肿瘤分子病理诊断基础2

1、肿瘤分子病理诊断基础闫庆国闫庆国分子病理与分子病理诊断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变化肿瘤的分子病理诊断技术肿瘤的分子病理诊断技术肿瘤分子病理诊断的应用肿瘤分子病理诊断的应用肿瘤的分子病理诊断技术肿瘤的分子病理诊断技术Molecular pathology protocalsMolecular pathology protocalslDNA DNA extraction from paraffin-embedded tissuesextraction from paraffin-embedded tissueslDNA extraction from fresh or

2、 frozen tissues DNA extraction from fresh or frozen tissues lRNARNA extraction from fresh or frozen tissues extraction from fresh or frozen tissueslSingle-strand conformation polymorphism Single-strand conformation polymorphism analysis of analysis of mutations in exons 4-8 of the TP53 genemutations

3、 in exons 4-8 of the TP53 genelCleavaes fragment length polymorphism analysis for Cleavaes fragment length polymorphism analysis for genotyping and mutation detectiongenotyping and mutation detectionlDetection of telomerase by Detection of telomerase by in situ hybridization in situ hybridization an

4、d by and by the polymerase chain reaction based telomerase activity the polymerase chain reaction based telomerase activity assayassaylDetection of Detection of microsatellite instabilitymicrosatellite instabilitylPolymerase chain reaction Polymerase chain reaction clonality assays clonality assays

5、based on X-based on X-linked geneslinked geneslFluorescent in situ hybridizationFluorescent in situ hybridization: evaluation for ploidy and : evaluation for ploidy and gene amplificationgene amplificationlHer-2/neu oncogene Her-2/neu oncogene amplificationamplification determined by fluorescence de

6、termined by fluorescence in situ hybridizationin situ hybridizationlA nested A nested reversed transcription-polymerase chain reaction reversed transcription-polymerase chain reaction assay to detect BCR/ab/assay to detect BCR/ab/lDetection of t(15;17)(q24;q21),inv(16)/t(16;16)(p13;q22), Detection o

7、f t(15;17)(q24;q21),inv(16)/t(16;16)(p13;q22), and t(8;21)(q22;q22) anomalies in acute myeloid leukemiaand t(8;21)(q22;q22) anomalies in acute myeloid leukemialDetection of t(14;18)(q32;q21)-Associated BCL-2/JH Gene Detection of t(14;18)(q32;q21)-Associated BCL-2/JH Gene Fusion in Non-Hodgkins Lymph

8、omaFusion in Non-Hodgkins LymphomalDetection of Breast Cancer Cells Using Immunomagnetic Beads Detection of Breast Cancer Cells Using Immunomagnetic Beads and Reverse Transcription-Polymerase Chain Reactionand Reverse Transcription-Polymerase Chain ReactionlMolecular Detection of Molecular Detection

9、 of Circulating Prostate Cancer CellsCirculating Prostate Cancer CellslMethods to Detect Methods to Detect Clonal Gene Rearrangements Clonal Gene Rearrangements in Lymphomas in Lymphomas and Leukemiasand LeukemiaslMonitoring of Bone Marrow Transplant EngraftmentMonitoring of Bone Marrow Transplant E

10、ngraftmentlDirect Molecular Diagnosis of Multiple Endocrine Neoplasia Type Direct Molecular Diagnosis of Multiple Endocrine Neoplasia Type 1 1lMolecular Detection of Multiple Endocrine Neoplasia Type 2Molecular Detection of Multiple Endocrine Neoplasia Type 2lAssay for Detecting the I1307K Susceptib

11、ility Allele within the Assay for Detecting the I1307K Susceptibility Allele within the Adenomatous Polyposis Coli GeneAdenomatous Polyposis Coli GenelDetection of Detection of Human Papillomaviruses Human Papillomaviruses by Polymerase Chain by Polymerase Chain Reaction and In Situ HybridizationRea

