Differentiation between malignant and benign follicular adnexal tumours of the skin by DNA image cytometry

Summary Background We have demonstrated in previous studies that DNA image cytometry (DNA ICM) can be helpful in detecting malignancy in sebaceous tumours of the Muir–Torre syndrome and sweat gland tumours. However, little is known about DNA ICM in cutaneous adnexal tumours with follicular differentiation. Objectives To study a larger series of benign and malignant follicular adnexal tumours with DNA ICM. Methods We studied 13 malignant follicular tumours (seven trichilemmal carcinomas, five malignant proliferating pilar tumours, one pilomatrix carcinoma) and 55 benign follicular tumours (four tumours of the follicular infundibulum, seven Winer's pores, eight trichilemmomas, two trichofolliculomas, 16 trichoepitheliomas, 13 pilomatrixomas, five trichoblastomas) by DNA ICM. All cases were clear‐cut as malignant or benign, respectively, on histopathological criteria. The stemline interpretation according to Böcking et al. (DNA distribution in gastric cancer and dysplasia. In: Precancerous Conditions and Lesions of the Stomach, Zhang YC, Kawai K, eds. Berlin: Springer‐Verlag, 1993: 103–20) was performed in all cases. In addition, 5[c]‐exceeding events (5cEE) and the 2[c] deviation index (2cDI) were calculated, except in one histopathologically benign tumour, which revealed euploid polyploidization, as the analysis of 5cEE and 2cDI is not valid in that case. Results A 2cDI threshold of 0·24 proved to be the most reliable marker for the distinction between malignant and benign follicular tumours. On the basis of this feature, all malignant and benign tumours were correctly classified. A specificity of 100% was achieved by all three interpretation methods, but the sensitivity of 2cDI for the detection of malignant tumours was superior to the analysis of 5cEE (sensitivity 77%) and to the stemline interpretation (sensitivity 23%). Conclusions DNA ICM may be helpful in distinguishing between malignant and benign follicular tumours.     

Phacomatosis Pigmentokeratotica: A Mosaic RASopathy with Malignant Potential

Phacomatosis pigmentokeratotica (PPK) is a rare epidermal nevus syndrome characterized by the co‐occurrence of a nevus sebaceous arranged along the lines of Blaschko with a speckled lentiginous nevus (SLN). We report a novel KRAS mutation in a patient with a large nevus sebaceous and an SLN who subsequently developed a vaginal botryoid rhabdomyosarcoma, an association not previously reported in the literature. This case expands our knowledge of the genetic basis for phacomatosis, in which mutations in HRAS have been previously described, although this report provides evidence that activating mutations in KRAS or HRAS may cause PPK. This report confirms that PPK is a mosaic RASopathy with malignant potential and raises the question of whether screening for other RAS‐associated malignancies should be performed for all children with PPK.     

CDKN2A mutations with p14 loss predisposing to multiple nerve sheath tumours,melanoma, dysplastic naevi and internal malignancies: a case series and review of the literature

An inherited germline mutation in CDKN2A is the most common cause of familial atypical multiple mole melanoma (FAMMM) syndrome. Although it is well known that CDKN2A mutations confer an increased risk for melanoma and pancreatic carcinoma, the association with an increased risk for nerve sheath tumours and other tumour types is under‐recognized. We report a family with a missense mutation (c.151–1G>C) at the acceptor splice site of intron 1 of CDKN2A, resulting in loss of function of both tumour suppressor proteins p16^INK4 and p14^ARF. This mutation is associated with a clinical phenotype of FAMMM syndrome in which patients develop numerous benign and malignant mutations, brain tumours, sarcomas and other solid tumours, in addition to melanoma and dysplastic naevi. Our proband initially presented with multiple nerve sheath tumours, leading to diagnostic confusion with Neurofibromatosis type 1. Loss of p14 expression results in increased MDM2‐mediated degradation of the tumour suppressor protein p53, and predisposes mutation carriers to multiple benign and malignant neoplasms. This article highlights the importance of considering CDKN2A mutations in patients with dysplastic naevi, melanoma and multiple nerve sheath tumours, specifically those with histological features of both neurofibromas and schwannomas. We also present a discussion of medical management for patients with this high‐risk cancer susceptibility syndrome.     

