MolecularDiagnosisofCancer-12-12-13final.pptx

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1、癌症的分子诊断癌症的分子诊断顾海华顾海华, Ph.DCell: 15058751720email: 癌症的分子诊断癌症的分子诊断用癌症的分子特证来对癌症组织和细用癌症的分子特证来对癌症组织和细胞进行检测，以此来对癌症分类并为胞进行检测，以此来对癌症分类并为制定有效的治疗方案提供依据制定有效的治疗方案提供依据有效的治疗方案有效的治疗方案1）彻底治愈）彻底治愈 （早发现）（早发现）2）控制病情并且延缓死亡期）控制病情并且延缓死亡期3）靶向治疗：有效治疗和降低副作用）靶向治疗：有效治疗和降低副作用4）降低治疗费用）降低治疗费用1. 癌症的发病机理和特征癌症的发病机理和特征 2. 癌症的分子诊断的重要

2、性癌症的分子诊断的重要性 4. 癌症分子诊断和个体化治疗癌症分子诊断和个体化治疗3. 用于癌症分子诊断的技术用于癌症分子诊断的技术癌症的发生是由于细胞里癌症的发生是由于细胞里DNA的突变而引起的的突变而引起的体细胞突变体细胞突变生殖细胞生殖细胞突变突变Genome Instability of CancerWikipediaDiverse Somatic Mutations Uncovered by Exome Sequencing in Colorectal （结直肠）（结直肠）Tumors* Single base mutations (92%) * Insertion, Deletion

3、s, or Duplications (The remaining 8%).Wood et al, 2007, SCIENCE, VOL 318 :1108-* Mutated gene mountains (5). * Mutated gene hills (large numbers80)DNA和和RNA变异的检测是变异的检测是癌症的分子诊断中一个重要部分癌症的分子诊断中一个重要部分Eight Hallmarks of Cancer避免免疫避免免疫击毁击毁持续细胞分裂的信号传导持续细胞分裂的信号传导躲避生长抑制体躲避生长抑制体激活侵袭和转移激活侵袭和转移抵抗细胞死亡抵抗细胞死亡诱导血管生成

4、诱导血管生成细胞能量代谢调节失控细胞能量代谢调节失控促进永久复制促进永久复制致癌基因致癌基因EGFR,BCR-ABL, Her2抑抑癌基因癌基因 BRCA1/2, PT53Enhanced Glucose Uptake癌症分子诊断与癌症特征癌症分子诊断与癌症特征致癌基因致癌基因BCR-ABL2. Why is 癌症分子诊断癌症分子诊断 needed?a.预测：遗传性癌症（预测：遗传性癌症（BRCA1/2,TP53)b.早期诊断：及时治疗早期诊断：及时治疗c. 预后：预后：Breast cancer: ER+; TNBCd. 验证对治疗的反应验证对治疗的反应e. 传统的肿瘤检测特异性和灵敏性差传

5、统的肿瘤检测特异性和灵敏性差f. 肿瘤的异质性肿瘤的异质性-个体化治疗个体化治疗 传统的肿瘤检测和治疗方法传统的肿瘤检测和治疗方法1）视，摸，）视，摸，X光射线光射线, CT, MRI, 活检，病理活检，病理切切 片评估片评估2)手术后的化疗，放疗手术后的化疗，放疗 副作用大，易复发，抗药性副作用大，易复发，抗药性 (de novo) 用于癌症分子诊断的技术用于癌症分子诊断的技术PCR/DNA sequencingcDNA Microarrays* FISH（荧光原位杂交）（荧光原位杂交）High throughput sequencing Genome wide sequencing* RN

6、Aseq (transcriptome profiling)* Exome-sequencing* CGH（比较基因组杂交）（比较基因组杂交）a. Detecting DNA and RNA mutations and copy number changes in cancer cells CNV: CGH, FISHRNA-Seq (Transcriptome Profiling)a. Cloonan, N. Nat. Methods 5, 613619 (2008)b. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wo

