2015临床病理高峰论坛提供课件applications of diagnostics to guide precision medicine for gi malignancies

1、Dongfeng Tan, MDNov 14,2015Guangzhou,Applications of Diagnostics to Guide Precision Medicine for GI Malignes2015Objectives*Introduction and Methodologies*Gastric Tumors and Their Molecular Features*Colorectal Cancer and Molecular Testing*New Trends of Applications of Molecular Diagnostics in Gastroi

2、ntestinal TumorsII. Gastric Cancer: Pathological/MolecularFeaturesPathological FeaturesAnatomic site Tumor size Tumor type Histologic grade Depth of invasionAngiolymphatic invasion Perineural invasionMargin involvement (proximal, distal, radial)Lymph node involvement (total involved, total examined)

3、 Other features (gastritis, dysplasia, specific types of infection) Molecular FeaturesAnatomic and Pathologic Markers in GCAVariableFavorableIntermediateUnfavorableTumor stageHistologic type Differentiation Vascular Invasion Mitotic Index Lymphocyte infiltrationTumor giant cellsIintestinal Well Abse

4、nt Low PresentAbsentIIAdenoModerately-III-IVsignet ringPoorly Present High AbsentPresentMajor Pathways in Gastric CarcinogenesisSoft tissue of the StomachUsing GIST as A MC-kit(CD117) + in 90-95% of GIST (IHC)Unequivocal C-kit staining, use DOG1(IHC) Mutation analysis: c-KIT gene and PDGFRA geneUp t

5、o 10% (-) in GISTJ Clin Pathol 2008;61:722J Clin Oncol 2008;26:5360C-Kit Gene Mutations 4个外显子: 11、9、13、17号 11号外显子基因突变最常见(70-80%)。突变的类型：缺失性突变、错义点突变、序列重复。 9号外显子基因突变，几乎均为编码Ala502- Tyr503的6个核苷酸序列重复。主要发生于小肠GIST。 13号外显子基因突变，错义突变，较少见，1-2%。这种突变与恶性行为有关。Bad outcome 17号外显子编码c-kit的催化酪氨酸激酶2（磷酸转移酶）结构域，在GIST中突变率较低

6、。引起格列卫耐药(second mutation)多发于17号外显子。PDGFRA Mutations: do not respond toTKIPDGFRA基因突变发生率较低（5-7%），主要有三种，分别涉及18号，12号，14号外显子。C-kit基因突变的GIST中，有30-40%的病例PDGFRA基因突变。PDGFRA基因突变主要涉及18号外显子（>80%）， 为错义点突变（D842V），这种突变对格列卫耐药(primary resistant)。14号外显子的错义点突变如为胃上皮样间质瘤， 提示预后较好。12号外显子的错义点突变导致Asp替换Val561III. Genetics

7、 of Colorectal Cancer andMolecular Tests15-20%80-85%10-15%1%80+%2 to 5%APCGermlineMutationAcquiredAPC, p53,DCC, kras,LOH,.MMR Germline MutationMLH1 MSH2 MSH6 PMS2 Epigenetic Hyper- methylation of promoter region of mismatch repair (MMR) gene MLH1 Most cancers occur inolder peopleSporadicFAPHNPCCSpor

8、adic MSI(+)MSI(Microsatellite Instability)CIN(Chromosome Instability)Tests of dMMR for Precision Colon CancerMedicineCarcinoma risk assessment: HNPCC (LynchSyndrome) and Microsatellite Instability (MSI)Criteria ofCRCdetermination of molecular type ofAssessment of CRC PrognosisSelecting and Monitorin

9、g Therapy Selecting Patient for ImmunotherapyResults of MSI TestPMS2+MLH1+MSH2-MSH6-Lynch Syndrome (HNPCC)Most common hereditary CRC syndromeUp to 5 % of CRCsAutosomal dominant, penetrance 80%HNProgresses rapidly, early detection is criticalfor affected pt, and their 1st-D relativesSusceptibility to