Assessment of clinical parameters associated with mutational status in metastatic malignant melanoma: a single‐centre investigation of 141 patients

Background Inhibitors of the mutated, constitutively activated BRAF protein have shown efficacy in the treatment of metastatic melanoma in clinical trials. Mutation analysis especially of the BRAF, NRAS and KIT genes is essential to identify patients suitable for targeted therapies and has been introduced into routine patient care. Objectives To correlate mutational status with clinical parameters including age, skin type, number of melanocytic naevi, primary tumour location, chronic sun damage and exposure to ultraviolet (UV) irradiation. The overall aim was to define subgroups with an increased or decreased likelihood of gene mutations. Additionally, the impact of activating BRAF mutations on clinical course was investigated. Methods In a single‐centre, retrospective approach, mutation analysis was performed on patients with metastatic malignant melanoma. Clinical parameters were correlated with molecular findings. The total sun‐burden score was assessed using a validated standardized questionnaire. Results The analysis included 141 patients with metastatic melanoma. Forty‐four per cent of patients had activating BRAF mutations and were significantly younger than patients with wild‐type BRAF or with NRAS mutations. KIT mutations were detected in only 3% of the patients. BRAF‐mutated melanomas developed preferentially in intermittently sun‐exposed areas of the body, and patients had significantly more melanocytic naevi. Once patients had progressed into stage IV disease, survival times were identical for those with BRAF‐mutated and BRAF wild‐type tumours. Conclusions Mutations of the BRAF gene are correlated with younger age, a higher number of melanocytic naevi and a tumour location in intermittently UV‐exposed skin. Signs of chronic photodamage are not indicative of mutational status. Patients with metastatic melanoma with BRAF mutations showed a nonsignificant tendency to progress later to stage IV disease, but once metastases were present the prognosis was identical to that with BRAF wild‐type tumours.     

Brooke–Spiegler syndrome: focus on reflectance confocal microscopy findings of trichoepithelioma and flat cylindroma

Brooke–Spiegler syndrome (BSS) is a rare, autosomal dominant disorder characterized by multiple adnexal tumours, especially trichoepitheliomas, cylindromas and occasionally spiradenomas. These lesions usually begin to appear in the second or third decade of life. Malignant transformation of pre‐existing tumours may occur. In vivo reflectance‐mode confocal microscopy (RCM) is a noninvasive method that can be used to visualize the epidermis and the upper dermis at almost histological resolution. It has been used to evaluate several skin conditions, especially malignant lesions, and has been reported to be useful for differentiating between malignant and benign skin tumours. Only a few studies have reported on the use of confocal microscopy to characterize the features of benign adnexal neoplasms. We present a patient with BSS who presented to our clinic with multiple skin tumours. The possible utility of RCM for identifying adnexal neoplasms is emphasized in this report, which also describes the observed microscopic features.     

FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis

Background Benign lichenoid keratoses (BLKs) are solitary skin lesions which have been proposed to represent a regressive form of pre‐existent epidermal tumours such as solar lentigo or seborrhoeic keratosis. However, the genetic basis of BLK is unknown. Objectives FGFR3, PIK3CA and RAS mutations have been shown to be involved in the pathogenesis of seborrhoeic keratosis and solar lentigo. We thus investigated whether these mutations are also present in BLK. Methods After manual microdissection and DNA isolation, 52 BLKs were screened for FGFR3, PIK3CA and RAS hotspot mutations using SNaPshot^® multiplex assays. Results We identified 6/52 (12%) FGFR3 mutations, 10/52 (19%) PIK3CA mutations, 6/52 (12%) HRAS mutations and 2/52 (4%) KRAS mutations. FGFR3 and RAS mutations were mutually exclusive. One BLK showed a simultaneous PIK3CA and HRAS mutation. In nine BLKs with a mutation, nonlesional control tissue from the epidermal margin and the dermal lymphocytic infiltrate were wild‐type, indicating that these mutations are somatic. To demonstrate that these findings are specific, 10 samples of lichen planus were analysed without evidence for FGFR3, PIK3CA or RAS mutations. Conclusions Our results indicate that FGFR3, PIK3CA and RAS mutations are present in approximately 50% of BLKs. These findings support the concept on the molecular genetic level that at least a proportion of BLKs represents regressive variants resulting from former benign epidermal tumours such as seborrhoeic keratosis and solar lentigo.     

