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.     

Novel LRRFIP2-RAF1 fusion identified in an acral melanoma: A review of the literature on melanocytic proliferations with RAF1 fusions and the potential therapeutic implications

A small subset of cutaneous melanomas harbor oncogenic gene fusions, which could potentially serve as therapeutic targets for patients with advanced disease as novel therapies are developed. Fusions involving RAF1 are exceedingly rare in melanocytic neoplasms, occurring in less than 1% of melanomas, and usually arise in tumors that are wild type for BRAF, NRAS, and NF1. We describe herein a case of acral melanoma with two satellite metastases and sentinel lymph node involvement. The melanoma had a concomitant KIT variant and LRRFIP2-RAF1 fusion. This constellation of molecular findings has not been reported previously in melanoma. We review the existing literature on melanocytic neoplasms with RAF1 fusions and discuss the potential clinical implications of this genetic event.     

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 mutations might be more common than supposed in vulvar melanomas

Data on BRAF, NRAS and KIT mutations are scarce in patients with vulvo‐vaginal melanomas and are associated with important therapeutic issues. We investigated their prevalence in a cohort of patients with female lower genital tract melanomas between 2003 and 2017. Of the 22 patients, 5 (22.7%) harboured a BRAF mutation, which was much higher than the rate of 5% reported in the literature. One patient, who was tested negative on the primary melanoma, had a NRAS mutation in a cutaneous metastasis. Our data provide a rationale for prospective and repeated mutations testing in female lower genital tract melanomas.     

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.     

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.     

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.     

Distant metastasis due to heavily pigmented epithelioid melanoma with underlying BRAF V600E,NOTCH1, ERBB3, and PTEN mutations

Pigment‐synthesizing melanoma (PSM) describes a morphologically and genetically diverse group of melanomas. In contrast, pigmented epithelioid melanocytoma (PEM) encompasses a spectrum of indolent tumors now classified as borderline/intermediate melanocytic tumors. Herein, we report a case of widely metastatic heavily pigmented epithelioid melanoma with fatal outcome in a 36‐year‐old woman. Next‐generation sequencing identified somatic (tumoral) mutations in BRAF V600E, PTEN, NOTCH1, and ERBB3. By contrast, GNAQ and GNA11 were wild type. Prkar1α and p16 expression were maintained. Identification of mutations in NOTCH1 and ERBB3 may support the diagnosis of heavily pigmented epithelioid melanoma. In contrast, PRKCA fusion genes and PRKAR1A mutations support the diagnosis of PEM. Given the heterogeneity, potential overlap (loss of Prkar1α expression), and evolving genetic profiles of these two distinct groups of tumors, careful appraisal of molecular profiles in the light of histomorphology and clinical history is necessary for distinction between PEM and PSMs including heavily pigmented epithelioid melanomas, with significant potential impact on prognosis and therapy.     

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.     

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.     

Detecting copy number alterations of oncogenes in cell-free DNA to monitor treatment response in acral and mucosal melanoma

BackgroundReliable biomarkers are necessary for assessment of treatment responses. Acral and mucosal melanomas are commonly associated with copy number (CN) alterations rather than specific point mutations, with CN alterations inKIT, CDK4, and CCND1 occurring frequently. Cell-free DNA is released to peripheral blood by both normal and tumor cells, and therefore contains the same genetic alterations present in the source tumor.ObjectiveTo investigate the usefulness of detecting CN alterations in oncogenes in cell-free DNA for monitoring treatment response in acral and mucosal melanomas.MethodsWe isolated cell-free DNA from peripheral blood and assessed the CN alterations in the cell-free DNA. Using droplet digital PCR, we examined CN alterations ofKIT, CDK4, and CCND1 in tumors from 37 melanoma patients (acral, n = 27; mucosal, n = 10) and peripheral blood from 24 melanoma patients (acral, n = 17; mucosal, n = 7).ResultsCN gain was detected in at least one of the genes examined in 62.9 % (17/27) of acral melanomas and 70 % (7/10) of mucosal melanomas. CN gains were also detected in the plasma of some patients. Furthermore, plasma CN ratio was correlated with clinical condition. This correlation was especially clear in patients with high CN ratios in tumors and high tumor burdens.ConclusionPlasma CN ratios may be useful for evaluating treatment responses in patients with acral and mucosal melanoma.     

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.

