BRAF(V600E)基因突变与儿童先天性色素痣的增殖活性及组织病理学特征的相关性研究

目的:明确先天性色素痣(congenital melanocytic nevi, CMN)中BRAF(V600E)基因突变对其增殖活性和组织病理学的影响。方法:回顾分析2018年12月至2020年12月我院皮肤科诊治的185例CMN患儿，所有患儿均进行基因检测、病理检查和Ki67免疫组化检测。根据基因检测结果是否存在BRAFV600E突变，将入选患儿分为突变组和对照组，然后再根据性别、年龄、皮损大小和皮损部位进行配对后进一步研究。结果:突变组Ki67指数、痣细胞累及深度、痣细胞巢个数、痣细胞巢大小与对照组比较差异均有统计学意义(均P<0.05)。与BRAF V600E阴性的CMN相比，BRAF(V600E)阳性CMN通常在表真皮交界部位形成黑素细胞巢，且巢较大。痣细胞累及深度和痣细胞巢个数与Ki67指数之间成正相关(均P<0.05)。结论:BRAF(V600E)基因突变使CMN的增殖活性明显升高，并对其组织病理学有特殊的影响。     

The association of BRAF V600E gene mutation with proliferative activity and histopathological characteristics of congenital melanocytic nevi in children

BackgroundA lot of congenital melanocytic nevi (CMN) carry the somatic mutation in the oncogene BRAF V600E. But the detailed histopathologic characteristics and the proliferative activity of CMN with BRAF V600E gene mutation have not been systematically documented.ObjectiveTo identify the proliferative activity and histopathological features correlating them with BRAF V600E gene mutation status in CMN.MethodsCMN were retrospectively identified from the laboratory reporting system. Mutations were determined by Sanger sequencing. The CMN were divided into a mutant group and control group according to whether there was BRAF gene mutation and were strictly matched according to gender, age, nevus size, and location. Histopathological analysis, analysis of Ki67 expression by immunohistochemistry and laser confocal fluorescence microscopy were performed.ResultsThe differences in Ki67 index, the depth of nevus cell involvement and the number of nevus cell nests between the mutant group and the control group was statistically significant, with p-values of 0.041, 0.002 and 0.007, respectively. Compared with BRAF V600E negative nevi, BRAF V600E positive nevi often exhibited predominantly nested intraepidermal melanocytes, and larger junctional nests, but the difference in this data sets were not statistically significant. The number of nests (p = 0.001) was positively correlated with the proportion of Ki67 positive cells.Study limitationsA small sample of patients were included and there was no follow-up.ConclusionsBRAF V600E gene mutations were associated with high proliferative activity and distinct histopathological features in congenital melanocytic nevi.     

BRAF mutations are common somatic events in melanocytic nevi

We determined mutations in the BRAF, N-ras, and CDKN2A genes in 27 histologically diverse melanocytic nevi and corresponding surrounding tissues from 17 individuals. Mutations in the BRAF and N-ras gene were found in 22 nevi (81%) from 16 individuals (94%). The predominant BRAF mutation T1799A (V600E) was detected in 18 nevi; 1 nevus had a novel A1781G (D594V) mutation in the same gene and 3 nevi had mutations in codon 61 of the N-ras gene. In 4 individuals both nevi carried a BRAF mutation, whereas in 2 other individuals 1 nevus showed a BRAF mutation and the second nevus had an N-ras mutation. In 2 individuals normal skin distant from nevi showed a BRAF mutation. No mutations were detected in the CDKN2A gene. The mutations in the BRAF and N-ras genes, in this study, were not associated with histologic type, location, skin type, size, or numbers of nevi. Our results suggest that mutations in the BRAF gene and to some extent in the N-ras gene represent early somatic events that occur in melanocytic nevi. We hypothesize the dual effect of solar ultraviolet irradiation on melanoma, through mutagenesis and by increasing the number of melanocytic nevi, many of which carry a BRAF or N-ras mutation.     