Widespread microsatellite instability in sebaceous tumours of patients with the Muir-Torre syndrome

Muir-Torre syndrome (MTS) is an autosomal dominant disorder characterized by the presence of at least one sebaceous gland tumour and a minimum of one visceral malignant tumour. Recently, microsatellite instability (MSI) has been detected in the tumours of patients with MTS and germline mutations of the hMSH2 and hMLH1 mismatch repair genes have been detected in some patients with this syndrome. To determine if the tumours of patients with MTS have widespread genomic instability and whether loss of heterozygosity (LOH) in the chromosomal regions containing hMSH2 and hMLH1 is detectable. MSI and LOH were examined at 10 dinucleotide repeats on chromosomes 2p, 3p, 5q, 9p, 17p and 18q. Data were obtained from six sebaceous gland tumours and two adenocarcinomas of the colon from three patients of two Muir-Torre families. MSI was detected at more than half of the loci tested in all sebaceous tumours examined. In addition, there was LOH at D2S119 in one sebaceoma and one sebaceous carcinoma from one patient. The colon carcinomas from two patients showed MSI at five of the 10 loci analysed. These results show that widespread MSI is a feature of tumours in patients with MTS. In addition, the finding of LOH at D2S119, a marker located in the vicinity of hMSH2, in sebaceous tumours of one patient indicates that this gene may have a pathogenetic role in this patient.     

Naevus sebaceus: a mosaic RASopathy

Epidermal naevi are common cutaneous mosaic disorders that occur in 0.1–0.3% of live births. They are subdivided into keratinocytic and organoid naevi, the latter including naevus sebaceus (NS). Typically, NS develops as a yellowish‐orange plaque on the scalp, and represents a hamartoma containing epidermal, sebaceous and apocrine elements. The histological features of NS sampled in childhood include hyperkeratosis, acanthosis, increased sebaceous lobules, and primitive hair follicles. During puberty, most lesions develop more prominent sebaceous and apocrine components. Subsequently, secondary tumours may occur in around 25% of NS; most lesions are benign (e.g. trichoblastomas, syringocystadenoma papilliferum or other basaloid proliferations), although malignant tumours arising within NS can occur (< 1%). Recently, somatic mosaicism has been shown, with activating Ras mutations in HRAS or KRAS in NS lesional keratinocytes (but not in adjacent nonlesional skin or dermal fibroblasts). These mutations lead to constitutive activation of the RAF–MEK–ERK and phosphoinositide 3‐kinase signalling pathways, and result in increased cellular proliferation. Similar but more extensive mosaicism underlies Schimmelpenning–Feuerstein–Mims syndrome. The most common mutation is c.37G>C (p.Gly13Arg) in HRAS, which is present in > 90% of NS. This mutation also seems to be present in NS cases that develop secondary tumours, although no additional mutations (second hit) or other genetic events have yet been identified. Treatment of NS often involves prophylactic surgical excision, but the recent identification of key epidermal signalling abnormalities underlying the cell proliferation means that future development of new medical treatments for NS that target the aberrant signalling pathways may also be feasible.     