12、ction and In Situ HybridizationlMolecular Methods for Detecting Molecular Methods for Detecting Epstein-Barr Virus Epstein-Barr Virus (Part I): (Part I): Hybridization to Epstein-Barr VirusEncoded RNA (EBER) Hybridization to Epstein-Barr VirusEncoded RNA (EBER) TranscriptsTranscriptslMolecular Metho

13、ds for Detecting Epstein-Barr Virus (Part II): Molecular Methods for Detecting Epstein-Barr Virus (Part II): Structural Analysis of Epstein-Barr Virus DNA as a Marker of Structural Analysis of Epstein-Barr Virus DNA as a Marker of ClonalityClonalitylMolecular Methods for Detecting Epstein-Barr Virus

14、 (Part III): Molecular Methods for Detecting Epstein-Barr Virus (Part III): EBV Viral Load by Competitive Polymerase Chain ReactionEBV Viral Load by Competitive Polymerase Chain ReactionlMolecular Detection of Kaposis SarcomaAssociated Molecular Detection of Kaposis SarcomaAssociated Herpesvirus/Hum

15、an Herpesvirus-8Herpesvirus/Human Herpesvirus-8lDiagnostic Applications of Quantitative Polymerase Chain Diagnostic Applications of Quantitative Polymerase Chain Reaction for Reaction for CytomegalovirusCytomegalovirus, Polymerase Chain Reaction , Polymerase Chain Reaction System That Detects System

16、 That Detects Herpes Simplex Virus Herpes Simplex Virus in Cerebrospinal in Cerebrospinal Fluid and Discriminates Genotypes 1 and 2Fluid and Discriminates Genotypes 1 and 2lDetection and Typing of Detection and Typing of Hepatitis CHepatitis C Virus ViruslDetection and Speciation of Detection and Sp

17、eciation of Mycobacteria Mycobacteria in Formalin-in Formalin-Fixed, Paraffin-Embedded Tissue SectionsFixed, Paraffin-Embedded Tissue SectionslUltrasensitive Quantitation of Human Immunodeficiency Ultrasensitive Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by the AMPLICOR and CO

18、BAS Virus Type 1 RNA in Plasma by the AMPLICOR and COBAS AMPLICOR AMPLICOR HIV-1HIV-1 MONITORTM Tests MONITORTM TestslMolecular Diagnosis of Molecular Diagnosis of HereditaryHereditary Thrombotic Disorders Thrombotic DisorderslPrenatal Genotyping Prenatal Genotyping of the RhD Locus to Identify of t

19、he RhD Locus to Identify Fetuses at Risk for Hemolytic Disease of the NewbornFetuses at Risk for Hemolytic Disease of the NewbornlMolecular Diagnosis of Hereditary HemochromatosisMolecular Diagnosis of Hereditary HemochromatosislGenotyping Genotyping of Apolipoprotein Eof Apolipoprotein ElGenotyping

20、 Genotyping for Functionally Important Human CYP2D6for Functionally Important Human CYP2D6* *4 4 (B) Mutation Using TaqMan Probes(B) Mutation Using TaqMan Probes常用的共性技术常用的共性技术u 核酸提取技术核酸提取技术u 电泳技术电泳技术 u 凝胶成像凝胶成像u PCR PCR 技术技术 分子病理诊断的代表性技术分子病理诊断的代表性技术 ISH In situ hybridizasionISH In situ hybridizasion

21、 FISHFISH Gene mutation analysisGene mutation analysis Clonality analysisClonality analysis FISH: Method FISH: Method Metaphase FISHMetaphase FISHPerformed on metaphase chromosome preparation.Performed on metaphase chromosome preparation.Advantage: Excellent resolution.Advantage: Excellent resolutio

22、n.Disadvantage: Collect live cells and culture in vitroDisadvantage: Collect live cells and culture in vitro. . Interphase FISHInterphase FISHPerformed on isolated nucleus or whole section.Performed on isolated nucleus or whole section.Advantage: Excellent correlation with histological Advantage: Ex

