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。     

BRAF、NRAS癌基因与恶性黑素瘤的研究

近年来,世界范围内黑素瘤的发生率正逐渐升高,发病年龄也愈来愈早,其死亡率也随之增加.环境因素和遗传易感因素是恶性黑素瘤发病的主要机制.遗传易感性的研究发现,与散发性恶性黑素瘤发病密切相关的两个遗传易感基因是位于RAS-RAF-MEK-ERK途径的BRAF、NRAS基因.其中BRAF基因是黑素瘤中突变率最高的基因,也是目前黑素瘤特异性靶向治疗研究的热点基因.概述BRAF、NRAS基因突变在恶性黑素瘤形成、发生、发展中所起的重要作用,并评述BRAF基因突变在黑素瘤临床诊治上的应用价值.     

肢端型黑素瘤NRAS基因突变检测及预后分析

目的 分析肢端黑素瘤临床及病理特点,检测肢端型黑素瘤NRAS基因突变情况,探讨NRAS基因突变与疾病预后的关系。方法 收集经病理确诊的55例肢端型黑素瘤患者的临床及病理资料,提取其石蜡包埋组织及15例色素痣石蜡包埋组织DNA,采用PCR及DNA直接测序法检测NRAS基因突变。采用Cox比例风险回归模型进行单因素和多因素分析。结果 55例肢端型黑素瘤患者中,6例（10.9%）发生NRAS突变,突变位于61密码子,以Q61R为主。NRAS基因1、2外显子及15例色素痣组织未见上述突变。6例NRAS突变患者中,4例发生淋巴结转移。多因素Cox回归模型分析中,临床分期晚（RR = 2.54,95% CI：1.062 ~ 6.066）、未手术切除（RR = 2.98,95% CI：1.316 ~ 3.525）、存在NRAS突变（RR = 2.73,95% CI：0.932 ~ 3.257）为预后不良的独立影响因素（均P < 0.05）。结论 肢端型黑素瘤患者NRAS突变可能与淋巴结转移相关,临床分期、治疗方法、NRAS突变与预后有关。NRAS突变可能为肢端型黑素瘤提供一个新的预后评估指标。     

活性诱导性胞嘧啶脱氨基酶表达与黑素瘤侵袭转移、预后的相关性分析

目的 探讨活性诱导性胞嘧啶脱氨基酶（AID）与黑素瘤侵袭转移、预后的关系和临床意义。方法 免疫组化SP法检测AID蛋白在80例黑素瘤、23例色素痣石蜡包埋组织切片中的表达,结合临床病理生物特性进行分析。 结果 黑素瘤AID蛋白的阳性表达率53.75%（43/80）,色素痣的表达率13.04%（3/23）,差异有统计学意义（P < 0.05）。AID蛋白的表达与黑素瘤淋巴结转移、Clark分级、浸润深度及预后密切相关（P < 0.05）,在年龄、性别、民族之间差异无统计学意义（均P ＞ 0.05）。19例发生BRAF突变黑素瘤组织中,AID蛋白17例阳性表达,其中15例BRAFV600E突变的黑素瘤AID蛋白均阳性表达。 结论 AID蛋白可能诱导了黑素瘤BRAF突变,并参与黑素瘤的侵袭、转移,与预后相关。     

恶性黑素瘤中BRAF癌基因突变研究新进展

恶性黑素瘤是死亡率较高的皮肤肿瘤之一,研究发现恶性黑素瘤中存在高频BRAF癌基因突变.紫外线是引起突变的高危因素,不同种族、发病部位以及病理类型的恶性黑素瘤间BRAF突变率存在差异.此突变与遗传、预先存在的色素痣、肿瘤侵袭性及预后之间的关系还存在争论.BRAF突变相关的MAPK信号传导分子是治疗恶性黑素瘤的靶点.综述近几年的研究,以认识BRAF突变与恶性黑素瘤的关系.     

