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。     

儿童阴茎分裂痣8例临床病理特征分析

目的 探讨儿童阴茎分裂痣的临床表现、皮肤镜及病理学特征。方法 回顾2018年6月—2022年10月首都儿科研究所附属儿童医院皮肤科诊治的8例阴茎分裂痣患儿的临床表现、皮肤镜、组织病理学特征以及治疗方法。结果 8例阴茎分裂痣患儿临床表现为龟头背侧及包皮内板可见境界清楚的黑褐色斑，两处皮损与冠状沟距离大致相等，呈镜像对称分布。龟头处皮损平均最大直径为5.7 mm(4～10 mm);包皮内板处皮损平均最大直径为6.6 mm(4～8 mm)。皮肤镜特征主要表现为球状模式，皮损内可以看到大小不等的色素小球。其中，包皮内板还可以看到球-均质模式及球-网状模式。组织病理检查(龟头部)示：表皮大致正常，真-表皮交界部位可见较多痣细胞巢，细胞排列规则，细胞内可见较多黑色素颗粒，病理类型均属于交界痣。而包皮部位皮损病理类型均为复合痣。8例患儿均在局麻下进行手术，完整切除皮损，术后随访均无复发。结论 阴茎分裂痣具有独特的临床表现和皮肤镜特征；冠状沟两侧的皮损具有不同的组织病理学特征；该部位皮损容易摩擦刺激，而且部位隐匿，应引起重视，建议早期手术切除。     

BRAF V600E突变蛋白在皮肤黑素瘤中的表达

目的 探讨皮肤黑素瘤BRAF V600E突变蛋白表达情况,分析免疫组化法检测V600E突变的灵敏度和特异度。 方法 应用抗BRAF V600E单克隆抗体的免疫组化法检测103例皮肤黑素瘤、40例色素痣石蜡包埋组织切片中BRAF V600E突变蛋白表达水平。采用SPSS 17.0统计软件进行统计分析,率的比较采用χ2检验。 结果 BRAF V600E突变蛋白阳性表达率在皮肤黑素瘤中为20.4%（21/103）,色素痣中为5.0%（2/40）,两组差异有统计学意义（χ2 = 5.06,P < 0.05）。黑素瘤BRAF V600E突变蛋白的表达率在不同年龄组［＜ 60岁组表达率为29.8%（14/47）, ≥ 60岁组为12.5%（7/56）］、不同民族［维吾尔族组为30.2%（13/43）,汉族组为13.3%（8/60）］、不同发病部位［肢端为13.6%（6/42）、黏膜为11.8%（4/29）、非肢端为45.8%（11/32）］、不同Clark分级［Ⅰ ~ Ⅲ级组为8.6%（4/42）,Ⅳ ~ Ⅴ级组为12.4%（17/61）］组间表达差异均有统计学意义（P < 0.05）,而在不同性别、有无淋巴结转移组间表达差异均无统计学意义（P ＞ 0.05）。免疫组化检测恶性黑素瘤中BRAF V600E突变灵敏度为100%（15/15）,特异度为98.5%（65/66）。 结论 BRAF V600E突变蛋白在皮肤黑素瘤中高表达,在维吾尔族人群表达率高于汉族人群;免疫组化法检测BRAF V600E突变具有准确、快速等特点。     

新疆80例恶性黑素瘤BRAF基因突变分析

目的 探讨BRAF基因突变与恶性黑素瘤临床表现的关系。方法 PCR及DNA直接测序法对新疆80例恶性黑素瘤及30例正常皮肤石蜡包埋组织BRAF基因11、15外显子进行检测。结果 80例恶性黑素瘤19例发生BRAF基因突变,突变率为23.8%（19/80）;有17例突变发生于15外显子,突变率为89.5% （17/19）,其中V600E突变占BRAF基因15外显子突变的88.2% （15/17）;2例突变位于11外显子,突变率10.5%（2/19）;30例正常皮肤组织均未发现BRAF基因突变。患者平均发病年龄为57.5岁,年龄在60岁以下患者BRAF基因突变率显著高于60岁以上（χ2 = 6.613,P < 0.05）。黏膜、肢端、非肢端突变率分别为：18.2%（4/21）,14.7%（5/34）,41.7%（10/24）,差异具有统计学意义（χ2 = 6.167,P < 0.05）。BRAF基因突变与恶性黑素瘤患者性别、民族、有无淋巴结转移无明显相关性（P > 0.05）。结论 BRAF基因仍为新疆地区恶性黑素瘤热点突变基因,且以该基因15外显子V600E突变为主。BRAF基因突变与恶性黑素瘤患者发病年龄、发病部位密切相关,而与民族、性别、有无淋巴结转移无相关性。     