7、ld, B. Nat. Methods 5, 621628 (2008)c. Shendure, J., Mitra, R.D., Varma, C. & Church, G.M. Nat. Rev. Genet. 5, 335344 (2004).nature methods | VOL.5 NO.7 | JULY 2008 | 585转录组转录组分分析析1. Discover novel transcripts: New alternative splicing species（新的选择性的剪接RNA产物） Fusion transcripts due to chromosomal tra

8、nslocation New transcriptional units New mutations and SNP (单核苷酸多态性)2. Highly related genes can be analyzed easily3. Detect low abundant transcripts with quantitation (digital; spiked with known concentration of RNA standard)4. Results can be reproduced between laboratories and across platformsAdvan

9、tages of RNA-Seq over Microarrays for Gene Expression AnalysisExome Sequencing (AKA targeted exome capture)180,000 exons in the genome. These constitute about 1% of the human genome or about 30 megabases.WikipediaAG GUAG GUTCTCCACAComparative Genomic Hybridization（CGH）Wikipedia检测基因组中不同检测基因组中不同DNA拷贝数

10、变化拷贝数变化Chromosomal Region Gain and Loss in A Neuronal Tumor Sample by CGHWikipediaWikipedia检测染色体上基因拷贝数变化检测染色体上基因拷贝数变化b. Detecting altered properties of cancer cells * Immunohistochemistry (IHC): * Cell specific secreted proteins/peptides: ELISA: PSA in prostate cancer * PET-CT (higher glucose uptake

11、): 影像成像技术影像成像技术 * Circulating tumor cells (CTC) (metastasis): 流式细胞仪流式细胞仪 Cellsearch CTC Test (Approved by FDA)用于癌症分子诊断的技术用于癌症分子诊断的技术Immunohistochemistry (IHC)（免疫免疫组组化化）H202 ELISA (Enzyme-Linked Immunosorbent Assay) (酶联免疫吸附试验酶联免疫吸附试验)Detection of Tumor Markers by ELISA CA-125 (a glycoprotein) is comp

12、onent of ocular surface, the respiratory tract and the female reproductive tract epithelia. Its level is elevated in blood of ovarian cancer patients ( but not very specific). PSA is a member of the kallikrein-related peptidase family and is secreted by the epithelial cells of the prostate gland. It

13、s level is elevated in blood of prostate cancer patients.1. PET-CT: 0.5 cm的结节的结节 (90%) Cancer cells take up a lot of glucose 2. 常规常规CT: 1cm 的结节的结节 PET-CT (Positron emission tomographycomputed tomography) (正电子发射断层扫描正电子发射断层扫描)2-18 F-fluoro-2-deoxy-D-glucose (FDG)2-氟氟-2-脱氧-D- 葡萄糖PET-CT (正电子发射断层扫描)检测肿瘤的

14、原理检测肿瘤的原理脂肪和核酸合成的底物脂肪和核酸合成的底物 有氧有氧 肝糖肝糖CELLSEARCH CTC Test (Veridex)A 7.5-mL sample of blood is placed in a special tube, centrifuged to separate solid blood components from plasma, then placed in the CellTracks AutoPrep System. Using ferrofluid nanoparticles with antibodies that target epithelial c

15、ell adhesion (EpCAM), CTCs are magnetically separated from the bulk of other cells in the blood.Use antibodies to stain cytokeratins (CK8,18,19) to confirm the presences of CTC.Use CD45 antibody to deplete contaminated bloods.The presence of CTCs in the peripheral bloods is associated with decreased

16、 progression free survival and decreased overall survival in patients treated for metastatic breast or metastatic colorectal cancer.The Presence of CTCs in the Peripheral Bloods can Predict Overall Survival of Breast Cancer 癌症分子诊断和个体化治疗癌症分子诊断和个体化治疗1. 肿瘤细胞基因组的不稳和异质性肿瘤细胞基因组的不稳和异质性2. 制定更好的治疗方案和方法制定更好的治