31例维吾尔族黏膜黑素瘤临床与c-kit基因突变研究

目的 分析维吾尔族黏膜黑素瘤临床特点,检测c-kit基因突变,探讨与黏膜黑素瘤临床特征之间的关系。 方法 收集经病理确诊的31例维吾尔族黏膜黑素瘤患者的临床资料,采用PCR及DNA直接测序法进行c-kit基因突变检测。 结果 维吾尔族黏膜黑素瘤男女性别比为1 ∶ 1.2,平均年龄61.35岁。60 ~ 70岁为高发年龄段,51 ~ 59岁为次高发年龄段。头颈部为最常见的发病部位,其中以鼻腔黏膜居多;其次为泌尿生殖道和直肠黏膜。31例黏膜黑素瘤有4例发生c-kit基因突变（12.9%,4/31）,突变均位于11外显子,以L576P突变为主。4例突变中,3例发生于直肠黏膜,1例发生于尿道黏膜。直肠黏膜与其他黏膜部位c-kit基因突变率分别为3/7、4.17%（1/24）。发生淋巴结转移患者c-kit基因突变率高于无淋巴结转移者（P = 0.043）。c-kit基因突变与性别、年龄无相关性（P > 0.05）。 结论 维吾尔族黏膜黑素瘤好发于老年人,发病部位以头颈部黏膜为主。c-kit基因突变与黏膜黑素瘤发生部位、有无淋巴结转移密切相关。     

人恶性黑素瘤细胞株A375中色素上皮衍生因子基因的突变

目的 探讨色素上皮衍生因子（PEDF）基因在恶性黑素瘤细胞系中的突变情况。方法 采用聚合酶链反应--单链构象多态性分析（PCR-SSCP）对人恶性黑素瘤细胞株A375和正常人黑素细胞中PEDF基因的所有8个外显子进行突变检测。对SSCP分析有异常泳动条带样本的PCR产物进行DNA 测序。结果 人恶性黑素瘤细胞株A375的第3到第7外显子都出现DNA泳动变位,其中以外显子5,6最为明显。PEDF基因第5,6外显子均存在突变。第5外显子的突变类型以单个碱基的缺失为主,第6外显子的突变类型则以单个碱基的置换为主。结论 PEDF基因的突变可能在恶性黑素瘤的发病中起一定作用。     

皮肤恶性肢端黑素瘤中c-kit基因的表达及突变分析北大核心CSCD

目的 分析皮肤恶性肢端黑素瘤患者中c-kit基因突变的频率与类型及表达情况.方法 应用PCR、基因测序、免疫组化染色等方法检测115例皮肤恶性黑素瘤（CMM）患者、30例肢端黑素细胞痣患者及15例健康人皮肤组织标本中c-kit基因序列及c-kit蛋白表达情况.采用x2检验、Mann-WhitneyU检验、Spearman相关检验等统计学方法分析数据.结果 93例肢端CMM中84例（90.3％）表达c-kit蛋白,22例非肢端CMM中18例（81.8％）表达.58例肢端侵袭性CMM中,29例真表皮交界处肿瘤细胞c-kit蛋白表达强阳性,而8例真皮内呈侵袭浸润性生长的肿瘤细胞则不表达或弱表达c-kit蛋白;健康人皮肤组织中c-kit蛋白表达较弱;在30例肢端黑素细胞痣中19例（63.3％）阳性表达.c-kit蛋白在93例肢端CMM组的阳性表达率（90.3％）显著高于肢端黑素细胞痣组（63.3％）,两组差异有统计学意义（x2=12.14,P＜ 0.05）;71例侵袭性CMM中,c-kit蛋白在58例肢端CMM中阳性表达55例（94.8％）,在13例非肢端CMM中阳性表达11例,在肢端CMM的表达高于非肢端CMM,两组差异有统计学意义（x2=4.18,P＜ 0.05）.在原位、侵袭性、转移性肢端CMM组织中,c-kit蛋白表达水平与肿瘤的临床病理特征均未见明显相关（均P＞0.05）.115例CMM中检测到4例患者存在c-kit基因点突变,其中3例为L576P突变,1例为K642E突变,且均为肢端CMM,免疫组化显示弥漫性c-kit蛋白强阳性表达.结论 在侵袭性CMM中,c-kit蛋白在肢端CMM中的表达高于非肢端CMM.肢端CMM中检测到c-kit基因点突变,均为肢端型较常见的突变类型,但突变率较国外文献低.     

