Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis

Background There have been conflicting data regarding the prevalence and clinicopathological characteristics of BRAF and NRAS mutations in primary cutaneous melanoma. Objectives To solve this controversy, this study used a meta‐analysis to evaluate the frequencies of BRAF and NRAS mutations, and the relationship between these mutations and clinicopathological parameters of cutaneous melanoma. Methods Data from studies published between 1989 and 2010 were combined. The BRAF and NRAS mutations were reported in 36 and 31 studies involving 2521 and 1972 patients, respectively. The effect sizes of outcome parameters were calculated by odds ratios (OR). Results BRAF and NRAS mutations were reported in 41% and 18% of cutaneous melanomas, respectively. The mutations were associated with histological subtype and tumour site, but not with age and sex. The BRAF mutation was frequently detected in patients with superficial spreading melanoma (OR = 2·021; P < 0·001) and in melanomas arising in nonchronic sun‐damaged skin (OR = 2·043; P = 0·001). In contrast, the NRAS mutation was frequently evident in patients with nodular melanoma (OR = 1·894; P < 0·001) and in melanomas arising in chronic sun‐damaged skin (OR = 1·887; P = 0·018). Conclusions This pooled analysis shows that the incidences of BRAF and NRAS mutations in cutaneous melanomas differ according to histological type and tumour location based on the degree of sun exposure.     

Genetic alterations in RAS‐regulated pathway in acral lentiginous melanoma

Studies integrating clinicopathological and genetic features have revealed distinct patterns of genomic aberrations in Melanoma. Distributions of BRAF or NRAS mutations and gains of several oncogenes differ among melanoma subgroups, while 9p21 deletions are found in all melanoma subtypes. In the study, status of genes involved in cell cycle progression and apoptosis was evaluated in a panel of 17 frozen primary acral melanomas. NRAS mutations were found in 17% of the tumors. In contrast, BRAF mutations were not found. Gains of AURKA gene (20q13.3) were detected in 37.5% of samples, gains of CCND1 gene (11q13) or TERT gene (5p15.33) in 31.2% and gains of NRAS gene (1p13.2) in 25%. Alterations in 9p21 were identified in 69% of tumors. Gains of 11q13 and 20q13 were mutually exclusive, and 1p13.2 gain was associated with 5p15.33. Our findings showed that alterations in RAS‐related pathways are present in 87.5% of acral lentiginous melanomas.     

Mutational profiling of acral melanomas in Korean populations

The proportion of acral melanoma (AM) is much higher in Asian populations than in Caucasian populations. Although mutational profiles associated with AM have been discovered in Caucasian populations, knowledge of its genetic alterations in Asian populations is limited. To describe the molecular nature of AM in Korean patients, we performed mutational profiling of AM and matched normal tissues in patients. Fifty‐one formalin‐fixed paraffin‐embedded AM samples and 32 matched pairs from patients’ saliva DNA were analysed by next‐generation sequencing. Only mutations confirmed via digital droplet PCR or in BRAF, KIT and NRAS, the most frequently altered cancer genes in cutaneous melanoma, were considered as positive. The relationship between mutational status and clinicopathological features were examined. Of the 47 AM patients screened, alteration of BRAF, NRAS and KIT genes was observed in 6.4%, 4.3% and 8.5%, respectively. We also tested matched normal tissues of patients to identify tumor‐specific mutations. Examination of the mutational profile in a cohort of 28 primary melanomas and matched normal controls found BRAF mutations in two cases (7.1%), KIT mutations in three cases (10.7%) and CTNNB1 mutations in one case (3.6%). The BRAF, NRAS and KIT mutation status did not correlate with clinicopathological characteristics. Our results show that KIT, NRAS and BRAF hotspot mutations occur at a low frequency in Korean populations. We also observed a case with the CTNNB1 mutation, which raises the possibility that other pathways are associated with AM development.     

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.     

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.     

