasini C, Pippione M (1995) Agminated Spitz nevi occurring within a congenital speckled lentiginous nevus. Am J Dermatopathol 17:594–8 Betti R, Inselvini E, Palvarini M et al. (1997) Agminated intradermal Spitz nevi arising on an unusual speckled lentiginous nevus with
localized lentiginosis: a continuum? Am J Dermatopathol 19:524–7 Bolognia JL (1991) Fatal melanoma arising in a zosteriform speckled lentiginous nevus. Arch Dermatol 127:1240–1 Groesser L, Herschberger E, Ruetten A et al. (2012) Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome. Nat Genet 44:783–7 Groesser L, Herschberger E, Sagrera A et al. (2013) Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell. J Invest Dermatol 133:1998–2003 Hafner C, Groesser L (2013) Mosaic RASopathies. Cell Cycle 12:43–50 Hafner C, Toll A, Gantner S et al. (2012) Keratinocytic epidermal nevi are associated with mosaic RAS mutations. J Med Genet 49:249–53 Happle R (1993) Mosaicism in human skin. Understanding the patterns and mechanisms. Arch Dermatol 129:1460–70 Happle R (2010) Speckled lentiginous nevi: no longer one single disorder. Arch Dermatol 146:204 Levinsohn JL, Tian LC, Boyden LM et al. (2012) Whole-exome sequencing reveals somatic mutations in HRAS and KRAS, which cause nevus sebaceus. J Invest Dermatol 133:827–30 Sarin KY, Sun BK, Bangs CD et al. (2013) Activating HRAS mutation in agminated spitz nevi arising in a nevus spilus. JAMA Dermatol 149:1077–81 Sun BK, Saggini A, Sarin KY et al. (2013) Mosaic activating RAS mutations in nevus sebaceus and nevus sebaceus syndrome. J Invest Dermatol 133:824–7 Vente C, Neumann C, Bertsch H et al. (2004) Speckled lentiginous nevus syndrome: report of a further case. Dermatology 209:228–9 Vidaurri-de la Cruz H, Happle R (2006) Two distinct types of speckled lentiginous nevi characterized by macular versus papular speckles. Dermatology 212:53–8
Decreased Expression of the Chromatin Remodeler ATRX Associates with Melanoma Progression Journal of Investigative Dermatology (2014) 134, 1768–1772; doi:10.1038/jid.2014.45; published online 27 February 2014
TO THE EDITOR ATRX is a member of the SWI/SNF family of chromatin remodelers, originally identified as mutated in patients with Alpha Thalassemia/Mental Retar-
dation, X-linked syndrome. The protein product contains several highly conserved domains, including an ADD (ATRX-DNMT3-DNMT3L) domain that binds methylated histone H3 at lysine 9
Abbreviations: ATRX, alpha thalassemia/mental retardation, X-linked; IHC, immunohistochemistry Accepted article preview online 27 January 2014; published online 27 February 2014
1768 Journal of Investigative Dermatology (2014), Volume 134
and an ATPase domain responsible for its remodeling activities (Ratnakumar and Bernstein, 2013). Recently, wholegenome sequencing studies identified ATRX mutations in multiple tumors, including those of neural crest cell origin: neuroblastoma, low-grade glioma, and glioblastoma (Heaphy et al., 2011a; Jiao et al., 2011; Cheung et al., 2012;