变性梯度凝胶电泳和单链构象多态性分析检测皮肤癌p16、p53基因突变

目的：探讨p16、p53基因突变在皮肤癌发生中的作用并比较变性梯度凝胶电泳(DGGE)和单链构象多态性分析(SSCP)检测基因突变的敏感性。方法：分别采用聚合酶链反应(PCR)-DEEG和PCR—SSCP，对40例皮肤癌患者手术切除组织的p16基因1、2外显子和p53基因5～8外显子进行突变检测。结果：仅2例鳞状细胞癌(SCC)出现p16基因外显子2突变，皮肤癌p16基因1、2外显子的突变率为5％。p53基因5—8外显子的突变率为35％，其中SCC的突变率为36％，基底细胞癌(BCC)为33％，采用DGGE检测的突变率为33％，SSCP检测的突变率为25％。结论：p16、p53基因突变参与皮肤癌的发病机制；p16基因的突变率低于p53基因；DGGE检测基因突变的敏感性高于SSCP；DGGE结合SSCP有助于提高突变检出率。     

BRAF基因突变与皮肤恶性黑素瘤

BRAF基因,位于染色体7q34编码一个67 000～99 000的丝,苏氨酸蛋白激酶,其产物在丝裂原活化蛋白激酶信号途径中起重要调控作用.国内外的研究者先后发现并揭示出BRAF基因变异与皮肤恶性黑素瘤的关系.随着探索的深入,部分学者以BRAF基因作为治疗靶向进行研究,并初步取得成果.对BRAF基因及其突变的研究可能在未来恶性黑素瘤诊断、治疗以及预后等方面产生临床价值,为恶性黑素瘤的治疗开辟新的方向.     

皮肤黑素瘤94例临床病理特点与基因突变的关系

目的:探讨皮肤黑素瘤（CMM）临床病理特点及与易感基因突变的关系。方法:回顾分析新疆维吾尔自治区人民医院2009年1月至2019年12月确诊的94例CMM临床及组织病理学特征。48例留存黑素瘤石蜡组织标本,采用Sanger测序法检测黑素瘤组织中BRAF、NRAS、c-KIT基因及人端粒酶逆转录酶（hTERT）基因启动子...     

Prevalence of exon 15 BRAF mutations in primary melanoma of the superficial spreading, nodular, acral, and lentigo maligna subtypes

DNA was extracted from 52 thick primary melanomas and mutations sought in exon 15 of the BRAF (v-raf murine sarcoma viral oncogene homolog B1) gene using denaturing high performance liquid chromatograph (dHPLC) fragment analysis, sequencing, and allele-specific PCR. Exon 15 BRAF mutations were found in 13 of 52 (25%) primary melanomas. These comprised five of 17 (29%) superficial spreading melanomas, three of 11 (27%) nodular melanomas, two of 13 (15%) acral lentiginous melanomas, one of one (100%) mucosal melanoma and two of 10 (20%) lentigo maligna melanomas. In common with other groups, our findings show a relative concentration of the exon 15 BRAF mutation in superficial spreading and nodular melanomas, but add further evidence that this mutation not necessary for malignant transformation of the melanocyte.     

Tandem BRAF mutations in primary invasive melanomas

The RAS/RAF/MAPK pathway likely mediates critical cell proliferation and survival signals in melanoma. BRAF mutations have been found in a high percentage of melanoma cell lines and metastases; however, only a few studies with a limited number of specimens have focused on primary melanomas. We examined BRAF exon 15 mutational status in 37 primary invasive melanomas of varying thicknesses, which had undergone a standardized pathology review. BRAF mutational status was determined using direct manual sequencing of PCR products, followed by resequencing separately amplified DNA aliquots to confirm each mutation. BRAF exon 15 mutations were found in 17 of 37 (46%) primary melanomas. Tumor-specific tandem mutations, encoding either V599K, V599R, or V599E, were found in 5 of 17 (29%) melanomas with BRAF exon 15 mutations. Cloning of BRAF double base-pair substitutions confirmed that both base changes were on the same allele and can result in a positive charge at codon 599. BRAF mutations, including tandem mutations, were frequently found in both thin and thick primary melanomas, implying that these mutations can occur early in the progression of melanoma. The finding of tandem mutations in thin melanomas makes it more likely that they arise as a simultaneous rather than sequential event.     