Laser therapy of giant congenital melanocytic nevi

Giant congenital melanocytic nevi (GCMN) are rare disfiguring potentially malignant lesions present at birth. The approach of these patients is based on two main considerations: attempt to minimize the risk of malignancy, and obtain an acceptable cosmetic result. Sometimes they are too large to be removed by multiple surgical excision or by use of osmotic expander. The objective of treatment of giant congenital nevi is to obtain ablation without side effects or after-effects from aesthetics. But for the moment such treatment doesn't exist. The aim of this review was to access treatment of GCMN with lasers as an alternative to surgery. Lasers should only be regarded as a treatment option for GCMN that cannot be surgically excised. For the moment laser therapy of GCMN should be restricted to well controlled studies or to individual patients in whom surgical procedures are not possible or would result in unacceptable scarring. Today ultrashort high energy pulsed CO2 laser and the normal mode ruby laser are the two lasers available. But the results are too unforeseeable, and painful with these two lasers. The combined use of normal mode and Q-switched ruby lasers or ultrashort high energy pulsed CO2 laser and Q-switched ruby or Nd:YAG lasers can give us a solution. In the future perhaps new Q-switched laser could give us a better way of treatment, with less pain, and no scars. We need an improvement of the technology in this field and hopefully the picosecond systems will be available in the future.     

Neonatal erosions and ulcerations in giant congenital melanocytic nevi

We report 10 cases of neonatal erosions or ulcerations in giant congenital melanocytic nevi. Histopathologic examination, performed in eight cases, demonstrated benign findings. Clinical follow-up for an average of 3.8 years (range 2 weeks to 8 years) has not revealed the development of cutaneous melanoma. We conclude that erosions or ulcerations in giant melanocytic nevi in neonates are often benign and do not necessarily signify the presence of cutaneous melanoma.     

Curettage of giant congenital melanocytic nevi in neonates: a decade later

BACKGROUND: Currently, there is tremendous uncertainty regarding how giant congenital melanocytic nevi (GCMN) should be treated. Our approach to patients with GCMN is based on 2 main considerations: (1) obtain an acceptable cosmetic result to decrease the psychosocial inconvenience to the patient, and (2) attempt to minimize the risk of malignancy. For the past 10 years we have treated GCMN by curettage in the neonatal period. We report our experience and results. OBSERVATIONS: Sixteen neonates with GCMN were treated by curettage between 1990 and 2000. Biopsy specimens were obtained and the patients received close clinical follow-up. In most patients cosmetic and functional results were good, and, to date, no melanoma has been observed in this series. CONCLUSIONS: Curettage offers an adequate alternative to surgical excision when performed during the first 2 weeks of life. Patients and parents are pleased with the cosmetic and functional results and thereby suffer less from the psychosocial inconvenience caused by these lesions. Careful long-term follow-up of these children is essential to monitor final cosmetic outcome and reduce the potential for malignancy.     

Lack of association between BRAF mutation and MAPK ERK activation in melanocytic nevi

The mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase signaling pathway can be activated through mutations of V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) oncogene, frequently found in melanoma (60%), common nevi (CN) (73-82%), and atypical nevi (AN) (52-80%). MAPK activation has been reported between 0 and 22% in nevi, and 86% of primary melanoma, without any knowledge of BRAF mutational status. We studied the correlation of MAPK activation status, BRAF mutation, and B-Raf expression in CN, AN, and melanoma. Using immunohistochemistry, phosphorylated (active) MAPK and B-Raf expression was studied in 24 CN, 21 AN, and 26 primary cutaneous melanomas (PM). BRAF mutations at codon 600 were assessed by PCR-RFLP. Active MAPK was detected in 29% of CN, 48% of AN, and 85% of PM. BRAF mutation was found in 67% of CN, 62% of AN, and 58% of PM. In all, 23% of CN, 54% of AN, and 93% of PM with BRAF mutation have activated MAPK. All lesions expressed B-Raf. BRAF mutation does not seem to be sufficient to produce MAPK activation in melanocytic nevi, and it is suggested that other events are needed to induce MAPK activation, that is, B-Raf overexpression, inhibition of MAPK phosphatases, or suppression of RAF kinase inhibitors.     