BRAF V600E mutation and the tumour suppressor IGFBP7 in atypical genital naevi

Background Atypical genital naevi (AGN) are naevi of special sites with atypical histological features that overlap with those of malignant melanoma. Activating BRAF mutations, identified in the majority of banal melanocytic naevi and cutaneous melanomas, are reportedly uncommon in naevomelanocytic proliferations in nonsun‐exposed sites. We have recently shown that constitutive activation of the BRAF‐MEK‐ERK signalling pathway in oncogenic BRAF‐positive naevi increases expression and secretion of IGFBP7, which induces senescence and apoptosis. Objectives To ascertain the frequency of BRAF V600E mutations in AGN compared with banal naevi without atypia. An additional aim was to assess the expression of IGFBP7 in oncogenic BRAF‐positive AGN. Methods Genomic DNA was isolated per protocol from seven genital naevi without atypia and 13 AGN for BRAF genotyping. Immunohistochemical staining for IGFBP7 was performed on all cases. Results The BRAF V600E mutation was identified in 43% of genital naevi without atypia and 23% of AGN (P = 0·61). In both groups, IGFBP7 expression was maintained in 67% of BRAF V600E‐positive cases. Conclusions The prevalence of BRAF V600E in AGN suggests that ultraviolet exposure is not essential for generating the mutation. The BRAF V600E mutational status appears to be of limited diagnostic utility in distinguishing genital naevi that exhibit atypia from those that do not. Similar to oncogenic BRAF‐positive common naevi without atypia, enhanced expression of the tumour suppressor IGFBP7 in oncogenic BRAF‐positive AGN supports that they are biologically inert.     

Prognostic and Clinicopathologic Associations of BRAF Mutation in Primary Acral Lentiginous Melanoma in Korean Patients: A Preliminary Study

Background

In the majority of melanomas, the RAS/RAF/MEK/ERK signaling pathway is constitutively activated, due to oncogenic mutations in the BRAF and NRAS genes. The BRAF mutation has been mainly described in Caucasian melanomas. However, there is a lack of study evaluating the status, and the clinical significance, of BRAF mutation in the Asian population.

Objective

This study was aimed to determine the frequency of BRAF mutation, and to evaluate the correlation of BRAF status with clinicopathologic features and outcomes, in Korean primary acral lentiginous melanoma (ALM) patients.

Methods

ALM samples (n=36) were analyzed for the BRAF V600E mutation, by dual-priming oligonucleotide (DPO) based real-time polymerase chain reaction. The clinicopathologic features and prognosis of the patients were analyzed with BRAF mutation status.

Results

The incidence of BRAF V600E mutation was 19.4% (7/36). The BRAF V600E mutations were not associated with clinicopathologic features, except for the age factor. All of the BRAF-mutant patients survived without recurrence or metastasis, and have a better clinical outcome than BRAF wild-type patients.

Conclusion

In Korean primary ALM, a low frequency of BRAF mutation was shown; and BRAF mutation presented with a favorable prognosis. These results indicate that other distinctive genetic mechanisms may have more important roles in the development and progression of disease. Further multicenter study with large sample size is firmly needed, to confirm the results of our preliminary study.     

Increase in NRAS mutant allele percentage during metastatic melanoma progression

One‐fifth of cutaneous melanomas have dominant gain‐of‐function mutations of the NRAS oncogene. We report the first two cases of increasing NRAS mutant allele frequency in melanoma metastases and show that the chromosomal mechanism of this homozygosity is an increased polysomy of chromosome 1. We observed an increase in NRAS mutant allele percentage (NRAS‐MA%) in the metastatic melanoma progression from 2 patients with melanomas harbouring a NRAS mutation (p.Q61K in case 1 and p.Q61R in case 2). In case 1, we observed a NRAS‐MA% increase from 18% within the first metastatic node to 81%, 92% and 85% respectively in the three subsequent metastases: lymph node, brain and subcutaneous metastases biopsied 1, 6 and 17 months, respectively, after the initial lymph node biopsy. In case 2, we observed an increase in NRAS‐MA% from 40% within the primary melanoma to 63% within the metastatic lymph node. FISH analysis showed the same results in both cases: a frequent polysomy of chromosome 1 in metastasis samples with NRAS mutant allele percentage >60%, while most cells were disomic in the samples with well‐balanced heterozygous mutations. The percentage of NRAS mutant allele may increase during metastatic progression and may be associated with chromosomal instability. Further studies are needed to evaluate the prognostic impact of the NRAS homozygous status and/or polyploidy in metastatic cutaneous melanomas.     