Ki67启动子调控的肿瘤增殖腺病毒对黑素瘤细胞的杀伤作用

目的：研究Ki67启动子调控增殖腺病毒Ki67-ZD55对黑素瘤A375细胞的杀伤作用。方法：将PBS、ZD55-EGFP和Ki67-ZD55分别作用于A375细胞。FIT—PCR法检测Ki67-promotermR—NA的表达;荧光显微镜观察Ki67-ZD55增殖过程;MTr法检测细胞存活率;WesternBlot法检测E1A蛋白及线粒体途径凋亡相关蛋白BAX、Bcl—XL、MCL-1和Caspase-3的表达。结果：RT—PCR检测结果显示Ki67-ZD55能够在A375细胞中表达Ki67-promoter序列;荧光显微镜观察结果显示Ki67-ZD55增殖明显;MTT结果表明Ki67-ZD55组对A375细胞的增殖具有明显抑制作用,抑制效果大于ZD55-EGFP组（P〈0．05）;WesternBlot法检测结果表明E1A、BAX的表达量增加,Bcl—XL、MCL-1的表达量均降低,Procaspase-3发生了明显剪切。结论：Ki67-ZD55能够明显抑制A375细胞增殖,促进A375细胞凋亡。     

痣样黑素瘤三例临床及组织病理学分析

【摘要】 目的 探讨痣样黑素瘤的临床和组织病理学特征。方法 回顾性分析2000—2020年西京皮肤医院诊断的3例痣样黑素瘤患者的临床和组织病理资料。结果 3例痣样黑素瘤患者中,女2例,男1例,皮损初始表现为黑斑、丘疹。2例在手术切除后皮疹复发增大成斑块或新发结节样皮损。组织病理学检查：表皮及真皮内上皮样黑素细胞增生,细胞有异型性,部分细胞核深染。免疫组化结果显示,皮损内瘤细胞Melan-A、S100表达阳性;HMB45在真皮瘤细胞内弥漫阳性,局部阴性;Ki67增殖指数升高,细胞周期蛋白D1表达活跃。结论 痣样黑素瘤易误诊为色素痣或脂溢性角化病;对于组织学诊断为色素痣,但临床出现复发或者转移的患者,需高度警惕痣样黑素瘤的可能。     

幼年黄色肉芽肿伴发多发性咖啡斑5例临床及遗传学分析

目的 探讨幼年黄色肉芽肿伴发多发性咖啡斑的临床表现与基因突变情况。方法 对5例幼年黄色肉芽肿伴发多发性咖啡斑患儿的临床资料与基因检测结果进行回顾性分析并复习相关文献。结果 5例患儿幼年黄色肉芽肿发病中位年龄为8个月，最小1个月，最大10岁，其中4例小于1岁。男女比例为2∶3。4例幼年黄色肉芽肿皮损多发，头面部多见；1例累及眼部。随访1～3年后幼年黄色肉芽肿大部分自行缓解。5例患儿多发性咖啡斑平均发病年龄为2.4个月，皮损分布全身，随访1～3年5例均出现腋窝和/或腹股沟雀斑。全身体检可见2例眼距宽，1例先天性斜视。5例患儿基因检测均可见NF1基因突变，1例来源于母亲，4例为自发突变。2例移码突变，1例剪接，1例无义，1例错义。5例均为致病性突变。结论 幼年黄色肉芽肿伴发多发性咖啡斑对于1型神经纤维瘤病(neurofibromatosis type1,NF1)诊断有预测价值，5例患儿均有NF1基因突变，突变类型多样。     

起源于巨大先天性痣细胞痣的增生性结节1例及相关文献复习

目的:探讨起源于巨大先天性痣细胞痣的增生性结节的组织病理特点及其与先天性痣细胞痣恶变的鉴别诊断。方法:对1例起源于婴儿巨大先天性痣细胞痣的增生性结节的临床表现、组织形态学、免疫组化及荧光原位杂交(FISH)特点进行分析并复习相关文献。结果:患儿男,4个月。自出生起全身多处皮肤可见大片黑斑;出生后3个月背部皮肤斑块上散在多个黑色疱状小结节,逐渐增大,个别结节出现破溃。皮损组织病理检查示皮内先天性痣细胞痣背景中可见形态较一致的尤因氏肉瘤细胞样细胞局灶结节状增生,核质比高,核分裂象2~4/mm~2,有坏死。免疫组化示结节状增生细胞D型细胞周期蛋白(cyclinD)1(50%+)、增殖核抗原(Ki-67)(5%~10%+)、HMB-45(灶状+)、melan-A(灶状+)及S-100蛋白(弥漫+)。FISH显示增生细胞具有分布于多条染色体的多个基因位点(包括6q23、6p25、6p11.1-q11.1、9p21、9q21、11q13、11p11.1-q11.1、17q12、17p11.1-q11.1)拷贝数增多。结论:起源于巨大先天性痣细胞痣的增生性结节常表现为细胞形态幼稚和增生活跃,Ki-67和FISH检测有助于其与痣恶变的鉴别。     

