Jonathan L. Levinsohn

Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy.

Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2). TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs.

Whole-Exome Sequencing Reveals Somatic Mutations in HRAS and KRAS, which Cause Nevus Sebaceus

ACKNOWLEDGMENTS We thank the patients and their families for taking part in this project. We also thank S Aasi, R Khosla, and P Lorenz for their valuable assistance. Bryan K. Sun, Andrea Saggini, Kavita Y. Sarin, Jinah Kim, Latanya Benjamin, Philip E. LeBoit and Paul A. Khavari Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA; Department of Dermatology, University of California, San Francisco, San Francisco, California, USA and Department of Dermatology, University of Rome Tor Vergata, Rome, Italy E-mail: bryansun@stanford.edu

Somatic HRAS p.G12S mutation causes woolly hair and epidermal nevi.

TO THE EDITOR Woolly hair nevus (WHN) is a mosaic disorder characterized by distinct patterns of tightly curled scalp hair which can appear concurrently with epidermal nevi (EN) at other sites (Peteiro et al., 1989; Venugopal et al., 2012). Woolly hair is also found in congenital disorders resulting from mutations affecting diverse cellular components including intermediate filament, adherens junction, and signal transduction proteins (Harel and Christiano, 2012). Embryonic somatic mutation causes mosaic disorders which appear in patterns of ectodermal progenitor dorsovental migration. Somatic mutations causing mosaic disorders including Proteus syndrome (Lindhurst et al., 2011), port-wine stains (Shirley et al., 2013), and EN (Levinsohn et al., 2013; Sun et al., 2013) have been found using exome sequencing. Recognizing that exome sequencing would permit identification of mutations causing WHN, we ascertained two cases. Our first (WHN100, Figure 1a-d) was a 10 year-old girl without history of developmental delay who had regions of slightly curly hair over her occipital scalp from infancy which progressively curled with no scalp surface change and lie alongside areas of straight hair. She has hyperpigmented patches on her neck, trunk, and arms, with more keratotic lesions on her distal extremities, and acanthosis nigricans in both axillae. There was linear palmar keratoderma (PPK) and hyperkeratosis over most metacarpophalangeal and some proximal interphalangeal joints. Given concurrent PPK and woolly hair, clinical concern for Naxos or Carvajal syndromes led to regular cardiology evaluations that found no abnormalities. Figure 1 Clinical features of index cases with woolly hair nevi. On the scalp, woolly hair nevus presents with a portion of the scalp exhibiting patches of curly, thin, hair intermixed with regions of normal, straight hair, as observed in WHN100 and WHN101. On ... Our second case (WHN101, Figure 1e-h) was a 6 year-old girl whose hair developed at age one and consisted of a mixture of poker-straight hair and curly, thin hair. In infancy, she developed linear dyspigmentation on the right arm and trunk, which became more raised and scaly on the distal extremities over time. She had normal development, with no cardiac or ophthalmic abnormalities found on routine physical examination, cardiac MRI and serial electrocardiograms. Clinical suspicion of mosaic Naxos or Caravajal syndrome motivated clinical sequencing of DSP, DSC1, DSG1, JUP, PKP2, and TMEM43; no mutations were found. To determine the genetic basis of WHN, we performed paired whole exome sequencing of DNA isolated from affected tissue and blood in both cases (Supplementary Figure 2). Data was analyzed to identify somatic single nucleotide variants (SNVs), deletions and insertions (Supplementary Methods). A somatic heterozygous HRAS c.34G>A, p.G12S substitution was found in each (Figure 2a). There was no evidence of loss of heterozygosity (LOH) (Supplementary Figure 3) or secondary mutation somatic mutation, suggesting that HRAS mutation alone is sufficient to cause WHN. Sanger sequencing confirmed mutation presence in affected tissue (Figure 2b, c). To determine if this mutation causes woolly hair, we prepared DNA from hair bulbs of straight and curly hair obtained from affected individual WHN101, finding the HRAS p.G12S mutation in curly hair only (Figure 2d, Supplementary Figure 1). Figure 2 Somatic HRAS p.G12S mutation causes WHN. (a) In WHN100 and WHN101, exome sequencing of affected tissue and blood was performed. Tissue-specific SNVs are annotated bychromosome, position, base change, protein consequence, and numbers of reference and non-reference ... Consistent with somatic mosaicism in an epidermal progenitor, prior cases of WHN have been reported with concurrent keratinocytic epidermal nevi (KEN). KEN result from somatic mutations in HRAS, KRAS, PIK3CA, FGFR3, and NRAS (Hafner et al., 2012) including the HRAS p.G12S mutation found in WHN (Hafner et al., 2011). Furthermore, Costello syndrome (CS), in which patients present with developmental delay, high birth weight, feeding difficulties, failure to thrive, cardiac anomalies, and curly hair, results from germline heterozygous HRAS mutations, including p.G12S (Gripp and Lin, 2012; Siegel et al., 2012). The timing of somatic mutation during embryonic development determines extent of cutaneous involvement and presence of other systemic abnormalities (Moss et al., 1993). Notably, somatic activating HRAS mutations are found in most cases of nevus sebaceus (NS), a mosaic lesion which typically appears on the scalp and features alopecia, papillomatosis, and marked sebaceus hyperplasia (Groesser et al., 2012; Levinsohn et al., 2013; Sun et al., 2013). These features contrast with those of WHN in which hair is present but curly, and sebaceous hyperplasia is absent. Given that WHN and NS are both caused by somatic HRAS mutations, we hypothesize that their phenotypic divergence may derive from relative potency of the mutant allele with respect to MAP kinase activation. HRAS mutations in WHN and NS fall within the finger loop of HRAS, replacing glycine residues with larger amino acids which prevent GTP hydrolysis (Malumbres and Barbacid, 2003). Though comparison of the WHN p.G12S mutation and the common NS p.G13R mutation has not been performed, HRAS codon 12 serine substitutions have been shown to be less activating than arginine, aspartic acid or valine substitutions (Fasano et al., 1984). To evaluate the frequency of HRAS mutation in NS, we screened 116 archival scalp NS lesions for HRAS and KRAS mutation. We found 88 HRAS and 9 KRAS mutations. HRAS p.G13R was present in 85 NS and p.G12S was not found (Supplementary Table 2). In prior reports, 64 additional samples were screened, and HRAS p.G12S mutations were not found (Levinsohn et al., 2013; Sun et al., 2013). In one report, 3 specimens with HRAS p.G12S mutations were identified; in 2 there was a concurrent HRAS p.G13R mutation, and in one, the lesion was on the ear, a site at which it could be difficult to distinguish EN and NS (Groesser et al., 2012). These data combined with evidence from CS suggest that more strongly activating RAS mutations may cause the alopecia and sebaceous hyperplasia found in NS, and the more mildly activating p.G12S mutation causes woolly hair phenotypes. In summary, we find somatic HRAS c.34G>A, p.G12S mutation in affected tissue from two cases with mosaic woolly hair and EN. Consistent with reports of WHN and in KEN, the identified p.G12S mutation causes an EN phenotype on the body, but the finding of curly hair on the scalp suggests that WHN represents a mosaic RASopathy with phenotype determined by location, either due to distinct epidermal progenitor types or site-specific mesenchymal interactions. We hypothesize that in contrast to strongly activating RAS mutations found in NS which drive hair follicle progenitors toward sebocyte differentiation, the more weakly activating mutation found in WHN permits an intermediate phenotype with abnormal curly hair growth but without sebaceous hyperplasia.