Comparative analysis of BRAF,NRAS and c‐KIT mutation status between tumor tissues and autologous tumor cell‐lines of stage III/IV melanoma

In the last decade, advances in molecular biology have provided evidence of the genotypic heterogeneity of melanoma. We analysed BRAF, NRAS and c‐KIT alterations in tissue samples from 63 stage III/IV melanoma patients and autologous cell‐lines, using either allele‐specific or quantitative PCR. The expression of BRAF V600E protein was also investigated using an anti‐BRAF antibody in the same tissue samples. 81% of FFPE samples and tumor cell‐lines harboured a genetic alteration in either BRAF (54%) or NRAS (27%) oncogenes. There was a strong concordance (100%) between tissue samples and tumor cell‐lines. The BRAF V600E mutant‐specific antibody showed high sensitivity (96%) and specificity (100%) for detecting the presence of a BRAF V600E mutation. The correlation was of 98% between PCR and immunohistochemistry results for BRAF mutation. These results suggest that BRAF and NRAS mutation status of tumor cells is not affected by culture conditions.     

Dermoscopy and confocal microscopy for metachronous multiple melanomas: morphological,clinical, and molecular correlations

Background

Cutaneous melanoma is one of the most frequent malignancies of the skin in Caucasian populations. Patients who develop cutaneous melanoma are at increased risk of developing a second primary melanoma. The estimated incidence of multiple primary melanoma (MPM) ranges from 1.2% to 8.2% of cases, with a high preponderance of melanomas occurring metachronously.

Objectives

The aim of this study was to describe dermoscopic, microscopic, clinical, and molecular correlations between first and subsequent melanomas in patients with metachronous MPMs.

Materials & Methods

Twenty-four paired melanomas from 12 MPM patients were evaluated for architectural characteristics based on dermoscopy and confocal microscopy, as well as for mutations in BRAF and NRAS genes by Sanger-based sequencing analysis. Specific scores used for classifying features of dermoscopy (global pattern; 7-point check list; ABCD Stolz score) and confocal microscopy (Segura and Pellacani) were compared with genetic and histological data.

Results

Consistency in dermoscopic patterns between the primary and subsequent cutaneous melanomas were observed in about two thirds of cases, whereas concordant features based on confocal microscopy were found in only about two fifths of cases. The majority of patients (7/12; 58%) presented consistent BRAF/NRAS mutation patterns between first and subsequent primary melanomas. A significant association between BRAF mutations and Pellacani scorewas evident.

Conclusions

Similarities between the index melanoma and subsequent cutaneous melanomas were observed with regards to dermoscopic features and, to a much less extent, confocal microscopy findings. Our data further indicate that the Pellacani score may be used to predict BRAF mutations.

     

BRAF,KIT and NRAS mutations and expression of c‐KIT,phosphorylated extracellular signal‐regulated kinase and phosphorylated AKT in Japanese melanoma patients

To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal‐regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c‐KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma.     

Variation of mutant allele frequency in <Emphasis Type="Italic">NRAS</Emphasis> Q61 mutated melanomas

Background

Somatic mutations of BRAF or NRAS activating the MAP kinase cell signaling pathway are present in 70% of cutaneous melanomas. The mutant allele frequency of BRAF V600E (M%BRAF) was recently shown to be highly heterogeneous in melanomas. The present study focuses on the NRAS Q61 mutant allele frequency (M%NRAS).

Methods

Retrospective quantitative analyze of 104 NRAS mutated melanomas was performed using pyrosequencing. Mechanisms of M%NRAS imbalance were studied by fluorescence in situ hybridization (FISH) and microsatellite analysis.

Results

M%NRAS was increased in 27.9% of cases. FISH revealed that chromosome 1 instability was the predominant mechanism of M%NRAS increase, with chromosome 1 polysomy observed in 28.6% of cases and intra-tumor cellular heterogeneity with copy number variations of chromosome 1/NRAS in 23.8%. Acquired copy-neutral loss of heterozygosity (LOH) was less frequent (19%). However, most samples with high M%NRAS had only one copy of NRAS locus surrounding regions suggesting a WT allele loss. Clinical characteristics and survival of patients with either <60% or ≥60% of M%NRAS were not different.

Conclusion

As recently shown for M%BRAF, M%NRAS is highly heterogeneous. The clinical impacts of high M%NRAS should be investigated in a larger series of patients.

     

Distinct MAPK and PI3K pathway mutations in different melanoma types in Taiwanese individuals

Background

A number of studies investigating mutations of genes involved in MAPK and PI3K pathways in melanoma patients have been performed, most of which were based on Caucasian populations.

Objectives

We sought to identify BRAF, NRAS, MEK1, PI3K, and PTEN mutations and further determine possible correlations with clinicopathological parameters in Taiwanese patients with acral, non-chronic sun damaged (NCSD), or mucosal melanoma.