儿童局灶性真皮发育不良2例及PORCN基因突变分析

目的:研究2例儿童局灶性真皮发育不良的临床及基因学特点。方法:总结局灶性真皮发育不良患者的临床资料、组织病理学特点并进行基因突变研究。结果:两例患者均为女性患儿,出生即发病。皮损均表现为全身皮肤局灶性萎缩变薄,口唇、肛周等部位大量乳头瘤样增生,呈黄红色、红色,沿Blaschko线分布色素沉着、色素减退。手足并指(趾)、缺指(趾)畸形,严重者右手"龙虾爪样"畸形。组织病理均提示真皮脂肪组织上移,附属器、胶原纤维减少。基因筛查1例存在PORCN基因c.1186CT突变,导致p.R396X替代(第396位精氨酸被终止密码子取代)。另1例存在PORCN基因c.808-811 del GGGG突变,造成移码突变,该突变为新发现突变。结论:局灶性真皮发育不良的诊断主要依据典型的临床表现和皮损组织病理表现,2例患儿均存在PORCN基因突变。     

兄弟同患Blau综合征基因突变分析

目的:探讨兄弟同患Balu综合征的2例患儿CARD15基因突变情况。方法:收集一对同患Blau综合征的同胞兄弟的临床资料,提取患儿及其血缘父母外周血DNA,应用聚合酶链式反应(PCR)技术对样品DNA进行扩增,采用基因芯片捕获目标基因CARD15,采用高通量测序仪进行测序。将测序结果与数据库(NCBI dbSNP,Hap Map,1000 human denome dataset和database of 100 Chinese healthy adults)进行比对,并对找出的可疑突变进行注释及筛选。采用Sanger法验证发现的基因突变。结果:患儿皮损及皮损组织病理表现均符合结节病,均不伴有关节损害及眼部损害。CARD15基因突变分析提示兄弟俩CARD15基因均存在c.1000CT(p.R334W)杂合致病突变(p.R334W),突变遗传自患儿父亲。该突变位点在既往研究中的其他Blau综合征患者中曾被报道过。结论:临床上皮损表现为鱼鳞病、毛发红糠疹及湿疹样皮炎的患儿要考虑Blau综合征可能,基因诊断是明确Blau综合征病因的有效手段。     

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.     

先天性色素痣的诊疗进展

先天性色素痣又称为先天性黑素细胞痣,在出生时或出生后不久出现,并具有独特的临床和组织病理学特征。先天性色素痣根据尺寸可分为四类:小痣、中痣、大痣和巨痣。约1%的新生儿患有先天性色素痣,其中大多数属于小/中型痣,大/巨痣比较罕见。本文将从临床、并发症、病理、基因、治疗等多方面对先天性色素痣进行总结,以期提高临床诊疗水平。     

Histogenesis of congenital and acquired melanocytic nevi based on histological study of lesion size and thickness

The histogenesis of melanocytic nevi is poorly understood. It is important to determine the differences and similarities in histogenesis between congenital and acquired nevi. To clarify the histogenic differences between acquired melanocytic nevi (AMN) and congenital melanocytic nevi (CMN), diameter and depth of nevus cells (tumor thickness) were examined in histological specimens from 80 cases of CMN and 71 cases of AMN, and these nevi were classified according to Mark's pathological CMN criteria. In all cases, giant CMN nevus cells were found in the lower marginal portion of excised specimens. The mean diameter and lesional thickness were significantly higher in CMN than in AMN. AMN diameter showed a significant correlation (r = 0.567, P < 0.05) with lesional thickness, while no such relation was observed in CMN. In addition, a significant correlation between lesion diameter and thickness was observed in small (<10 mm) non-Mark's type CMN (r = 0.626, P < 0.05). CMN may be classified into three subtypes: (i) caused by increased proliferation of melanoblasts during the course of migration from the neural crest to the epidermis; (ii) proliferation of nevus cells after arrival at the epidermis, and nevus cell distribution affected by adnexa and dermal differentiation; and (iii) arising after completion of skin development before birth.     

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.     