Somatic V600E BRAF Mutation in Linear and Sporadic Syringocystadenoma Papilliferum

Jonathan L. Levinsohn(1),(2),(3), Jeffrey L. Sugarman(4), Kaya Bilguvar(1),(5), Jennifer M. McNiff(2),(3), The Yale Center for Mendelian Genomics, and Keith A. Choate(1),(2),(3) (1)Department of Genetics, Yale University, New Haven, Connecticut (2)Department of Pathology, Yale University, New Haven, Connecticut (3)Department of Dermatology, Yale University, New Haven, Connecticut (4)Departments of Dermatology and Family Medicine, University of California, San Francisco, San Francisco, California

Identification of a gain-of-function STAT3 mutation (p.Y640F) in lymphocytic variant hypereosinophilic syndrome

To the editor: Hypereosinophilic syndrome (HES) is a heterogeneous group of disorders characterized by (1) persistent peripheral eosinophilia, (2) target organ pathology mediated by infiltrating eosinophils, and (3) the absence of known infectious or allergic causes of hypereosinophilia.[1][1],[2][

Somatic Mutations in NEK9 Cause Nevus Comedonicus

Acne vulgaris (AV) affects most adolescents, and of those affected, moderate to severe disease occurs in 20%. Comedones, follicular plugs consisting of desquamated keratinocytes and sebum, are central to its pathogenesis. Despite high heritability in first-degree relatives, AV genetic determinants remain incompletely understood. We therefore employed whole-exome sequencing (WES) in nevus comedonicus (NC), a rare disorder that features comedones and inflammatory acne cysts in localized, linear configurations. WES identified somatic NEK9 mutations, each affecting highly conserved residues within its kinase or RCC1 domains, in affected tissue of three out of three NC-affected subjects. All mutations are gain of function, resulting in increased phosphorylation at Thr210, a hallmark of NEK9 kinase activation. We found that comedo formation in NC is marked by loss of follicular differentiation markers, expansion of keratin-15-positive cells from localization within the bulge to the entire sub-bulge follicle and cyst, and ectopic expression of keratin 10, a marker of interfollicular differentiation not present in normal follicles. These findings suggest that NEK9 mutations in NC disrupt normal follicular differentiation and identify NEK9 as a potential regulator of follicular homeostasis.

A Somatic p.G45E GJB2 Mutation Causing Porokeratotic Eccrine Ostial and Dermal Duct Nevus

IMPORTANCE Recent data demonstrated somatic mutations in GJB2 that were present in affected porokeratotic eccrine ostial and dermal duct nevus (PEODDN) tissue but absent in unaffected skin. Recognizing that PEODDN lesions can also appear in individuals with keratitis-ichthyosis-deafness syndrome and finding somatic mutations in their cohort, the authors concluded that somatic GJB2 mutation may cause PEODDN. By using whole-exome sequencing, we show that somatic GJB2 mutation alone is sufficient to cause PEODDN. OBSERVATIONS We performed whole-exome sequencing of paired blood and affected tissue samples isolated from a PEODDN lesion of a primary school-aged female patient with bands of hyperkeratotic-affected skin on the upper and lower extremities and trunk, and identified a single, protein-damaging p.Gly45Glu GJB2 mutation present in tissue samples but not in blood samples. CONCLUSION AND RELEVANCE Our results prove that somatic GJB2 mutation is sufficient to cause PEODDN. Dominantly inherited GJB2 mutations, including the p.Gly45Glu found in our case, have been shown to cause the severe multisystem disorder keratitis-ichthyosis-deafness syndrome. GJB2 encodes connexin 26, a gap junction protein, which permits intercellular ion and macromolecule flux. Individuals with somatic mosaicism are at risk for transmitting systemic disease to their offspring, and all individuals with PEODDN lesions should be counseled regarding the risk of having a child with keratitis-ichthyosis-deafness syndrome.