Melanoma arising in a nevus of Ito: novel genetic mutations and a review of the literature on cutaneous malignant transformation of dermal melanocytosis

Dermal melanocytosis refers to a spectrum of benign melanocytic proliferations that includes Mongolian spot, nevus of Ota and nevus of Ito. These lesions most commonly occur in persons of Asian or African descent and are often present at birth or develop during childhood. Very rarely, dermal melanocytoses undergo malignant transformation. There have been only 13 reports in the literature of primary cutaneous melanoma arising in dermal melanocytoses. We report a case of a Chinese woman with melanoma arising in a congenital nevus of Ito. We performed targeted next‐generation sequencing of the tumor which revealed mutations of GNAQ and BAP1, suggesting that alterations in these two genes led to malignant transformation of the nevus of Ito. We also provide a summary of reports in the literature regarding primary cutaneous melanoma arising in the context of dermal melanocytosis.     

Early excision of congenital melanocytic nevi under tumescent anesthesia and skin expansion by intracutaneous double butterfly sutures

Background: Excision of large congenital melanocytic nevi is recommended because of the risk of malignant transformation and for aesthetic reasons. Our treatment concept includes 1) excision in one or more steps preferably before the age of one year, 2) skin expansion through high‐tension wound closure with intracutaneous double butterfly sutures and 3) performing surgery with tumescent or general anesthesia. Patients and Methods: A retrospective survey was conducted among all parents with children treated for congenital melanocytic nevi in 2004 or 2005 with a maximum age of 6.0 years. The parents were asked to evaluate the trauma associated with their child's surgery and the aesthetic result. Results: Fifty‐nine families were contacted, of whom 51 replied. Forty‐two parents assessed surgery‐associated trauma as light or very light. Thirty‐nine parents rated the aesthetic results as good to very good. Severe complications did not occur. Conclusion: The proposed surgical concept is a safe procedure with good aesthetic results and low surgery‐associated trauma for the child.     

BRAF, NRAS and HRAS mutations in spitzoid tumours and their possible pathogenetic significance

Background The relationships between so‐called spitzoid tumours have proven difficult to understand. Objectives To address three questions: does spitzoid tumour morphological similarity reflect molecular similarity? Does Spitz naevus progress into spitzoid melanoma? Are ambiguous spitzoid tumours genuine entities? Methods BRAF, NRAS and HRAS mutations were analysed using single‐strand conformational polymorphism analysis and sequencing. Results Both Spitz naevi and spitzoid melanoma had a lower combined BRAF and NRAS mutation frequency compared with common acquired naevi (P = 0·0001) and common forms of melanoma (P = 0·0072), respectively. To look for evidence of progression from Spitz naevi to spitzoid melanoma, HRAS was analysed in 21 spitzoid melanomas, with no mutations identified. The binomial probability of this was 0·03 based on an assumption of a 15% mutation frequency in Spitz naevi with unbiased progression. Under these assumptions, HRAS mutations must be rare/absent in spitzoid melanoma. Thus, Spitz naevi seem unlikely to progress into spitzoid melanoma, implying that ambiguous spitzoid tumours cannot be intermediate degrees of progression. In addition, the data suggest that HRAS mutation is a potential marker of benign behaviour, in support of which none of three HRAS mutant spitzoid cases metastasized. Conclusions First, the morphological similarity of spitzoid tumours reflects an underlying molecular similarity, namely a relative lack of dependence on BRAF/NRAS mutations. Second, Spitz naevi do not appear to progress into spitzoid melanoma, and consequently ambiguous spitzoid tumours are likely to be unclassifiable Spitz naevi or spitzoid melanoma rather than genuine entities. Third, HRAS mutation may be a marker of Spitz naevus, raising the possibility that other molecular markers for discriminating Spitz naevi from spitzoid melanoma can be discovered.     