17、疗方案和方法c. NSCLC: EGFR mutation, EML4-ALKb. Breast Cancer: ER, Her2a. CML: -Imatinib resistance- BCR-ABL mutationsMost of the cancers are driven by mutations of different genes Only some rare cancer (i.e. CML) is mainly dependent on a single gene mutationFigure 2.23a The Biology of Cancer ( Garland Sc

18、ience 2007)Chromosomal Translocation in Chronic Myelogenous Leukemia (CML) Figure 4.15a The Biology of Cancer ( Garland Science 2007)BCR-ABL is the Result of Reciprocal Chromosomal TranslocationBCR-ABLFigure 4.15b The Biology of Cancer ( Garland Science 2007)Different Form of BCR-ABL is Associated w

19、ith Distinct Types of LeukemiaDifferent parts of BCRSame ABL(B)癌症分子诊断癌症分子诊断Needs PCR to identity differentFusion conjunctions and differentForms of BCR-ABLImatinib Makes CML from a Deadly Cancer into a True Chronic Disease (The best example of targeted cancer therapy)JNCI J Natl Cancer Inst (2011) 1

20、03 (7): 553-561. Gleevec/Imatinib for Treating Chronic Myelogenous Leukemia (CML)NCI/NIH websiteA Small molecule inhibitor/ATP Competitor BCR-ABLBCR-ABLImatinib 抗药性抗药性 1. Activation of the BCR-ABL Independent pathways ODS/T kinaseDBLSH3 SH2NLSDNA BDABDY kinase | Imatinib 抗药性抗药性 1. Activation of the

21、BCR-ABL Independent pathways (minor)2. Mutations in ABL of BCR-ABL (major) Imatinib 抗药性点突变体抗药性点突变体 Sawyers, Nature Medicine 15, 1158 - 1161 (2009) Dasatinib is highly active in most imatinib-resistant BCR-ABLABL Kinase DomainImatinib Gate keeper抗药性点突变体抗药性点突变体(T315I)C-src kinase domainT315IABL Kinase

22、 Domain产生空间位阻产生空间位阻失去氢键失去氢键T315I BCR-ABL is resistant to Imatinib and Dasatinib，but sensitive to Ponatinib.乳腺癌乳腺癌 （Breast Cancer）1)The second leading cause of cancer death in woman with 400,000 death worldwide.2) Three major types of breast cancer in clinica) Luminal type (官腔）官腔） (50-70%): estrogen

23、receptor (ER) and progestrone receptor (PR) (IHC for detection)b) Her2 positive (20-25%): Her2/ErbB2 gene amplification (FISH for detection)c) Triple negative breast cancer (15%) (TNBC) (三阴性乳腺癌）三阴性乳腺癌）: ER, PR, and Her2 negative3) Targeted Breast therapies a) Tamoxifen or aromatase inhibitor （Letroz

24、ole) for ER+ breast cancer b) Herceptin or lapatinib for Her2+ breast cancer c) Only chemotherapy and radiation therapy for TNBC without targeted therapyProblems in Targeted Therapy for BreastCancer and Management of TBNC: 1) Resistance to target therapies: De-novo resistance Acquired resistance 2)

25、Only chemotherapy for TNBC (三阴性乳腺癌）三阴性乳腺癌）: No targeted therapy Faster relapse and metastasis Has the highest death rate among all types of breast cancerBasal-like 1 Basal-like 2 Immunomodulatory Mesenchymal-like Mesenchymal Stem-likeLuminal ARLehmann 2011Lehmann et al, 2011, JCILuminal AR无肿瘤复发的存活无肿

26、瘤复发的存活DHTCo-activatorsCo-repressorsDHTBicalutamideBicalutamide（AR抑制剂）抑制剂） Inhibits LAR-TNBC Tumor Growth in Mice1）全世界肺癌发病例超过百万人。肺癌死亡率是所有癌）全世界肺癌发病例超过百万人。肺癌死亡率是所有癌症中最高的。症中最高的。85%左右的肺癌是非小细胞肺癌（左右的肺癌是非小细胞肺癌（NSCLC)。2）EGFR 因为在绝大多数的因为在绝大多数的NSCLC表达较高，以此被作治表达较高，以此被作治疗靶标。疗靶标。3）Iressa/Gefitinib 是是EGFR的小分子抑制剂。可对

