Prevalence of BRAF V600E mutation in Chinese melanoma patients: large scale analysis of BRAF and NRAS mutations in a 432-case cohort

Background

Mutations of NRAS and BRAF have been described in Caucasian melanomas. However, the status and the clinical significance of BRAF and NRAS mutations in the Asian population have not been investigated on a large scale.

Methods

Melanoma samples (n = 432) were analysed for mutations in exons 11 and 15 of the BRAF gene, and exons 1 and 2 of the NRAS gene in genomic DNA by polymerase chain reaction (PCR) amplification and Sanger sequencing. Mutations of BRAF and NRAS genes were correlated to clinicopathologic features and prognosis of the patients.

Results

The incidence of somatic mutations within the BRAF and NRAS genes was 25.5% (110/432) and 7.2% (31/432), respectively. Among the 110 patients with BRAF mutations, 98 patients (89.1%) had V600E mutations. Melanomas without chronic sun-induced damage (Non-CSD) were more likely (P < 0.01) to show BRAF mutations while NRAS mutation frequency was unbiased between melanoma subtypes. Patients with genetic mutations in BRAF (P < 0.01) or NRAS (P = 0.04) gene are more likely to have ulceration as compared to patients without BRAF or NRAS mutations, respectively. Both BRAF (P = 0.003) and NRAS mutations (P = 0.031) are inversely correlated to overall survival.

Conclusions

BRAF mutation is frequent while mutations in NRAS gene are rare. The most prevalent BRAF mutation type is V600E. Patients with mutations in BRAF or NRAS gene are frequently present with ulceration, and mutation in BRAF or NRAS gene is indicator for poor prognosis. Our study may warrant a clinical trial of kinase inhibitors targeting BRAF V600E in Chinese and Asian melanoma patients.     

BRAF基因V600E突变在甲状腺病变中的表达

目的 分析BRAF基因V600E突变在中国汉族人群甲状腺病变中的表达情况.方法 240例甲状腺病变中,乳头状癌(PTC)129例(其中经典型PTC 121例,滤泡型PTC 8例).滤泡癌12例,髓样癌4例,甲状腺隙瘤30例,结节性甲状腺肿30例,Lp状腺乳头状增生35例.采用常规酚.氯仿法抽提87例新鲜组织样本的总DNA,改良试剂盒法抽提153例石蜡组织样本的总DNA,经过PCR、测序,检测BRAF V600E突变.结果 在240例甲状腺病变中,BRAF V6OOE突变61例,发生率为25.4%.61例BRAF V600E突,变患者均为PTC,占PTC的47.3%.BRAF V600E突变在经典型PTC中的表达率(49.6%)与在滤泡型PTC中的表达率(12.5%)比较,差异有统计学意义(P<0.05).BRAF V600E突变与PTC患者临床病理参数间的差异无统计学意义(P>0.05).结论 BRAFV600E突变与PTC的发生、发展可能有着重要联系,BRAF V600E突变可以作为PTC诊断的特异性标记;改良的试剂盒法抽提石蜡组织DNA具有高效、简便、价格栩对低廉的优点.

Abstract：

Objective To evaluate the expression of BRAF V600E mutation in 240 Chinese patients with thyroid lesions. Methods Two hundred and forty Chinese patients with thyroid lesions, including 129 papillary thyroid carcinomas (PTC) , 12 follicular carcinomas, 4 medullary carcinomas, 30 adenomas, 30 nodular goiters, and 35 papillary hyperplasia. DNA was extracted from thyroid biopsy and paraffin embedded thyroid tissues, and the expression of BRAF V600E mutation was detected by polymerase chain reaction and DNA sequencing assays. Results The presence of BRAF V600E mutation was found in 61 of the total group of 240 cases (25. 4%). It was only detected in PTC (47. 3%), and not detected in other types of malignant and benign thyroid lesions. There was a statistically significant difference between the expression of BRAF V600E mutation in classic type PTC (49. 6% ) and in follicular type PTC (12. 5% ,P <0. 05) , but statistical data did not show any correlation between BRAF V600E mutation and clinicopathologic parameters in PTC (P > 0. 05). Conclusions BRAF V600E mutation has a significant correlation with PTC and the detection of BRAF V600E mutation may be used as an important prognostic marker of PTC. Our new method of DNA extraction from paraffin embedded tissues is efficient and inexpensive.     