Novel filaggrin mutation but no other loss-of-function variants found in Ethiopian patients with atopic dermatitis

Background Filaggrin is a key protein involved in maintaining skin barrier function and hydration. Mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris (IV) and are a major predisposing factor for atopic dermatitis (AD) in individuals of European and Asian descent. It has been proposed that FLG mutations are population specific and a difference in the spectra of mutations between different ancestral groups has been described. However, it is unknown whether FLG mutations in the African population are a causative genetic factor for IV and predispose to AD, or whether other mechanisms are more prominent. Objectives The present aim was to investigate the role of FLG mutations as predisposing factors for IV or AD among individuals from Ethiopia. Methods A case series of Ethiopian patients with AD (n = 103) and IV (n = 7) together with controls (n = 103; subjects without past or present history of AD, dry skin or atopic manifestations) was collected at the outpatient dermatology clinics at ALERT Dermatology Hospital, Tikur Anbessa Hospital and Gondar University Hospital, Ethiopia. AD was diagnosed by a dermatologist using the U.K. Working Party’s diagnostic criteria. The IV diagnosis was based on clinical examination and genetic testing of the steroid sulphatase gene to exclude X‐linked recessive ichthyosis. Patients were studied with direct sequencing (n = 40) and/or allelic discrimination (n = 110). Immunohistochemical analysis was performed for filaggrin expression in the skin of patients (n = 7) and controls (n = 2). Results The Ethiopian patients and controls were genotyped for the four previously described common European FLG null mutations (R501X, 2282del4, S3247X, R2447X) and no carriers were found. In one patient with AD a novel heterozygous 2‐bp deletion, 632del2, leading to a premature stop codon was revealed by direct sequencing. No additional carrier of this deletion or other mutations was found. In addition, no difference in filaggrin expression was detected in AD or IV skin compared with healthy control skin. Conclusions Our results indicate that FLG loss‐of‐function‐variants are less common in patients with IV and AD in the Ethiopian population, suggesting that other factors may be of importance in the pathogenesis in this ethnic group.     

Molecular analysis of atypical deep penetrating nevus progressing to melanoma

Deep penetrating nevi (DPN) are dermal-based, heavily pigmented melanocytic proliferations primarily resulting from mutations in B-catenin and BRAF or, less commonly, NRAS. DPNs are considered to be intermediate grade tumors which are stable with low risk of malignant transformation. The precise risk for transformation is unknown. Only rare cases of DPN progressing to melanoma have been described. We present a case of a 53-year-old female with a blue-black thigh lesion, on histopathology illustrating a melanocytic proliferation with morphology most consistent with a DPN progressing to melanoma. Targeted next generation sequencing performed on both the atypical melanocytic proliferation and melanoma components showed NRAS and CTNNB1 mutations but no evidence of TERT promoter mutation or chromosomal copy number aberrations. The melanoma had additional mutations including a hotspot TERT promoter mutation as well as unbalanced chromosomal copy number aberrations. This report details the progression of DPN to melanoma through a prominent ultraviolet signature and acquisition of genetic aberrations. While the vast majority of DPNs are benign stable nevi, there are rare examples, which may progress to melanoma. This report documents a case and shows the molecular evolution by which the tumor transformed to melanoma.     

Expression of AID in malignant melanoma with BRAFV600E mutation

BRAF‐activating somatic mutations often exist in malignant melanoma. The underlying molecular mechanism of somatic BRAF mutation inductions remained to be clear. Activation‐induced cytidine deaminase (AID), a member of a cytidine deaminase family, and APOBEC3B induce somatic mutations and recently have been indicated to be involved in the pathomechanism of several kinds of cancers. The aim of this study was to explore the expression level of AID and APOBEC3B in BRAF‐mutation‐ containing malignant melanoma. Immunohistochemical study demonstrated that 9 of 10 malignant melanomas with high AID expression had BRAF^V600E mutation. Eight of them developed multiorgan metastases or multiple lymph node metastases afterwards. Although the size of the patient panel was small, the results indicate that there might be an association between AID expression and BRAF mutation in melanoma.     