Materials & Methods

Forty melanocytic nevi, 24 dysplastic nevi, and 175 melanomas from Taiwanese patients were analysed for mutations in BRAF, NRAS, MEK1, PI3K, and PTEN genes by PCR and direct sequencing. Immunohistochemical analysis of the respective proteins in nevi and melanomas were also performed to determine possible clinicopathological characteristics.

Results

In addition to the classic BRAF^V600E mutation, a novel BRAF^V600L mutation was identified in acral and sinonasal melanomas. A significantly reduced frequency of NRAS and BRAF mutations was noted compared with Caucasian-based studies. Increased immunohistochemical scores for pan-RAS and MEK1 and less nodal metastasis were associated with enhanced survival rates in acral and NCSD melanoma patients.

Conclusions

Our findings suggest that oncogenic events may differ among melanomas in Asian patients geographically (e.g. between Japanese and Taiwanese patients). Moreover, distinct genetic alterations were noted among acral, NCSD, and mucosal melanoma patients in our study, and the expression of biomarkers correlated with clinical survival rates differentially among the various melanoma groups.

     

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.     

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.     

Beyond the BRAFV600E hotspot: biology and clinical implications of rare BRAF gene mutations in melanoma patients

BRAF gene mutations can be found in approximately 50% of melanomas, but the most common BRAF mutation leads to substitution at residue 600 of the protein, from valine to glutamic acid. BRAF^V600E occurs in up to 95% of all melanoma cases and can be successfully blocked by using a combination of BRAF‐ and MEK inhibitors. The wider availability of next‐generation sequencing is revealing more non‐V600 BRAF mutations, and the clinical implications of these mutations are widely unknown. In this review, we will discuss the biology of the MAPK pathway and the different types of BRAF mutations as well as their effect on MEK activation. Current literature will be reviewed including in vitro data, case reports and case series.     

Sensitivity and Usefulness of VE1 Immunohistochemical Staining in Acral Melanomas with BRAF Mutation

BackgroundAcral melanomas are known to have a low frequency of BRAF mutation, in contrary to higher KIT mutation. Recently, VE1 immunostaining was reported to have a good correlation with BRAF mutation status.ObjectiveWe aimed to evaluate the clinicopathological features of BRAF-mutated acral melanomas and validate the correlation of the VE1 immunohistochemical stains in those cases.MethodsThe clinical features (age, sex, anatomical site), and histopathological characteristics of 41 patients with acral melanoma were evaluated. We performed a next-generation sequencing to detect BRAF mutation status. We also determined the correlation of VE1 immunohistochemical staining with BRAF mutation status.ResultsAmong 19 acral melanomas with BRAF mutation, common histopathological subtype was acral lentiginous melanoma (8/19, 42%) and nodular melanoma (8/19, 42%) and superficial spreading melanoma (3/19, 16%) followed. VE1 immunostaining results were positive in all 15 cases with BRAF V600E mutation (sensitivity 100%), and negative in 4 cases of BRAF non-V600E mutation. However, VE1 immunostaining was negative in all 22 patients with BRAF wild-type.ConclusionVE1 immunostaining had a good correlation with BRAF V600E mutation status.     

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.     

Mutated and amplified NRAS in a subset of cutaneous melanocytic lesions with dermal spitzoid morphology: report of two pediatric cases located on the ear

Extensive cytogenetic testing is slowly unveiling the complexity of the genomics of melanocytic tumors. NRAS mutations have been the first genetic abnormality described in malignant melanomas. We report the cases of two children, presenting a melanocytic lesion located on the ear. One appeared as a combined dermal clone inside a congenital nevus and the other as a centimetric purely dermal tumor. Both tumors were composed of spindled spitzoid melanocytes with atypical histologic features. aCGH and FISH revealed an amplification of the NRAS gene. Sequencing showed an exon 3 NRAS mutation. In the combined case, the amplification was limited to the spitzoid component, underscoring a possible phenotypic shift induced by the alteration. Similarly an overexpression of CyclinD1 and elevation of ki‐67 was found in the spitzoid component confirming a raise in proliferation. Such combination of mutation and copy number increase has been previously reported for the HRAS gene in a subset of Spitz nevi. Further studies must evaluate if mutated NRAS is also amplified in melanomas arising in this clinical setting. These combined alterations could represent an early event ultimately leading to malignancy.