The safety and efficacy of cobimetinib for the treatment of BRAF V600E or V600K melanoma

Introduction: In the recent years, melanoma patients’ outcome and survival improved, mainly because of systemic treatment improvement with targeted therapy and checkpoint blockade. Targeted therapy with BRAF and MEK inhibitors was approved to treat patients with unresectable or metastatic melanoma, harboring BRAF V600 mutations. This paper addresses the safety and efficacy of cobimetinib, when used in combination with vemurafenib, in the previous mentioned setting.

Areas covered: This article presents an overview on the rationale for clinical development of cobimetinib, as well as the mechanism of action, the efficacy and safety, and the most important trials that led to the approval of the combination therapy with vemurafenib. We searched the PubMed for published papers related to safety and efficacy of cobimetinib, and resistance mechanisms to BRAF inhibition. The abstract databases of the American Society of Clinical Oncology and European Society for Medical Oncology were also searched for updates on the mentioned clinical trials.

Expert commentary: Patients treated with targeted therapy experience a rapid tumor response. However, virtually all patients will develop resistance to treatment. Therapeutic combinations to overcome resistance mechanisms are currently addressed. In the future, targeted therapy strategy will include three or more drugs, probably from different therapeutic classes.     

BRAF V600突变阳性的晚期黑色素瘤治疗的临床研究进展

多数黑色素瘤具有BRAF V600E/K突变,因此V600成为黑色素瘤精准治疗的重要靶点,并通常可被BRAF抑制剂和MEK抑制剂联合阻断。免疫检查点抑制剂的出现也极大地改善了BRAF V600突变阳性的晚期黑色素瘤患者的治疗结局,探究这部分患者的最佳一线治疗及序贯治疗顺序的临床试验正在开展。本文就精准医疗时代BRAF V600突变阳性的晚期黑色素瘤患者治疗的最新研究进展进行综述。     

甲状腺乳头状癌BRAF V600E基因突变 及相关蛋白的表达

背景与目的:BRAF V600E基因突变可作为甲状腺乳头状癌靶向治疗的靶点,因此检测患者BRAF基因状态对于能否应用靶向药物治疗具有重要意义.观察BRAF V600E基因突变及突变蛋白VE1在甲状腺乳头状癌中的表达情况,并分析其与甲状腺乳头状癌的临床病理特征及其预后的关系.方法:采用DNA测序法及免疫组织化学法分别检测108例甲状腺乳头状癌、54例甲状腺腺瘤和54例结节性甲状腺肿标本中BRAF基因突变及其相关蛋白VE1的表达.结果:108例甲状腺乳头状癌基因突变率为67.6%,VE1表达率为64.8%,与甲状腺良性病变相比差异有统计学意义(P<0.05),与临床病理参数间无相关性.结论:甲状腺乳头状癌BRAF V600E基因突变率和BRAF V600E蛋白表达水平增高,可以作为鉴别甲状腺良、恶性肿瘤的有效指标.免疫组织化学法检测甲状腺乳头状癌BRAF V600E蛋白的表达与其基因突变的一致性高,可间接有效地反映BRAF V600E基因突变的状态.BRAF V600E基因突变及突变蛋白的表达与甲状腺乳头状癌患者预后无关.     

Contemporary treatment approaches for metastatic colorectal cancer driven by BRAF V600 mutations

The treatment of metastatic colorectal cancer (mCRC) harboring BRAF V600 mutations is challenging. These tumors are often refractory to standard treatment. Therefore, the patients may exhibit rapid clinical deterioration, depriving them of the chance to receive salvage therapy. In newly diagnosed patients with good performance status, the administration of an intensive chemotherapy regimen like FOLFOXIRI (5-fluorouracil, leucovorin, oxaliplatin, and irinotecan) along with the antiangiogenic agent bevacizumab can modify this aggressive behavior of the disease and improve patient clinical outcomes. The recently published results of the BEACON (Binimetinib, Encorafenib, and Cetuximab Combined to Treat BRAF-Mutant Colorectal Cancer) study demonstrated that a combination therapy consisting of BRAF, epidermal growth factor receptor, and mitogen-activated protein kinase kinase inhibitors could be a useful second-or third-line alternative. This review summarizes the current treatment strategies for BRAF-mutant mCRC.     