10、 CRC & extracolonic cancersGermline mutation in genes belonging to DNA MMR family- MLH1, MSH2, MSH6, or PMS2Mutations in these genes lead to defective DNA repair and microsatellite instabilityCharacteristics Lynch SyndromeHigh lifetime cancer risk:80% by 75 y.o.Early age at onset:45 yearsPredomi

11、nately in right colonExtracolonic cancers: GYN, GI (gastric, small bowel, pancreas), GU, liver, bile ducts, brain, sebaceous skin tumorsLynch综合征相关肿瘤 Sentinel Cancer结直肠内膜胃肝胆管输尿管小肠脑/神经系统MSI is Caused by Failure of MismatchRepair (MMR) Genesextrogenic (UV, Sancar) introgenic(random, Modrich)Normal DNA

12、repairBase pair mismatchT CT A CA G C T GT CT A CDefective DNA repair (MMR+)A G C T GT C T A CA G A T GT C G A CA G C T GDetection of Patients with MSI+ andLynch SyndromeHow do we identify patients?HistoryHistology TestsBethesda Guidelines(1996)CRC dx <50 y.o.CRC with MSI-H histology dx <60CRC

13、 with >1 FDR with an HNPCC-related tumor, with one cancer dx <50Synchronous or metachronous CRC, or other HNPCC-associated tumor(s) regardless of age CRC with >2 FDRs or SDRs with an HNPCC-associated tumor, regardless of ageMSI test in patients meeting any of the aboveJNCI. 2004;96(4):261-2

14、68.MSI Associated Histological FeaturesIntraepithelial lymphocytesCrohns like lymphocytic reaction- most sensitiveMucinous and signet ring cellsMedullary carcinoma, tumor heterogeneity Sensitivity and specificityJass J, Gut, 1998; Alexander, Am J Pathol, 2001; Jass J,Fam Cancer, 2004; Greenson, Am J

15、 Surg Pathol, 2008Adenoma and AdenocarcinomaLynch Syndrome PMS2/MLH1+ Adenoma Adenoma Adenocarcinoma Adenocarcinoma MSH2/MSH6+ MSH2/MSH6- Adenoma Adenocarcinoma Pathological characteristics of MSI-H in CRCSpecific morphology:Tumor-infiltrating lymphocytes Crohns-like lymphocytic reaction Mucinous ad

16、enocarcinoma Signet ring cell differentiation Medullary adenocarcinomaHG carcinoma with heterogeneous elementsAbnormal Immunophenotype:Aberrant CK7/CK20 expression: unreactive Aberrant CDX2 expression: unreactive BEREP4(EPCAM) expression: unreactiveDetection of Patients with MSI +and Lynch SyndromeH

17、ow do we identify patients?HistoryOffer some infoHistologyEvaluate MMR systemMSI Tumor DNA(PCR)IHC Tumor proteinJ Clin Oncol. 2008;26:5783Genet Med. 2009;11:35MSI screening test methodologiesParameterIHC analysisMSI assayAssessment levelProteinsDNASensitivity90-93%Up to 95%CostLess expensiveMore exp

18、ensiveAvailabilityMost pathology labsMolecular labsImplicates genesYesNoSampleTumor cellsTumor and normal cellsTurnaround timeSame dayA couple of daysVariability in resultsCapricious stainingDepending on tumorcellularityInterpretationExperienced pathologistRelatively straightforwardPanels of Immunos

19、tains (IHC)Assess MMR proteinsNormally presentIf protein is absent, gene is not being expressed (mutation or methylation)Helps direct gene testing by predicting likely involved geneMSH2MLH1Using twobodies, tocheck MSH6 and PMS2(short panel)PMS2MSH6No germline mutation in MLH1, MSH2, MSH6, PMS2consid

20、er family history, MSI analysisNo BRAF mutation, <5%Sequencing of MLH1 and/or PMS2BRAF mutationpresent, 14%BRAF mutationanalysisSequencing of MSH2 and/or MSH6All proteins staining, 80%, microsatellite stableMLH1 or PMS2 not staining, 15-20%, microsatellite unstableMSH2 or MSH6 not staining, 2%, m

