肿瘤分子病理诊断基础2

肿瘤分子病理诊断基础,闫庆国,分子病理与分子病理诊断肿瘤的分子病理变化肿瘤的分子病理诊断技术肿瘤分子病理诊断的应用,肿瘤的分子病理诊断技术,Molecular pathology protocals,DNA extraction from paraffin-embedded tissuesDNA extraction from fresh or frozen tissues RNA extraction from fresh or frozen tissuesSingle-strand conformation polymorphism analysis of mutations in exons 4-8 of the TP53 geneCleavaes fragment length polymorphism analysis for genotyping and mutation detectionDetection of telomerase by in situ hybridization and by the polymerase chain reaction based telomerase activity assayDetection of microsatellite instabilityPolymerase chain reaction clonality assays based on X-linked genes,Fluorescent in situ hybridization: evaluation for ploidy and gene amplificationHer-2/neu oncogene amplification determined by fluorescence in situ hybridizationA nested reversed transcription-polymerase chain reaction assay to detect BCR/ab/Detection of t(15;17)(q24;q21),inv(16)/t(16;16)(p13;q22), and t(8;21)(q22;q22) anomalies in acute myeloid leukemiaDetection of t(14;18)(q32;q21)-Associated BCL-2/JH Gene Fusion in Non-Hodgkins LymphomaDetection of Breast Cancer Cells Using Immunomagnetic Beads and Reverse Transcription-Polymerase Chain ReactionMolecular Detection of Circulating Prostate Cancer CellsMethods to Detect Clonal Gene Rearrangements in Lymphomas and Leukemias,Monitoring of Bone Marrow Transplant EngraftmentDirect Molecular Diagnosis of Multiple Endocrine Neoplasia Type 1Molecular Detection of Multiple Endocrine Neoplasia Type 2Assay for Detecting the I1307K Susceptibility Allele within the Adenomatous Polyposis Coli GeneDetection of Human Papillomaviruses by Polymerase Chain Reaction and In Situ HybridizationMolecular Methods for Detecting Epstein-Barr Virus (Part I): Hybridization to Epstein-Barr VirusEncoded RNA (EBER) TranscriptsMolecular Methods for Detecting Epstein-Barr Virus (Part II): Structural Analysis of Epstein-Barr Virus DNA as a Marker of ClonalityMolecular Methods for Detecting Epstein-Barr Virus (Part III): EBV Viral Load by Competitive Polymerase Chain Reaction,Molecular Detection of Kaposis SarcomaAssociated Herpesvirus/Human Herpesvirus-8Diagnostic Applications of Quantitative Polymerase Chain Reaction for Cytomegalovirus, Polymerase Chain Reaction System That Detects Herpes Simplex Virus in Cerebrospinal Fluid and Discriminates Genotypes 1 and 2Detection and Typing of Hepatitis C VirusDetection and Speciation of Mycobacteria in Formalin-Fixed, Paraffin-Embedded Tissue SectionsUltrasensitive Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by the AMPLICOR and COBAS AMPLICOR HIV-1 MONITORTM Tests,Molecular Diagnosis of Hereditary Thrombotic DisordersPrenatal Genotyping of the RhD Locus to Identify Fetuses at Risk for Hemolytic Disease of the