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。     

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 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.     

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.     

Varying proliferative and clonogenic potential in NRAS‐mutated congenital melanocytic nevi according to size

Congenital melanocytic nevi (CMN) are benign proliferations that may be associated with various consequences depending on their size. They are characterized by a specific molecular signature, namely a postzygotic somatic NRAS or BRAF mutation. We have recently reported that large CMN (lCMN), which are classically associated with an increased melanoma risk, harbour cell subpopulations with specific clonogenic and tumorigenic potential. We wished to ascertain whether cells displaying similar properties persisted postnatally in medium CMN (mCMN). Eighteen medium M1, nine large and one giant NRAS‐mutated CMN were prospectively included in the study. Subpopulations of mCMN cells expressed stem cell/progenitor lineage markers such as Sox10, nestin and Oct4, as was the case in lCMN. Nevertheless, conversely to lCMN, mCMN cells with clonogenic properties were rarer. In vitro, approximatively one in 1500 cells isolated from fresh mCMN formed colonies that could be passaged. In vivo, mCMN seemed to harbour cells with less proliferative potential than the larger lesions as lCMN biopsies displayed a threefold expansion compared to mCMN when xenografted in Rag2^?/? mice. Thus, our data revealed variations in clonogenicity and tumorigenic properties in NRAS‐mutated CMN according to size.     

Proliferative activity in melanocytic nevi from patients grouped by age with clinical follow‐up

Any mitotic activity in a melanocytic nevus is a source of concern about the biologic potential of that lesion, especially in an adult. Previously diagnosed benign melanocytic nevi in individuals from six different age groups were re‐examined; mitotic figures were counted in routine hematoxylin and eosin‐stained sections; Ki‐67 nuclear positivity was assessed by immunohistochemistry. Mitoses were seen in 0–14.3% of nevi in all groups of patients >1 year of age; 55.6% (5/9 cases) of nevi in patients <1‐year old had mitoses identified histologically. Ki‐67‐positive melanocytes were seen in all cases of those lesions in infants (less than 1‐year old) and only in a minority of lesions from the other age groups. The maximum and mean numbers of Ki‐67‐positive melanocytes per square millimeter were highest in patients <1‐year old (16.7 and 5.6, respectively), and decreased in all other groups. Follow‐up data were available in the majority of the patients. There were no examples of malignant melanoma in the various age groups. We conclude that proliferative activity in benign melanocytic nevi decreases with age, however, proliferative activity can be seen at any age and its significance must be judged in the context of other histopathologic features.     

Lack of BRAF(V600E) mutations in giant congenital melanocytic nevi in a Chinese population

Giant congenital melanocytic nevi (CMNs) are at an increased risk for malignant transformation. To explore the mutation frequencies of BRAF(V600E) (V-raf murine sarcoma virus oncogene homolog B1) and NRAS (neuroblastoma ras viral oncogene homolog) codon 61 in CMNs of Chinese, we selected 55 paraffin-embedded tissue blocks, including 37 cases of medium CMNs (1.5-20cm) and 18 cases of giant CMNs (>20 cm). Direct sequencing was performed to detect the BRAF(V600E) and NRAS codon 61 mutations. The BRAF(V600E) mutations were detected in 9 of 55 nevi (16.4%). In medium CMNs, 9 of 37 BRAF(V600E) mutations (24.3%) were detected. Notably, in giant CMNs, no BRAF(V600E) mutations were found. The difference between these frequencies is statistically significant (P = 0.0231). NRAS codon 61 mutations were detected in 13 of 55 nevi (23.6%), including 10 of 37 medium CMNs (27.0%) and 3 of 18 giant CMNs (16.7%). Additionally, the BRAF(V600E) and NRAS codon 61 mutations did not coexist in the same sample. Finally, we found that the NRAS codon 61 mutation was significantly related to the amount of sun exposure (0 of 18 CMNs from sites of intermittent sun exposure and 13 of 36 CMNs from sites of chronic continuous sun exposure, P = 0.0024). The paradoxically higher incidence of BRAF(V600E) mutations in medium-sized compared with giant CMNs suggests that the presence of the BRAF(V600E) mutation may play different roles between medium and giant CMNs in melanocytic tumorigenesis.     

Installation of a network for patients with congenital melanocytic nevi in German‐speaking countries

In 2005, an Internet‐based network for the support of patients with congenital melanocytic nevi in German‐speaking countries was started ( http://www.naevus‐netzwerk.de ). Along with detailed information for patients and parents, the home‐page includes a nevus registry which is based on an electronic questionnaire and which aims at providing data on the long‐term course of nevi estimated to reach > 10 cm in largest diameter. In the past, congenital melanocytic nevi have been subject to various mythological interpretations (“Tierfellnävus”, lit.”animal coat nevus”;”Muttermal”). Today an increasing body of reliable scientific data allows a differentiated reflection of the risk of malignant transformation and has led to progress in the diagnostic and therapeutic management. Recent findings from the literature and considerations from scientific meetings are reviewed.