BRAF V600E and Novel Somatic Mutations in Thyroid Cancer of Libyan Patients

Background: Thyroid cancer (TC) is a common endocrine malignancy that frequently harbours the oncogenic V600E BRAF mutation. This mutation has received considerable attention in recent years for its potential utility in the risk stratification and management of TC. This study aims to investigate BRAF mutational status in thyroid cancer of Libyan patients and their association with clinicopathological factors. Methods: 44 thyroid tissue samples were analysed for mutations in exon 15 of the BRAF gene by performing polymerase chain reaction and Sanger sequencing. The results of BRAF mutation screening were correlated to clinical and pathological characteristics of the studied thyroid cancer patients. Statistical analyses were performed using SPSS. Results: The BRAF exon 15 mutations were detected in 19 (43.2%) of the thyroid cancer cases. The V600E was the most frequent one found in 15/44 (34.1%) cases. We also detected 6 other variants in 7 patients (15.9%), the S616F, the W619R and the T599S. Three mutations were associated with V600E, the L584I, the D587Y and the synonymous L597L. None of these mutations were reported previously in thyroid cancers. No statistical association was found between BRAF mutations and clinicopathological factors except with papillary thyroid cancer type (p= 0,032). Conclusions: Novel BRAF mutations and V600E were frequently detected in thyroid cancer of Libyan patients; this suggests a potential role of these novel mutations in carcinogenesis and in anti-EGFR therapy resistance.     

Ppp6c haploinsufficiency accelerates UV-induced BRAF(V600E)-initiated melanomagenesis

According to TCGA database, mutations in PPP6C (encoding phosphatase PP6) are found in c. 10% of tumors from melanoma patients, in which they coexist with BRAF and NRAS mutations. To assess PP6 function in melanoma carcinogenesis, we generated mice in which we could specifically induce BRAF(V600E) expression and delete Ppp6c in melanocytes. In these mice, melanoma susceptibility following UVB irradiation exhibited the following pattern: Ppp6c semi-deficient (heterozygous) > Ppp6c wild-type > Ppp6c-deficient (homozygous) tumor types. Next-generation sequencing of Ppp6c heterozygous and wild-type melanoma tumors revealed that all harbored Trp53 mutations. However, Ppp6c heterozygous tumors showed a higher Signature 1 (mitotic/mitotic clock) mutation index compared with Ppp6c wild-type tumors, suggesting increased cell division. Analysis of cell lines derived from either Ppp6c heterozygous or wild-type melanoma tissues showed that both formed tumors in nude mice, but Ppp6c heterozygous tumors grew faster compared with those from the wild-type line. Ppp6c knockdown via siRNA in the Ppp6c heterozygous line promoted the accumulation of genomic damage and enhanced apoptosis relative to siRNA controls. We conclude that in the presence of BRAF(V600E) expression and UV-induced Trp53 mutation, Ppp6c haploinsufficiency promotes tumorigenesis.     

Vemurafenib for BRAF V600 mutated advanced melanoma: Results of treatment beyond progression

BackgroundSelective BRAF inhibition (BRAFi) by vemurafenib or dabrafenib has become approved standard treatment in BRAF V600 mutated advanced stage melanoma. While the response rate is high, the response duration is limited with a progression-free survival (PFS) of 5–6 months. Our observation of accelerated disease progression within some patients after stopping vemurafenib treatment has fostered the idea of treatment beyond progression (BRAFi TBP).MethodIn this retrospective study, we analysed 70 metastatic melanoma patients, treated at our institute, who experienced progression after prior objective response upon treatment with vemurafenib. Thirty-five patients that continued treatment beyond progression are compared with 35 patients who stopped BRAFi treatment at disease progression.ResultsMedian overall survival beyond documented progression was found to be 5.2 months versus 1.4 months (95% confidence interval (CI): 3.8–7.4 versus 0.6–3.4; Log-Rank p = 0.002) in favour of BRAFi TBP. In the multivariate survival analysis, stopping treatment at disease progression was significantly associated with shorter survival (hazard ratio: 1.92; 95% CI: 1.04–3.55; p = 0.04).ConclusionOur results suggest that continuing vemurafenib treatment beyond progression may be beneficial in advanced melanoma patients, who prior to progression responded to vemurafenib.     

