临床肿瘤学实践中的braf突变检测.pdf

BRAF Mutation Testing in Clinical Practice of Oncology Pei Hui M.D, Ph.D. Associate Professor of Pathology, Gynecology and Obstetrics Director of Clinical Molecular Diagnostics Laboratories Yale University School of Medicine The 8thForum of Mainland and Overseas Pathologists, April 23, 2012, Beijing 临床肿瘤学实践中的临床肿瘤学实践中的BRAFBRAF突变检测突变检测 Howard (Jack) West, MD EGFREGFR- -酪氨酸激酶抑制剂酪氨酸激酶抑制剂 抗抗EGFREGFR抗体类抑制剂抗体类抑制剂 EGFREGFR信号传导通路信号传导通路 KRASKRAS活化及下游效应分子活化及下游效应分子 BRAF, also known as v-Raf murine sarcoma viral gene homolog B1, is a proto-oncogene composed of 18 exons and 17 introns spanning approximately 200 Kb (199,622) on the long arm of chromosome 7 (7q34). B-RAF/BRAF  别名鼠类肉瘤滤过性病毒致癌同源体别名鼠类肉瘤滤过性病毒致癌同源体B1,B1,属于原癌基因属于原癌基因 位于位于1717号染色体长臂号染色体长臂3 3区区4 4带带 (7q34)(7q34)，含，含1818个外显子和个外显子和1717个内含子，个内含子， 长约长约200Kb(199,622bp)200Kb(199,622bp) Ziai J, Hui P. Expert Rev Mol Diagn. 2012 Mar;12(2):127-38. BRAFBRAF转录子和蛋白结构及相关生物学效应转录子和蛋白结构及相关生物学效应 BRAF (along with ARAF and CRAF/Raf-1) is a member of the RAF subfamily of serine/threonine kinases, critical mediators of the RAS/RAF/MEK/ERK pathway. BRAF (以及ARAF和CRAF/Raf-1) 是丝/苏氨酸激酶RAF家族 成员之一，是RAS/RAF/MEK/ERK通路的关键中介分子。

Apoptosis Proliferation Migration Differentiation 凋亡凋亡 增殖增殖 迁移迁移 分化分化 Transcription of BRAF occurs in all cell types and tissues. However, expression occurs at varying levels and mRNA, in a given tissue type, is present as one of several isoforms of the gene. BRAF在所有类型的组织和细胞中均有转录，但表达水平各 有差异,不同组织中BRAF可以转录为不同亚型的mRNA分子 Similarly, the BRAF protein exists in multiple isoforms, which range in size from 67 to 99 kDa and contain approximately 750–800 amino acids. 同样，BRAF蛋白也存在多种亚型，分子量为67-99kDa，包 含750–800个氨基酸残基。

BRAF Mutation Testing in Clinical Oncology Ziai J, Hui P. Expert Rev Mol Diagn. 2012 Mar;12(2):127-38. 临床肿瘤学之BRAF突变检测 Structural analysis of the BRAF protein, including recent elucidation of the kinase domain crystal structure, suggests the following explanation: 在BRAF蛋白的结构分析结果基础上，特别是近期 对激酶区晶体结构的阐明，可以提出以下解释： V600 sits four residues beyond the DFG motif and forms a hydrophobic interaction within the glycine- rich loop (P-loop) in the wild-type configuration Oncogenic Mutations in the B-Raf Kinase Domain. The kinase domain of B-Raf is shown in backbone representation and colored gray, except for the nucleotide binding loop (yellow), activation segment (blue), and the catalytic loop (magenta). A portion of the activation segment, represented by blue spheres, is disordered in the crystalstructure. The N- and C termini are denoted by N and C. The small-molecule inhibitor (BAY43-9006) cocrystallized with the B-Raf kinase domain is shown in full- sphere representation and colored black. Oncogenic mutations have been mapped onto the backbone representation as stars, color coded according to class. Mutations that strongly activate B-Raf kinase activity are colored green, those that result in intermediate activation are colored orange, and those that result in impaired B-Raf catalytic activity are colored red. In the nucleotide binding loop (yellow), mutations exist that, depending on the exact amino acid change, fall into more than one activation class. Such mutations are represented by bicolored stars. The positions of two sites of oncogenic mutation, Gly-465 (P loop), and Val-599 (activation segment), are indicated, as well as the position of Asp-593 of the DFG motif in the activation segment Highly conserved residues are important in stabilizing the glycine-rich loop (P-Loop)/activation segment to maintain an inactive conformation and V600 residue stabilizes DFG motif in an inactive conformation and prevents phosphoryl transfer to the DFG motif. Inactivation State of BRAF Activation: phosphorylation of T599 displaces the activation segment, allowing the Asp594- Phe595-Gly596 motif (DFG motif) to adopt an active conformation V600E mutation disrupts this hydrophobic interaction and facilitates BRAF phosphorylation leading to an increased activity of MEK–ERK signaling up to 700-fold. V600E Activation of BRAF Ziai J, Hui P. Expert Rev Mol Diagn. 2012 Mar;12(2):127-38. 不同人类肿瘤中 BRAF突变率、突变 方式及临床意义 Currently, more than 70 mutations of BRAF have been documented in multiple human tumor types. 89% of BRAF mutations cluster within the P- loop/activation segment of the BRAF protein. 如今，在多种人类肿瘤中已发现70多种BRAF突变。

89%的BRAF 突变集中在BRAF蛋白的P-环/活化片段 V600E as the most prevalent (90%) and most clinically relevant! V600E是最常见的（90%）、与临床 联系最为密切的BRAF突变类型！ For diagnostic purposes, more than 95% of clinically relevant mutations can be covered by assays examining exon 15, particularly the V600E mutation. 就诊断需要而言，检测BRAF基因第15外显子，特别是 V600E突变，可以涵盖超过95%的临床相关突变 In clinical practice, almost all available methodologies are PCR-based and essentially any types of tissue or cytological specimens can be used for BRAF mutation testing 几乎所有的BRAF突变临床检测方法都是以PCR为基础的，而 且所有组织和细胞学样本均可用于检测。

PCR Amplicon Design - Sufficiently short ! PCR扩增子设计——足够短！ IMPORTANT: BRAF mutation testing requires a much higher analytical sensitivity when。