21、icrosatellite unstableNewly diagnosed patients with CRCIHC analyses of MLH1, MSH2, MSH6, PMS2Reporting: MMR-INTACTImmunoperoxidase stains were performed withbodiesfor the DNA mismatch repair gene products MLH1, MSH2,MSH6 and PMS2.Intact nuclear expression is evident forall four of these proteins in

22、carcinoma cells and in non-neoplastic cells that serve as internal controls. Therefore,the likelihood ofdefective DNA mismatch repair/highlevels of microsatellite instability (MSI-H) in the tumor islow.Immunohistochemistry is approximately 90-95%sensitive for detection of MSI-H. Most of the MSI-Hcar

23、cinomas missed by immunohistochemistry occur in patients with Lynch syndrome/hereditary non-polyposis colorectal cancer (HNPCC) syndrome.Reporting: Loss of MLH1/PMS2Abnormal loss of MLH1 and PMS2 protein expression in carcinoma cell nuclei is evident with retention of expression in non-neoplastic ce

24、ll nuclei that serve as internal controls. Normal MSH2 and MSH6 expression are also retained in carcinoma cell nuclei. These findings indicate defective DNA mismatch repair/high levels of microsatellite instability (MSI-H) in the tumor. Loss of MLH1 function could be due to germline abnormality or s

25、omatic hypermethylation or mutation.To aid in differentiating these two possibilities, we will perform molecular studies for hypermethylation for the MLH1 promoter and for BRAF mutation, both of which are typically associated with sporadic MSI-H tumors. If clinically indicated, germline analysis of

26、the MLH1 gene could also be considered.Methylation or Germline MutationMSH6+MLH2+MLH1-PMS2-Reporting: Loss of MSH2/MSH6Abnormal loss of MSH2 and MSH6 protein expression in carcinoma cell nuclei is evident with retention of expression in non-neoplastic cell nuclei that serve as internal controls. Nor

27、mal MLH1 and PMS2 expression are also retained in carcinoma cell nuclei. These findings indicate defective DNA mismatch repair/high levels of microsatellite instability (MSI-H) in the tumor and are strongly suggestive of a germline MSH2 abnormality (Lynch syndrome/hereditary non-polyposis colorectal

28、 cancer, HNPCC). If clinically indicated, germline analysis of the MSH2 gene and EPCAM/TACSTD1 gene could also be considered.MMR基因及突变比率Accuracy of IHC & MSI: CRC and EC(False Negative dMMR)4 of 56 mutations (7%) missed by IHC-Variability in staining 3 of 56 mutations (5%) missed by MSI Mucin and

29、 scant tumor cellsMLH1:Internal control and negative tumor nuclei with some positive nucleoliMicrosatellite Panels by PCRNCI Panel (minimal): 5 markers2 mono-nucleotides: BAT25 & BAT263 dinucleotides: D5S346, D2S123 & D17S250MDAanel: 7 markers2 more mono-nucleotide: BAT40, TGFbetaR2Microsate

30、llite InstabilityBAT-26 and D2S123 - GenotypeScreening is Feasible for LS: Choosing theScreening TestMSI vs. IHCIHC isily available, while MSI requiresmolecular diagnostics facilitiesIHC with 4bodies is similar in cost to MSIwith 5 markersIHC directs gene testing saving money(IHC screening, then PCR

31、 confirmatory)Ethical issues surrounding IHCBoth IHC and MSI have limitationsPrognosis Significance of Identifying CRCwith MSI-HBetter prognosis: Patients with MSI+ CRCshave more favorable overall survival than those with MSS tumors(meta-analysis of 32 studies: 7,642 patients. J Clin Onc, 2005)Stron