NewbornMolecular Diagnosis of Hereditary HemochromatosisGenotyping of Apolipoprotein EGenotyping for Functionally Important Human CYP2D6*4 (B) Mutation Using TaqMan Probes,常用的共性技术,核酸提取技术,电泳技术,凝胶成像,PCR 技术,分子病理诊断的代表性技术,ISH In situ hybridizasionFISHGene mutation analysisClonality analysis,FISH: Method,Interphase FISHPerformed on isolated nucleus or whole section.Advantage: Excellent correlation with histological and immunophenotypic features.Multiple probes on the same section.Disadvantage: Nucleus and signal overlap truncation.,FISH: Protocols for interphase FISH,Slides preparationPrepare 2-4 m sections and dewax;Digest in 0.1% pepsin in 0.015N HCl for 20 min at 37C;Dehydrate and air dry slides.Hybridisation Apply probe to area of interest, place coverslip and seal with rubber cement;Denature probe and target DNA at 80C for 25 min;Hybridise in a moisture chamber at 45C for 2 days.Post-hybridisation washing Remove rubber cement and cover slip; Wash 1- 0.4x SSC and 0.3% IGEPAL CA-630 buffer in a water bath set at 72 C; Wash 2- 2SSC and 0.1% IGEPAL CA-630 buffer at room temp for 1 min;Wash 3 - 2SSC for 2-5 min at room temp;Apply anti-fade solution containing DAPI and coverslip.Read signals with a fluorescent microscope.,FISH: ProbesDual-colour break-apart probe independent of translocation partners,FISH: ProbesDual colour-dual fusion probe - translocation partners known,FISH: Probes Single colour probe,Examples of specimen types applicable to FISH,Fresh/frozen tissueCytology specimensBody fluids(urine)Intraoperative smearsCell culture preparationsFresh-frozen, parafin-embedded tissueThin sections(46)Disaggregated nucleiArchived unstained sectionsPreviously stained sections(e.g., negative immunohistochemistry controls),CISH,Detection of gene amplification, chromosome translocations and chromosome number Using conventional enzymatic reactions under the brightfield microscope on formalin-fixed, paraffin-embedded tissuesCompared to FISH, CISHs advantagesDetect under bright fieldThe morphological details are readily apparent The probe signals are not subject to rapid fading,21,22,突变检测原理：根据野生型与突变型所表现出的差异,测序法：经典、荧光、焦磷酸 （直接从序列上看差异）定量 PCR：探针法-染料法 ARMS-TaqMan （引物结合后反应的差异-间接） HRM、PNA-LNA （根据产物变性溶解过程的差异-间接）DHPLC （根据不同构象分子色谱分析的移动性差异-间接）PCR-RFLP （酶切后电泳看泳动性差异-间接）,直接测序法,焦磷酸测序根据已知序列，每合成一个核苷酸加一次样，测一次荧光强度,焦磷酸测序,EGFR Exon 21 第858密码子发生CTG到CGG的突变，即发生了L858R突变,ARMS amplification refractory mutation system,高分辨率熔解曲线分析,High Resolution Melting, HRMPCR产物中的突变位点改变了DNA熔解曲线的形状；纯合子和杂合子样本可以通过使用一种饱和型的DNA结合染料轻松的区分开来，并显示出清晰的熔解曲线图谱。