Detection of the BRAF V600E mutation in melanocytic lesions using the ligase detection reaction

BACKGROUND: BRAF is a member of the RAF kinase family, and it promotes signaling through the RAS-MAP kinase signal transduction cascade. Research has shown that a majority of melanomas and nevi exhibit an activating V600E (T1799A) mutation in BRAF exon 15. Additional studies of BRAF have demonstrated that the T1799A mutation is absent in uveal melanomas and Spitz nevi. METHODS: The ligase detection reaction (LDR) is a sensitive technique that can identify specific DNA mutations occurring at very low frequency within heterogeneous clinical samples, and it has previously been used to analyze mutations in paraffin-embedded tissue specimens. RESULTS: The LDR can readily detect mutations in DNA extracted from non-microdissected paraffin-embedded sections of common nevi, dysplastic nevi, and melanomas. In addition, this method demonstrated the absence of the V600E (T1799A) mutation within Spitz nevi. CONCLUSION: The LDR is a highly sensitive and specific test for detecting mutations in formalin-fixed tissue. The LDR can be easily adapted to detect any mutation associated with a cutaneous disorder. The absence of the BRAF V600E mutation in Spitz's nevi may serve as a molecular signature to distinguish these lesions from common nevi, dysplastic nevi, and some types of malignant melanoma.     

Chromosomal translocations as a mechanism of BRAF activation in two cases of large congenital melanocytic nevi

Genetic studies of melanocytic tumors have mainly demonstrated activation of oncogenes such as NRAS or BRAF through point mutations. In two cases of large congenital melanocytic nevi, we observed a chromosomal translocation involving the BRAF oncogene on chromosome 7q34, resulting in both cases in removal of the auto-inhibitory N-terminal regulatory domain (hence the Ras-guanosine triphosphate binding domain) of BRAF from its protein kinase domain. This is early evidence of BRAF activation through chromosomal translocation in melanocytic tumors. Because BRAF point mutations are rather rare in congenital melanocytic nevi and melanoma arising in non-sun-exposed area, the molecular mechanism of oncogenic activation as described here could be a recurrent molecular feature in these groups of melanocytic tumors.     

BRAF kinase gene V599E mutation in growing melanocytic lesions

Mutations in the BRAF-gene are found in benign and malignant melanocytic lesions, >90% being a V599E mutation. This mutation results in constitutively active kinase function and increased colony formation in vitro. The biological impact of this mutation in vivo is still debated. To address this question, we used our digital epiluminescence image archive and retrospectively selected 49 melanocytic lesions, which did not meet the criteria of melanoma at the initial presentation. Mean 12 months later these lesions were excised because of increased size or changed structure and BRAF(V599E) mutations were analyzed. Among 36 growing lesions, BRAF(V599E) mutations were found in 16 (11 melanomas and 5 nevi). Among 13 lesions with structural changes, BRAF(V599E) mutations were found in 4 (3 melanomas and 1 nevus). Thirty-five randomly selected additional lesions with no changes during follow-up served as controls, all nevi by histology, and two of them showed a BRAF(V599E) mutation. Statistics revealed odds for the presence of the BRAF(V599E) mutation being seven times higher in lesions with structural changes and 13 times higher in growing lesions as compared with lesions without changes. This raises the question if the V599E mutation determines lesions at risk developing into melanoma and if not, what are the mechanisms controlling growth stop in benign lesions?     