23、cellent correlation with histological and immunophenotypic features.Multiple probes on the and immunophenotypic features.Multiple probes on the same section.same section.Disadvantage: Nucleus and signal overlap truncation. Disadvantage: Nucleus and signal overlap truncation. FISH: Protocols for inte

24、rphase FISHFISH: Protocols for interphase FISHSlides 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

25、 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

26、 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 partnersNormal cell14 1414q32t(14;18)/IGH-BCL2 positive cell14 der (14)der (18) 18FISH: ProbesDual colour-dual fusion

27、 probe - translocation partners knownNormal cell14q3214 1418q211818t(14;18)/IGH-BCL2 positive cell14 der (14)der (18) 18 FISH: Probes Single colour probe CEP 8Chromosome specific to detect aneuplody17q11.2-q12 Her-2/neuCEP 17locus specific to detect copy number changes of a particular region (deleti

28、ons, gain or amplifications) Examples of specimen types applicable Examples of specimen types applicable to FISHto FISHFresh/frozen tissueCytology specimensBody fluids(urine)Intraoperative smearsCell culture preparationsFresh-frozen, parafin-embedded tissueThin sections(46)Disaggregated nucleiArchiv

29、ed unstained sectionsPreviously stained sections(e.g., negative immunohistochemistry controls)CISHDetection of gene amplification, chromosome translocations and chromosome number Using conventional enzymatic reactions under the brightfield microscope on formalin-fixed, paraffin-embedded tissuesCompa

30、red to FISH, CISHs advantages Detect under bright field The morphological details are readily apparent The probe signals are not subject to rapid fading2122突变检测原理：突变检测原理：根据野生型与突变型所表现出的差异根据野生型与突变型所表现出的差异 测序法：测序法：经典、荧光、焦磷酸经典、荧光、焦磷酸 （直接从序列上看差异）（直接从序列上看差异）定量定量 PCRPCR：探针法：探针法- -染料法染料法 ARMS-TaqMan ARMS-Ta

31、qMan （引物结合后反应的差异（引物结合后反应的差异- -间接）间接） HRM HRM、PNA-LNA PNA-LNA （根据产物变性溶解过程的差异（根据产物变性溶解过程的差异- -间接）间接）DHPLC DHPLC （根据不同构象分子色谱分析的移动性差异（根据不同构象分子色谱分析的移动性差异- -间接）间接）PCR-RFLP PCR-RFLP （酶切后电泳看泳动性差异（酶切后电泳看泳动性差异- -间接）间接） 直接测序法直接测序法焦磷酸测序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150200E SG CA A G

32、 T G C T G AT G CT C G TT/TG/G51015焦磷酸测序焦磷酸测序 EGFR Exon 21 EGFR Exon 21 第第858858密码子发生密码子发生CTGCTG到到CGGCGG的突变，即发生了的突变，即发生了L858RL858R突变突变 150200E ST G C G T G T C A A C T A C GT/GT/T51015150EST GCG T G T C A A CT A C GT/TT/T51015ARMSARMS amplification refractory mutation system高分辨率熔解曲线分析高分辨率熔解曲线分析 High

33、 Resolution Melting, High Resolution Melting, HRMHRMPCRPCR产物中的突变位点改变产物中的突变位点改变了了DNADNA熔解曲线的形状；纯熔解曲线的形状；纯合子和杂合子样本可以通过合子和杂合子样本可以通过使用一种饱和型的使用一种饱和型的DNADNA结合结合染料轻松的区分开来，并显染料轻松的区分开来，并显示出清晰的熔解曲线图谱。示出清晰的熔解曲线图谱。 克隆性分析克隆性分析u 概念：确定一群体细胞是单克隆性的还是多概念：确定一群体细胞是单克隆性的还是多克隆性的克隆性的u 意义：正常组织和反应性增生的病变是多克意义：正常组织和反应性增生的病变是多