     

Targeted next generation sequencing (NGS) to classify melanocytic neoplasms

This study piloted a pan-solid-tumor next generation sequence (NGS)-based laboratory developed test as a diagnostic aid in melanocytic tumors. 31 cases (4 “epithelioid” nevi, 5 blue nevi variants, 7 Spitz tumors [3 benign and 4 malignant] and 15 melanomas) were evaluated. All tumors [median diameter 7 mm (range 4-15 mm); median thickness 2.25 mm (range 0.25-12 mm)] yielded satisfactory results. The number of small nucleotide variants/tumor was significantly different between melanoma (median 18/tumor, range 4-71) and all other lesions (median 8/tumor, range 3-17) (P < 0.004) and malignant (median 16/tumor, range 4-71) vs benign lesions (median 7/tumor, range 3-14) (P = 0.01). BRAF, MET, NTRK1, and ROS fusions only occurred in benign Spitz tumors; EML4 fusion, BRAF, MAP2K1 and TERT mutations occurred in malignant Spitz tumors and/or melanoma. Amplifications and NRAS and NF1 mutations only occurred in melanoma. Most melanomas contained >1 pathogenic alteration. Developed NGS-based criteria correctly classified all malignant lesions in this series. 10/12 cases showed concordance with FISH; consensus diagnosis agreed with NGS classification in FISH-non-concordant cases. This pilot study suggests that NGS may be an effective diagnostic adjunct comparable to FISH, but further studies with larger numbers of cases are needed.     

Melanoma's high C>T mutation rate: is deamination playing a role?

The majority of melanoma mutations are C>T transitions, and most bear UV signatures. However, other process may contribute to the high C>T mutation rate. Okura et al., have demonstrated immunohistochemical evidence of deaminating enzymes, activation‐induced cytidine deaminase (AID) and apolipoprotein B mRNA‐editing enzyme catalytic polypeptide‐like 3B (APOBEC3B) in melanoma. Both have been implicated in cancer. While further validation is necessary, these findings warrant consideration of a role for deamination in melanomagenesis. Deamination primarily drives C>T transitions. Compared with trunk/extremity melanomas, acral melanomas display a significantly higher percentage of ‘spontaneous’ and ‘AID’ mutation signature events suggesting deamination may be particularly important in this subgroup.     

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.     

Characterization of KIT mutation in melanoma

Background/objectivesRecent studies have shown that the KIT mutational type appears to be a predictive marker for the efficacy of imatinib in treating melanoma. However, a wide range of KIT mutation rates was reported in different types of melanoma, suggesting that the mutation frequency of KIT may be associated with clinicopathological subsets of melanoma.MethodsTo characterize their relationship, we sequenced exons 11, 13, 17, and 18 of KIT in 80 of 85 melanomas collected from two hospitals and categorized KIT mutation by tumor type, age and sex of patients, and mutation hot spots of KIT.ResultsOur results showed that KIT mutation rates were 25%, 22%, and 8% in acral, mucosal, and cutaneous melanomas, respectively. Approximately 38% (5/13) of male patients with acral melanoma and 45% (5/11) of female patients with mucosal melanomas of the anorectal and genitourinary regions had a KIT mutation. Approximately 81% of KIT mutations occurred in L576P, K642E, V559A, and D820Y.ConclusionThis result shows that KIT mutation is enriched in a certain subset of melanoma patients and mutation hot spots do exist.     

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.     

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.     

Fluorescence in situ hybridization for distinguishing cellular blue nevi from blue nevus-like melanoma

Background: Blue nevus‐like melanomas are melanomas that arise in association with blue nevi or closely simulate the histopathologic appearance of a blue nevus, usually a cellular blue nevus (CBN). Although the majority of CBN can be readily distinguished from blue nevus‐like melanoma by conventional microscopy, there are a subset of cases where this distinction may be exceedingly difficult or impossible. Methods: In this study, we evaluated the ability of a fluorescence in situ hybridization (FISH) assay targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and the centromere of chromosome 6 (Cep6) to distinguish between CBN and blue nevus‐like melanoma. We identified five cases of blue nevus‐like melanoma and 12 cases of CBN. Results: The FISH assay was performed with 100% sensitivity and 100% specificity. Three of five cases met the 6p25/Cep6 criteria, all five met the 6p25 gain criteria and three of five met the 6q23/Cep6 loss criteria. None of the cases met criteria for gains in 11q13. None of the 12 CBN met any criteria for melanoma. Conclusions: A combined analysis of clinical aspects, histopathologic changes and FISH analysis could potentially contribute significantly to the ability of pathologists to discriminate between blue nevus‐like melanoma and blue nevi in challenging cases. Gammon B, Beilfuss B, Guitart J, Busam KJ, Gerami P. Fluorescence in situ hybridization for distinguishing cellular blue nevi from blue nevus‐like melanoma.