,克隆性分析,概念：确定一群体细胞是单克隆性的还是多克隆性的意义：正常组织和反应性增生的病变是多克隆起源，而绝大多数肿瘤是单克隆起源诊断与鉴别诊断意义,克隆性分析的方法,T、B细胞受体基因重排的克隆性分析基于X染色体连锁的体细胞克隆性分析细胞表型分析体细胞突变位点分析染色体中病毒DNA整合位点分析,Rearrangement of the immunoglobulin heavy chain gene generates antigen recognition diversity in normal B-cells. Three areas of the germline IgH gene are moved and brought together during B-cell maturation, the V (variable), D (diversity) and J (joining) regions,受体基因重排,Site-Specific Recombination in Eukaryotes: Immunoglobulin Gene Rearrangement During Development,26个英文字母可以组成数以亿万计的单词,AATEATBIRDABOUTHISTORYWELCOMENOVEMBERPATHOLOGYIMMUNOHISTOCHEMISTRY.多克隆性,WELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOMEWELCOME单克隆性,新的BIOMED-2系统,欧洲淋巴瘤合作组织制定；107条引物；T、B检测：14组项PCR；内对照、肿瘤对照：共30余组；InVivoScribe 公司试剂；3个工作日；筛选病例做，免疫组化能解决的不做。,BIOMED-2系统的优势,部分解决假阴性的问题假阴性的主要原因之一是设计的引物少，不能涵盖可能发生的各种重排。如果把一个字母理解为一段引物的话，引物不够，就相当于字母的种类不够，因为没有26个字母就不能保证拼出各种单词！方法似乎“烦琐”了，但是确实科学合理了，而且实际应用并没有真正烦琐多少，只是多做些PCR，多花些时间判断！,BIOMED-2系统的局限,本身并不能解决假阳性的问题条带必需在特定的预期的目标位置上；条带不是预期的假阳性条带；结果可重复；对于多于两个条带时（如三个）是寡克隆、亚克隆的肿瘤性克隆演进现象还是多克隆更要密切结合形态判断；由于TCR-不同克隆之间基因重排片段的大小差异较小，更易出现假阳性；配合其它方法区别单克隆与多克隆：毛细管凝胶电泳、梯度凝胶电泳、单链构象多态性、自动化荧光分析技术等。,BIOMED-2系统没有解决基因重排克隆性分析的所有问题,重排基因中的突变和缺失问题基因重排不能替代免疫组化，进一步的分型还是依靠免疫组化仍然要强调四结合：临床、形态、免疫表型、克隆性分析,肿瘤分子病理诊断的应用, 肿瘤的分类, 肿瘤诊断与早期诊断, 肿瘤预后的判断与检测, 肿瘤个体化治疗,核酸分子诊断示例之一: FISH,乳腺癌治疗药物Herceptin的选择,HER-2阳性占乳腺癌的2530；对三苯氧胺治疗无效，甚至病情进展（部分ER、PR阳性不宜用三苯氧胺治疗）；化疗方案选择：含蒽环类抗生素敏感；环甲氟联合化疗有抗性；单抗可逆转肿瘤浸润、转移。,HER-2的检测不是仅仅确定阳性或阴性，而是确定基因过表达（扩增）的程度。免疫组化：分级初筛FISH确定：具有高度重复性和可靠性,Abnormal 2+,Abnormal 3+,Normal 0,Normal,Normal 1+,Normal,免疫组化与FISH,51,比2 2013,52,核酸分子诊断示例之二: FISH用于鉴别诊断 Case 1 : BL,A 21/M presented with neck node swelling, 5 ml aspirate taken from submandibular LN. Clinical: Reactive LN, infection? neoplastic?,Diagnosis: Presence of t(8;14)(q24:q32)/MYC-IGH BL,Histology: Relatively monomorphic intermediate sized lymphocytes with high N:C ratio, occasional large tingible body macrophages. IHC: CD20+, CD10+, BCL6+, MIB-1 90% CD3-, CD30-, BCL2- Pre. Dx: High grade B-NHL. BL?,核酸分子诊断示例之三 协助诊断,詹某，女，55岁，发热，皮疹，肝脾大，肝功能异常。颈部淋巴结切片，会诊病例。,Ig与TCR重排克隆性分析,1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30,Case,斑某，男，40岁，发现颈部包块1月，行颈部淋巴结活检。,CD3,CD20,Ig与TCR重排克隆性分析,1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30,Case Study 15岁，阴道出血，HCG没有明显增高。子宫内容物送检。,核酸分子诊断示例之四: 倍体分析与特定基因表达结合,?,VERY EARLY COMPLETE MOLE非常早的完全性水泡状胎块,由于B超等检测技术进步，过去14周发现，现在提早到612周，早期诊断带来困难。 过去认为早期漏诊也没关系，现在临床发现持续的滋养叶细胞疾病与固有的生物学特性相关，而与孕周关系不大，因此，诊断早期水泡状胎块是非常重要的。,Early complete mole: confusion with partial mole and hydropic villi. How to recognize? 早期完全性水泡状胎块与部分性水泡状胎块及绒毛水肿的鉴别？,46 XpXp,23 X Sperm,Empty Egg,COMPLETE MOLE,Paternal Chromosome Only(Androgenetic),P57：来自父亲的等位基因是沉默的，只有来自母亲的等位基因才表达。完全性水泡状胎块P57阴性。,完全性与部分性的鉴别： P57、倍体分析可靠且客观完全性：二倍体，P57阴性正常：二倍体，P57阳性,PARTIAL MOLE,23 X Sperm,23 X Sperm,23X,Egg,69 XpXpXm,Paternal and MaternalChromosome (Triploid),P57：来自父亲的等位基因是沉默的，只有来自母亲的等位基因才表达。故部分性水泡状胎块P57阳性。,完全性与部分性的鉴别： P57、倍体分析可靠且客观不完全性：三倍体，P57阳性正常：二倍体，P57阳性,示例之五:分子靶向用药靶点检测,Example of the Resp