27、绝大多数的小分子抑制剂。可对绝大多数的的NSCLC是没有治疗效果的。是没有治疗效果的。4）Iressa/Gefitinib 只对有特定只对有特定EGFR突变体的突变体的NSCLC （20%）有疗效。这个发现为癌症的个体化治疗开创了先例。有疗效。这个发现为癌症的个体化治疗开创了先例。Allelic-PCR based diagnosis and sequencing 在这个发现中起在这个发现中起了关键作用。了关键作用。非小细胞肺癌非小细胞肺癌 (NSCLC) 的治疗的治疗1) FDA approved in May 2003 for NSCLC, Gefitinib is currently m

28、arketed in over 64 countries.2) In June 2005 the FDA withdrew approval for use in new patients due to lack of evidence that it extended life.Iressa/Gefitinib: Small Molecule ATP Competitor for EGFRCheng et al, 2012, Modern Pathology, 25:347- EGFR Signaling Pathways (蛋白酪氨酸激蛋白酪氨酸激酶酶受体受体信号通道信号通道)Pao W

29、et al; Nature Cancer Review, 2010, 10:760-敏感性突敏感性突变变体体抗抗药药性突性突变变体体敏感性突敏感性突变变体体: are activating mutations that make cancer cells are addictive to EGFR signalingand make the Gefitinib bind to ATP binding pocket of the mutant EGFR better抗抗药药性突性突变变体体: make Gefitinib unable to fit into ATP binding pocket

30、Lynch TJ et al; N Engl J Med 2004; 350:2129-蛋白酪氨酸激酶蛋白酪氨酸激酶抑制剂抑制剂Gefitinib敏感和抗药性的敏感和抗药性的EGFR点突变点突变Cheng et al, 2012, Modern Pathology, 25:347-蛋白酪氨酸激蛋白酪氨酸激酶酶抑制剂抑制剂Gefitinib 敏感和抗药性的敏感和抗药性的EGFR点突变点突变Pao W et al; Nature Cancer Review, 2010, 10:760-有敏感性有敏感性EGFR点突点突变变的病人群的病人群EGFR Mutations and its Associat

31、ion to Responses to Gefitinib Therapy 无无筛选筛选病人群病人群Pao W et al; Nature Cancer Review, 2010, 10:760-非小非小细细胞肺癌胞肺癌Gefitinib抗抗药药性的性的多种途径多种途径Cheng et al, 2012, Modern Pathology, 25:347-The Discovery of EML4 and ALK Fusion in NSCLC ALK: Receptor Tyrosine KinaseCrizotinib: a smallmolecule specific inhibitor

32、 for ALK (PF)Whole genome wide sequencing and RNA seqTreating EML4-ALK+ Advanced NSCLC with Crizotinib 1) 6个月疗程, 57% 的病人对治疗有反应。2) 6个月progression-free survival 有 72%。E. L. Kwak et al ., N. Engl. J. Med . 363, 16931703 (2010).Sci. Transl. Med. 2012, 4:120-Allelic-PCR based diagnosis/sequencing 引起引起Crizotinib抗抗药药性的性的多种多种ALK的点突的点突变变Sci. Trans. Med. 2012, 4:120-ImatinibGefitinibCrizotinibALK+肺癌肺癌Crizotinib抗抗药药性的性的多种途径多种途径ALK AmplificationpEGFRKit AmplicationCheng et al, 2012, Modern Pathology, 25:347-主要主要驱动驱动（ （Driver） ） 突突变变基因在基因在非小非小细细胞肺癌里出胞肺癌里出现现的的频频率率基因基因检测检测技技术术