黑色素瘤中线形程序性坏死与血管生成及预后关系的初步探讨*

目的:初步探讨线形程序性坏死（linearly patterne d programmed cell necrosis ,LPPCN ）与黑色素瘤血管生成的关系,并分析其临床病理意义,为抗肿瘤血管生成治疗寻找新的思路及靶点。方法:观察68例人体黑色素瘤标本中LPPCN 的形态学特点及分布特征;应用免疫组织化学方法检测组织中CD105 及TGF β 1 蛋白的表达情况;同时进行CD31和PAS 双重染色以观察血管生成拟态（Vasculo genic mimicry,VM）的分布。结果:1）LPPCN 在HE镜下形态为一簇胞质浓缩、胞核深染的肿瘤细胞,呈线形或网状分布,68例患者中LPPCN 阳性率为55.89%（38/68）。 2）血管生成拟态密度（VMD）及CD105 标记的微血管密度（microvessel density,MVD）在LPPCN 阳性组高于LPPCN 阴性组（P<0.05）;且二者与LPPCN 的密度均呈正相关。TGF β 1 蛋白的阳性表达率在LPPCN 阳性组高于阴性组,同时其在发生LPPCN 的肿瘤细胞中的表达明显高于周围细胞,差异均具有统计学意义（P<0.05）;且TGF β 1 阳性表达指数与CD105 标记的MVD呈正相关。3）LPPCN 与性别、年龄、发生部位、有无瘤栓、淋巴结转移及远处转移无关（P>0.05）;而与肿瘤大小、核分裂像数目、Breslow厚度密切相关（P<0.05）。 Kaplan-Meier 生存分析显示LPPCN 阳性组的生存率低于LPPCN 阴性组,差异有统计学意义（P<0.05）。 结论:黑色素瘤中存在LPPCN 且与血管生成关系密切,部分肿瘤细胞发生LPPCN 可能为肿瘤实质中VM及新生血管的形成提供空间基础;研究LPPCN 对判断患者的预后有一定的参考价值。      

BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease

Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis.     

BRAF基因突变与皮肤黑色素瘤临床病理特征关系的meta分析

目的 探讨BRAF突变频率以及该突变与皮肤黑色素瘤(CM)临床病理特征的关系.方法 使用Pubmed、Embase、维普、清华同方等数据库检索2013年4月以前相关文献,按纳入排除标准筛选文献,采用Revman5.0软件进行统计分析.结果 涉及BRAF突变有9项研究,共1134例患者.CM患者BRAF突变率为47.3％.50岁以上患者BRAF突变OR =2.30,95％ CI为1.36 ～3.91；浅表扩散性黑色素瘤(SSM)B RAF突变OR =2.11,95％CI为1.64～2.71;病灶在躯干时BRAF基因突变OR=2.35,95％ CI为1.71～3.21;浸润深度小于1mm时BRAF突变OR=1.56,95％ CI为1.08 ～2.25.结论 BRAF基因突变易发生于年龄50岁以上、SSM、病灶在躯干及浸润深度小于1mm的患者,而与性别无关.     

Dual inhibition of (V600E)BRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism

BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic ^V600EBRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease.     

Time to re‐consider the meaning of BRAF V600E mutation in papillary thyroid carcinoma

The BRAF V600E mutation, resulting from the BRAFT1799A transversion, is the most common genetic mutation in papillary thyroid carcinoma (PTC), with a mean frequency close to 50% among all cases. A large number of studies in the past decade have tried to dissect the relevance and the function of the V600E mutation in controlling oncogenesis and progression of thyroid cancer. However, several works published in the latest years have provided new evidence, in partial conflict with the previous knowledge, suggesting the need of reconsidering the meaning of the BRAF V600E mutation in PTC. In this work, we attempt to discuss some of the most recent molecular, preclinical and clinical evidence to construct a more exhaustive model of function for the BRAF V600E in development, progression and therapeutic approach of thyroid cancer.