32、g evidence that individuals with MSI+CRCs do not respond as well to 5-FU based chemotherapy regimens (Ribic, NEJM, 2003; Carethers, Gastroenterol, 2004; Jover, Gut, 2006; Jover, Eur J Cancer, 2008; Sargent, J Clin Onc 2008, ASCO.)Lynch Syndrome/HNPCC检测流程MSI：微序列不性MSI-H：高度微MSI-L：低度微序列不序列不MSS：微：检测阳性：检测

33、序列MSSMSI-LHNPCC可能性非常小,不建议进行MMR基因突变检测 MSI，PCR MMR，IHC-检测到BRAF突变或获得性MLH1甲基化，很可能是散发性结直肠癌MLH1基因突变检测检测说明：1、如果中有HNPCC先证者， MMR突变位点已知，可直接选择相应突变位点进行检测。2、本检测流程根据Bethesda指导原则设计。3、PMS2暂时不提供基因突变检测。 MSH6突变检测 MLH1基因突变检测 MSH2突变检测PMS2突变检测MSH6突变检测MSH2突变检测 hMLH1甲基化检测 BRAF 突变检测MSI-HPMS2表达异常MSI-HMSH6表达异常MSI-HMSH2和MSH6表达

34、异常MSI-HMLH1表达异常MSI-HIHC未检测到表达异常疑似HNPCC51Sentinel Cancer in Lynch Syndrome*Colorectal*Endometrioid Ca of Uterus/ Ovary*Stomach*PancreasMMR缺陷是PD-1抗体pembrolizumab疗效的生物标志物*Trial: 研究纳入了三组：MMR表达的肠癌25，MMR缺陷肠癌13，MMR缺陷其他肿瘤（10）。都有转移灶，且既往治疗失败* Results: 两组治疗效果的巨大差异：在MMR缺陷的肠癌病人中，客观缓解率（ORR）为62%，而MMR表达者无一缓解。疾病率（DC

35、R），MMR缺陷者为92%，vs表达者16%。*Mechanism: MMR缺陷的患者平均有1782个突变，而MMR表达的平均有73个突变。免疫系统发现并摧毁肿瘤的机会便增加2015/11/18Advances in Target TherapySummary: Applications of dMMR in MolecularDiagnostics & Precision Cancer MedicineCarcinoma risk assessment: HNPCC (LynchSyndrome)LS patients at high risk for second primary

36、cancers (CRC & extracolonic): Early Screening Assessment of CRC Prognosis: Better outcomeEvaluation of Treatment: MSI+ CRC patients not respond well to 5-FUGenetic Counseling and Cancer Prevention Selecting Patient for ImmunotherapyEGFR pathway: Mutation frequencies & loss ofprotein expressi

37、on in CRCCIMP-H and BRAF mutationK-RAS突变特点多形性癌K-RAS突变率最高突变与吸烟无关突变与有关突变与EGFR靶向治疗有关突变与化疗敏感度有关：G12V突变：铂类药敏感；G12D突变：敏感，紫杉醇不敏感G12C突变：紫杉醇培美曲塞敏感，铂类药不敏感哪些人对抗EGFR单抗敏感？KRAS+: 不敏感KRAS和BRAF突变几乎不同时发生（一个只发生其中一种突变），建议krasKRAS-/BRAF+: 敏感性差进行Braf检测KRAS-/BRAF-/NRAS+: 敏感性差KRAS-/BRAF-/ PI3K+(exon 20):敏感性差KRAS-/BRAF-/PI

38、3K-/PTEN+: 敏感性差KRAS, BRAF,NRAS,PI3K,PTEN：“5阴”敏感武忠弼（1919-2007）2015/11/18小结：需要进行分子检测的曲妥珠单抗（®）治疗(1) 乳(2)进展期胃或胃食管连接部：Her-2/neu (IHC and FISH)吉非替尼（厄洛替尼（®）治疗非小细胞肺癌：EGFR突变检测®）治疗(1)非小细胞肺癌(2）进展期胰：EGFR突变西妥昔单抗（爱必妥®）治疗（1）转移性结直肠癌（2）复发/转移性头颈部鳞状细胞癌:要求进行Kras突变检测，强烈建议检测Braf, Nras, PI3K.替比®）治