Dynamic evolution of a scalp congenital melanocytic nevus with poliosis and cutis verticis gyrata

Cutis verticis gyrata (CVG), characterized by cerebriform overgrowth of the scalp, is rarely observed in congenital melanocytic nevi (CMN). We describe a 13-year-old male with autism and a large CMN of the scalp with numerous satellite nevi whose scalp nevus exhibited evolution with poliosis and CVG. Given the potential association of CVG (independent of CMN) with seizures, neuropsychiatric, and ophthalmologic disorders, and nevus-associated CVG (cerebriform intradermal nevus) with melanoma, multidisciplinary evaluation of CMN patients with CVG is important to guide management and treatment.     

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.     

In vivo expression of the insulin-like growth factor-I (IGF-I) receptor in congenital pigmented nevi

Growth of normal melanocytes, nevus cells and primary melanoma cells is enhanced by insulin/insulin-like growth factor-I (IGF-I) in vitro. It has been shown that a melanoma cell line possesses the IGF-I receptor which plays a role in activation of the chemotactic response. Little is known about the in vivo expression of the IGF-I receptor and its role in melanocytic lesions. In an immunohistochemical study, we investigated the expression of IGF-I receptor in frozen sections of congenital pigmented nevi from 10 patients (ages 8 months to 4 yrs) using the monoclonal antibody αIR₃, which specifically recognizes the extracellular alpha subunit of the IGF-I receptor. The proliferative activity of the nevus cells was examined by staining with Ki67 monoclonal antibody (reactive with all actively cycling cells). IGF-I receptor was found to be widely expressed by the cell surface of the nevus cells. Membrane staining was occasionally stronger in the superficial portion of the congenital pigmented nevi. In contrast, Ki67-positive cells were only sparsely scattered throughout the nevi with some tendency to localization to the superficial portion.
This study indicates that in vivo the IGF-I receptor is widely expressed by congenital pigmented nevus cells. As opposed to keratinocytes, in which IGF-I receptor expression defines the proliferation pool of the normal and disordered epidermis, the IGF-I receptor is expressed by all nevus cells, irrespective of their proliferative status.
Further studies are needed to assess whether the IGF-I receptor expression can serve as a marker for increased risk for development of malignancy in various types of benign melanocytic lesions.     

The relationship between melanocytes and peripheral nerve sheath cells (Part I): melanocytic nevus (excluding so-called "blue nevus") with peripheral nerve sheath differentiation

Among thousands of specimens of melanocytic nevi, not including giant congenital melanocytic nevus or blue nevus, 42 melanocytic nevi that showed peripheral nerve sheath differentiation were collected. The patterns of melanocytic nevi with peripheral nerve sheath differentiation may be classified into three groups: 1) "neurotized and neural nevi" with nests of "neuroid cords" and "nevic corpuscles" (the most common pattern); 2) nerve fascicle-like structures with no relation to neurotized and neural nevi; and 3) palisading melanocytes of a nevus in nests of conventional melanocytic nevi (a rare pattern). Each pattern may represent a different expression of nerve sheath differentiation in melanocytic nevi. Some melanocytic nevi with nerve fascicle-like structures show discrete structures closely resembling authentic nerve fascicles, confirming a close relationship between melanocytes and peripheral nerve sheath cells (Schwann cells and probably perineurial cells in part) and suggesting derivation of the two types of cells from common precursor cells of the neural crest and their de novo development in the dermis rather than by Abtropfung of melanocytes from the epidermis. In addition, the high prevalence of Unna, Miescher, and superficial congenital nevi in melanocytic nevi with peripheral nerve sheath differentiation suggests a different character or process for these congenital melanocytic nevi than for Clark and Spitz nevi (junctional and compound types).     

先天性色素痣伴增生性结节10例临床病理分析

【摘要】 目的 探讨先天性色素痣伴增生性结节的临床特点及组织病理特征。方法 收集第四军医大学西京皮肤医院2015—2019年经临床和病理确诊的10例先天性色素痣伴增生性结节患者的临床及病理资料，并进行回顾性分析。结果 10例患者年龄2～45岁（平均15岁），9例增生性结节发生于婴儿，1例发生于成人。皮损位于四肢4例，头面部3例，躯干2例，躯干及四肢同时受累1例。临床表现为黑色斑片或斑块中出现1个或多个结节，6例增生性结节为多发，4例为单发，单个结节直径0.2～1.5 cm，仅1例出现溃疡。组织病理检查显示增生性结节内黑素细胞均存在成熟现象，核分裂象少，细胞无明显异型性，无坏死现象，免疫组化检查显示痣细胞弥漫表达Melan-A，不表达或仅部分表达HMB45，Ki67增殖指数 < 5%。结论 先天性色素痣伴增生性结节可发生于四肢、头面部及躯干；临床表现为原先天性色素痣皮损上的单发或多发结节；病理上增生性结节内黑素细胞可见成熟现象，免疫组化HMB45及Ki67染色有助于诊断，其预后有待长期随访。