34、克隆起源，而绝大多数肿瘤是单克隆起源隆起源，而绝大多数肿瘤是单克隆起源诊诊断与鉴别诊断意义断与鉴别诊断意义克隆性分析的方法克隆性分析的方法u T T、B B细胞受体基因重排的克隆性分析细胞受体基因重排的克隆性分析u 基于基于X X染色体连锁的体细胞克隆性分析染色体连锁的体细胞克隆性分析u 细胞表型分析细胞表型分析u 体细胞突变位点分析体细胞突变位点分析u 染色体中病毒染色体中病毒DNADNA整合位点分析整合位点分析Rearrangement of the immunoglobulin heavy chain gene generates antigen recognition diversit

35、y 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受体基因重排受体基因重排Site-Specific Recombination in Eukaryotes: Immunoglobulin Gene Rearrangement During Development2626个英文字母可以组成数以亿万计的单词个英

36、文字母可以组成数以亿万计的单词l A Al ATATl EATEATl BIRDBIRDl ABOUTABOUTl HISTORYHISTORYl WELCOMEWELCOMEl NOVEMBERNOVEMBERl PATHOLOGYPATHOLOGYl l IMMUNOHISTOCHEMISTRYIMMUNOHISTOCHEMISTRYl .l 多克隆性多克隆性l WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WEL

37、COMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl WELCOMEWELCOMEl 单克隆性单克隆性新的新的BIOMED-2BIOMED-2系统系统u欧洲淋巴瘤合作组织制定；欧洲淋巴瘤合作组织制定；u107107条引物；条引物；uT T、B B检测：检测：1414组项组项PCRPCR；u内对照、肿瘤对照：共内对照、肿瘤对照：共3030余组；余组；uInVivoScribe InVivoScribe 公司试剂；公司试剂；u3 3个工作日；个工作日；u筛选病例做，免疫组化能解决的不做。筛选病例做，免疫组化能解决的不做。BIOME

38、D-2BIOMED-2系统的优势系统的优势 部分解决部分解决假阴性假阴性的问题的问题u 假阴性的主要原因之一是设计的引物少，不能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。涵盖可能发生的各种重排。u 如果把一个字母理解为一段引物的话，引物不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，因为没有够，就相当于字母的种类不够，因为没有2626个个字母就不能保证拼出各种单词！字母就不能保证拼出各种单词！u 方法似乎方法似乎“烦琐烦琐”了，但是确实科学合理了，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，只是多做而且实际应用并没有真正烦琐多少，只是多做

39、些些PCRPCR，多花些时间判断！，多花些时间判断！BIOMED-2BIOMED-2系统的局限系统的局限 本身并不能解决假阳性的问题本身并不能解决假阳性的问题u条带必需在特定的预期的目标位置上；条带必需在特定的预期的目标位置上；u条带不是预期的假阳性条带；条带不是预期的假阳性条带；u结果可重复；结果可重复；u对于多于两个条带时（如三个）是寡克隆、亚克隆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；态判断；u由于由于TCR-TCR-不同克隆之间基因重排片段的大小差异不同克隆之间基因重排片段的大小差异较小

40、，更易出现假阳性；较小，更易出现假阳性；u配合其它方法区别单克隆与多克隆：毛细管凝胶电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、自动化荧光泳、梯度凝胶电泳、单链构象多态性、自动化荧光分析技术等。分析技术等。BIOMED-2BIOMED-2系统没有解决基因重排系统没有解决基因重排克隆性分析的所有问题克隆性分析的所有问题u 重排基因中的突变和缺失问题重排基因中的突变和缺失问题u 基因重排不能替代免疫组化，进一步的分型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还是依靠免疫组化u 仍然要强调四结合：临床、形态、免疫表型、仍然要强调四结合：临床、形态、免疫