黑素瘤BRAF V600E突变的临床检测方法进展

针对黑素瘤BRAF V600E突变的靶向治疗为黑素瘤的治疗提供了新方法。BRAF基因突变的准确检测是实施靶向治疗的前提。以Sanger测序法为"金标准"的基因测序是目前临床检测黑素瘤BRAF V600E突变的主要方法。近年来,针对BRAF V600E突变的单克隆抗体VE1在组织学上显示V600E突变蛋白,还可显示异质性肿瘤细胞,其高敏感性和特异性是对BRAF突变基因检测的重要补充。该文对BRAF V600E突变检测方法做一综述,为临床应用提供信息。     

BRAF V600E mutation as a prognostic factor in cutaneous melanoma patients

The prognostic significance of BRAF mutations in the natural course of melanoma is controversial. The aim of study was to assess the prognostic significance of BRAF V600E mutation in cutaneous melanoma patients. A total of 151 melanomas were included in the study. BRAF V600E mutation was detected using the real‐time PCR. BRAF V600E mutation rate was 51%. BRAF mutation rate was higher for young patients (61.4%) and upper limbs (63.2%), trunk (59.3%) and head and neck (59.2%) were the most frequently afflicted sites in BRAF‐mutant patients, whereas lower limbs were mostly affected in BRAF‐wild patients (77.8%). Likewise, acral melanomas rarely harbored BRAF mutation (17.1%). The disease‐free survivals regarding the entire and Stage III cohorts were longer in the BRAF‐mutant group than in the BRAF‐wild group (p = .006 and p = .004, respectively), whereas Stage I–II patients had no survival differences between BRAF statuses (p = .2). Likewise, BRAF‐mutant patients had better overall survival (OS) time compared to BRAF‐wild patients in all stages (p = .01), in Stage III (p = .01), and in Stage IV patients (p = .001). However, no differences between BRAF statuses were observed in Stage I–II melanomas (p = .3). In conclusion, BRAF V600E‐mutant melanomas show favorable prognostic impact on both disease‐free and OSs in all staged melanomas except local disease.     

High frequency of BRAFV600E mutation in acquired nevi and small congenital nevi, but low frequency of mutation in medium-sized congenital nevi

To investigate whether the frequency of the BRAF(V600E) (V-raf murine sarcoma virus oncogene homolog B1) mutation in melanocytic nevi is associated with sun exposure patterns, we examined 120 acquired melanocytic nevi excised from various anatomic sites, including glabrous skin, as well as 62 congenital nevi. We used a new mutation detection system based on the shifted termination assay, called Mutector, which was able to detect only 5% of heterozygous mutant cells within the samples. We detected the mutation in 105/120 (87.5%) acquired nevi and 43/62 (69.4%) congenital nevi. Notably, we found the mutation in 35/43 (81.4%) acquired nevi excised from glabrous skin and genitalia. These results strongly suggest that UV light is not necessarily required for the acquisition of the BRAF(V600E) mutation, and suggest that non-mutagenic effects of UV light to melanocytes may be more important in the nevogenesis. Additionally, we showed heterogeneous distribution of BRAF-mutated cells within the lesions of small congenital nevi by a combination of laser microdissection and direct sequencing. Finally, we found low frequency of BRAF(V600E) mutation (6/20, 30.0%) in medium-sized congenital nevi. Most of these nevi with wild-type BRAF had neroblastoma ras viral oncogene homolog mutations (9/14, 64.3%), suggesting different pathogenesis of medium-sized congenital nevi from acquired nevi and small congenital nevi.     

Rhabdomyosarcoma arising in a giant congenital melanocytic nevus

We report the case of a 6-week-old girl who presented with a pedunculated embryonal rhabdomyosarcoma arising in a giant congenital melanocytic nevus (GCMN) on her lower back. There was no associated leptomeningeal involvement. The patient underwent surgical resection of the rhabdomyosarcoma at age 2 months, with subsequent chemotherapy consisting of actinomycin D and vincristine. No recurrences or metastases of tumor have been noted at 5 months of age.