Multiple trichoepitheliomas – a novel mutation in the CYLD gene

Background Trichoepitheliomas are benign neoplasms with follicular differentiation. They may present as a solitary lesion or as multiple lesions. Multiple trichoepitheliomas are inherited in an autosomal dominant pattern within families, with both variable penetrance and expressivity. Recent investigations support that mutations in CYLD, the gene affected in familial cylindromatosis as well as in Brooke–Spiegler syndrome, are also responsible for multiple trichoepitheliomas. Objective The authors report the case of a 9‐year‐old African girl with multiple facial trichoepitheliomas in whom a mutation in the CYLD gene was hypothesised. Materials and methods After genomic DNA extraction from the peripheral blood, a molecular analysis of the CYLD gene was performed by PCR, DHPLC and automated sequencing. Results A novel heterozygous mutation in exon 18 of the CYLD gene (c.2449delT) was identified, with a deletion of one nucleotide resulting in a premature translational termination codon at amino acid position 831 on the affected allele (p.Cys817Valfs X15). Conclusions The predominating tumours define the classification of these three entities. Nevertheless, studies suggest that they can simply represent phenotypic variations of the same disease spectrum, sharing common genetic mutations.     

The BRAF and NRAS mutation prevalence in dermoscopic subtypes of acquired naevi reveals constitutive mitogen‐activated protein kinase pathway activation

Most people have a few brown spots on their skin known as melanocytic naevi or moles. Moles are harmless but can occasionally transform into a potentially lethal tumour called malignant melanoma. Dermatologists recognise different types of mole from their appearance under a magnifying lens (dermatoscope). Broadly speaking: “globular” naevi appear early in life and may transform into melanoma; “reticular” naevi develop in adults and may be associated with melanoma arising at a site where no mole was present; reticular naevi with “peripheral globules” (PG) are growing but not malignant. Mutations (mistakes) in BRAF and NRAS genes occur in both benign and cancerous moles (malignant melanoma). These genes are components of the MAPK pathway which is important in tumour development. To try to clarify the role of these genes, these researchers in Australia looked for a relationship between dermoscopic appearance and NRAS and BRAF mutations in benign moles. They examined, dermoscopically, 40 acquired (non‐congenital) moles in 27 people then removed them for analysis. Under the microscope, disordered cell structure was observed in most reticular naevi but rarely in globular and PG naevi. Using a new, highly sensitive technique (“droplet digital PCR”) able to detect mutations in single cells, they found that most globular and all PG naevi had a BRAF mutation whereas reticular naevi had either BRAF or NRAS mutations. They conclude that all moles harbour mutations in MAPK genes, confirming a fundamental role for the MAPK pathway in the development of melanocytic naevi as well as melanoma.     

Epidermolysis bullosa simplex with PLEC mutations: new phenotypes and new mutations

Background Genetic mutations in the plectin gene (PLEC) cause autosomal recessive forms of epidermolysis bullosa simplex (EBS) associated with either muscular dystrophy (EBS‐MD) or pyloric atresia (EBS‐PA). Phenotype–genotype analysis has suggested that EBS‐MD is due mostly to genetic mutations affecting the central rod domain of plectin, and EBS‐PA to mutations outside this domain. Objectives This study aimed to describe new phenotypes of patients with EBS‐MD and EBS‐PA, to identify novel PLEC mutations and to establish genotype–phenotype correlations. Methods Seven patients with a suspicion of EBS linked to PLEC mutations were included. A standardized clinical questionnaire was sent to the physicians in charge of each patient. Immunofluorescence studies of skin biopsies followed by molecular analysis of PLEC were performed in all patients. Results We report the first case of nonlethal EBS‐PA improving with age, the first multisystemic involvement in a patient with lethal EBS‐PA, and the first patients with EBS‐MD with involvement of either the bladder or oesophagus. Eleven novel PLEC mutations are also reported. Conclusions Our results confirm that EBS‐PA is linked to mutations in the distal exons 1–30 and 32 of PLEC. Long‐term survival is possible, with skin improvement, but a delayed onset of MD is probable. While EBS‐MD is linked to PLEC mutations in all exons, in most cases one of the mutations affects exon 31. The precocity of MD seems to be linked to the type and localization of the PLEC mutation(s), but no correlation with mucosal involvement has been found.