41、表型、克隆性分析克隆性分析肿瘤分子病理诊断的应用肿瘤分子病理诊断的应用uu 肿瘤的分类肿瘤的分类uu 肿瘤诊断与早期诊断肿瘤诊断与早期诊断uu 肿瘤预后的判断与检测肿瘤预后的判断与检测uu 肿瘤个体化治疗肿瘤个体化治疗核酸分子诊断示例之一核酸分子诊断示例之一: FISH: FISH乳腺癌治疗药物乳腺癌治疗药物HerceptinHerceptin的选择的选择u HER-2HER-2阳性占乳腺癌的阳性占乳腺癌的25253030；对三苯氧；对三苯氧胺治疗无效，甚至病情进展（部分胺治疗无效，甚至病情进展（部分ERER、PRPR阳性不宜用三苯氧胺治疗）；阳性不宜用三苯氧胺治疗）；u 化疗方案选择：含蒽环

42、类抗生素敏感；环化疗方案选择：含蒽环类抗生素敏感；环甲氟联合化疗有抗性；单抗可逆转肿甲氟联合化疗有抗性；单抗可逆转肿瘤浸润、转移。瘤浸润、转移。u HER-2HER-2的检测不是仅仅确定阳性或阴性，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。而是确定基因过表达（扩增）的程度。u 免疫组化：分级初筛免疫组化：分级初筛u FISHFISH确定：具有高度重复性和可靠性确定：具有高度重复性和可靠性Abnormal 2+Abnormal 3+Normal 0Normal Normal 1+Normal Abnormal lowamplificationAbnormal highamp

43、lification免疫组化与免疫组化与FISHFISH51比比2 20132 201352核酸分子诊断示例之二核酸分子诊断示例之二: FISH: FISH用于鉴别诊用于鉴别诊断断 Case 1 : BL A 21/M presented with neck node swelling, 5 ml aspirate taken from submandibular LN. Clinical: Reactive LN, infection? neoplastic?Diagnosis: Diagnosis: P Presence of t(8;14)(q24:q32)/MYC-IGH BLrese

44、nce of t(8;14)(q24:q32)/MYC-IGH BL FISH: MYC +, IGH +IGH-MYC +BCL2 BCL6 IGH break part +VeMYC break part +VeIGH-MYC fusion +Ve Histology: Relatively monomorphic intermediate sized lymphocytes with high N:C ratio, occasional large tingible body macrophages. IHC: CD20+, CD10+, BCL6+, MIB-1 90% CD3-, C

45、D30-, BCL2- Pre. Dx: High grade B-NHL. BL?核酸分子诊断示例之三核酸分子诊断示例之三 协助诊断协助诊断 詹某，女，詹某，女，5555岁，发热，岁，发热，皮疹，肝脾大，肝功能异常。皮疹，肝脾大，肝功能异常。颈部淋巴结切片，会诊病例。颈部淋巴结切片，会诊病例。IgIg与与TCRTCR重排克隆性分析重排克隆性分析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 30Case 斑某，男，斑某，男，4040岁，岁，发现颈部包块发现颈部包块1 1月，行颈部月，行

46、颈部淋巴结活检。淋巴结活检。CD3CD20IgIg与与TCRTCR重排克隆性分析重排克隆性分析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 StudyCase Study1515岁，阴道出血，岁，阴道出血，HCGHCG没有明显没有明显增高。子宫内容物送检。增高。子宫内容物送检。核酸分子诊断示例之四核酸分子诊断示例之四: : 倍体分析与特定基因表达结合倍体分析与特定基因表达结合 由于由于B B超等检测技术进步，过去超等检测技术进步，过去1414周发周发现，现在提早到现，现

47、在提早到6 61212周，早期诊断带来困周，早期诊断带来困难。难。 过去认为早期漏诊也没关系，现在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，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。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？状胎块及绒毛水肿的鉴别？ P57P57：来自父亲的：来自父亲的等位基因是沉默的，等位基因是沉默的，只有来自母亲的等位只有来自母亲的等位基因才表达。完全性基因才表达。完全性水泡状胎块水泡状胎块P57P57阴性。阴性。完全性与部分性的鉴别：完全