Mutations of the BRAF gene in benign and malignant melanocytic lesions

A single-point mutation in exon 15 of the BRAF gene has recently been reported in a high percentage in cultured melanoma cells and in 6 of 9 primary melanomas examined. To evaluate the impact of the T1796A BRAF mutation, we screened primary melanomas, various types of nevi and lesions where a melanoma developed in an underlying nevus. We could detect the mutation in 28 of 97 (29%) melanomas and in 39 of 187 (21%) nevi, including blue nevi (0/20) and Spitz nevi (0/69), which did not carry the mutation. In melanomas with an underlying nevus, either the mutation was present in both the laser-microdissected nevus cells and the laser-microdissected melanoma cells (3/14) or both lesions were negative for the BRAF mutation except one case. In conclusion, mutations in exon 15 of the BRAF gene are nonspecific for progression of a nevus to a melanoma. Other so far unknown cofactors seem to be of importance.     

BRAF point mutations in primary melanoma show different prevalences by subtype

To elucidate the biological significance of activating mutations of BRAF in human malignant tumors, we performed a mutation analysis using 43 cell lines established from tumors that had developed in several kinds of human organs. Because the same V599E point mutation was observed in three of six melanoma cell lines and no such mutations were observed in other types of cancers, we focused further on melanoma, performed mutation analyses of NRAS, KRAS, CTNNB1, and p16/p14(ARF) in these cell lines, and found one NRAS mutation and three p16/p14(ARF) mutations. We further searched for mutations of BRAF and NRAS in 35 primary sporadic melanomas from 35 Japanese patients and detected the V599E BRAF point mutation in only nine (26%) of them. Significant differences in mutation frequency were observed among four histological subtypes; four (50%) of eight superficially spreading melanoma and five (33%) of 15 acral lentiginous melanoma had the mutation, whereas none of 12 other types (six nodular melanoma, five lentigo melanoma, and one mucosal melanoma) had it. The BRAF mutation was observed frequently even in small lesions, indicating that activation of this gene may be one of the early events in the pathogenesis of some melanomas.     

BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin

BRAF mutations have recently been detected with a high frequency (66%) in cutaneous melanoma. All those mutations are activating, with a single substitution (T1796A) at codon 599 (V599E) accounting for over 90%. To investigate the stage in which those mutations occur in the currently proposed sequential malignant transformation of melanocytes, 22 benign melanocytic nevi, 23 melanocytic atypical nevi, and 25 primary cutaneous melanoma from 63 different patients were examined for BRAF mutations using DNA extracted from microdissected formalin-fixed and paraffin-embedded tissues, and a two-round PCR-RFLP-based strategy. A subset of samples was sequenced for mutation confirmation. Sixteen benign (73%) and eleven atypical (52%) melanocytic nevi, and thirteen melanoma (56%) demonstrated BRAF mutations at codon 599, and no statistically significant differences were detected among all three types of lesions. No mutations were demonstrated in microdissected epidermal keratinocytes adjacent to melanocytic lesions having BRAF mutations. No correlation was detected between BRAF mutational status and age, sun exposure, and Clark's level in malignant melanoma. However, comparing only atypical nevi and melanoma lesions the frequency of BRAF mutation is significantly greater in male (78%) than female (35%) patients (P = 0.0194). The previously described T1796A point mutation was detected in 17 of 18 mutated samples, and a novel mutation consisting of a substitution of valine for lysine (GT1795-96AA) was detected in one melanoma case. Our findings of a high frequency of BRAF mutations at codon 599 in benign melanocytic lesions of the skin indicate that this mutation is not sufficient by itself for malignant transformation.