39、疗转移性结直肠癌:KRAS突变检测，并推荐检测单抗（BRAF，NRAS，PI3K伊马替尼（格列卫®） 1）GIST（2）慢性粒细胞白血病（3）急性淋巴细胞白血病:c - kit和PDGFR突变检测指导GIST治疗；淋巴瘤检测Ph唑蒂尼治疗非小细胞肺癌：检测ALK融合基因转移性黑色素瘤：BRAF 突变检测5-FU and 5-FU衍生物不敏感.治疗多种癌：结直肠癌推荐检测MSI，MSI+对此类药IV. New Trends of Applications ofMolecular Diagnostics in Gastrointestinal TumorsWhere is the fut

40、ure?基因/DNA和修饰(基因组和表观基因组学)基因产物RNA和蛋白质(翻译转录和蛋白质组学)基因间接产物如代谢物和碳水化合物(新陈代谢和糖代谢)以上各因子间互相作用其他子生物学因素：如环境和表型（细胞、组织、器官和环境之间相互作用产生表型因素）Selected databases and tools for therapeutic targeting & drug discoveryCategoryResourceDescriptionOnline referenceCancer genome analysis(COSMIC)Database of cancer mutations

41、(literature-derived)GP/cosmic/The CancerGenome AtlasDatabase of cancer mutations(ex-vivo derived)Gene expression analysisOncomineCompendium of gene expression data with analysis tools.htmlGene ExpressionOmnibus (GEO)Repository of gene expression data with a curated dataset browser and analysis tool.

42、Array ExpressGene expression database hosted by the European Bioinformatics InstituteFunctional analysesGene Ontology (GO)Genes by biological process, function, and cellular componentKEGG: Kyoto Encyclopedia of GenomesSystems-level organization of genes, pathway and disease associationDatabase for A

43、nnotation, Visualization and Integrated Discovery (DAVID)Identifies enriched biological themesDrug sensitivityNCI-60 Drug Therapeutic Program (DTP) Human Tumor Cell Line Screen and COMPAREDrug sensitivities of >45,000 compounds and natural products in NCI-60 cancer cell line panelTargeted NGS Tes

44、t for CRCTargeted GeneSignificance-APC(Exon 1-16)BRAF (Exon 15, 16)Familial adenomatous polyposis syndrometo-EGFR Mab RxPrognosisKRAS (Exons 2, 3)MLH1 (Exons 1-19)MSH2 (Exons 1-16)MSH6 (Exons 1-10)MUTYH (Exons 1-16)NRAS (Exons 2, 3)PIK3CA (Exons 9, 20)PMS2 (Exons 1-15)PTEN (Exons 7, 8)to-EGFR Mab Rx

45、Lynch syndrome; 5FU therapy response Lynch syndrome; 5FU therapy response Lynch syndrome; 5FU therapy response Familial adenomatous polyposis syndrometo-EGFR Mab Rxto-EGFR Mab RxLynch syndrome; 5FU therapy responseto-EGFR Mab RxSTK11 ( Exons 1-10)Peutz-Jeghers syndrome-Targeted NGS for Hereditary Ca

46、ncersTargeted GeneSignificance-APCBRCA1 BRCA2Familial adenomatous polyposis syndromeHereditary breast cancer and ovarian cancer Hereditary breast cancer and ovarian cancerPrognosisHereditary diffuse gastric cancer syndrome Lynch syndrome; 5FU therapy response Lynch syndrome; 5FU therapy response Lyn

47、ch syndrome; 5FU therapy response Familial adenomatous polyposis syndrome Lynch syndrome; 5FU therapy response Peutz-Jeghers syndromeCDH1MLH1 MSH2 MSH6 MUTYH PMS2 STK11-Compensive Cancer Panel400 genes, 10,000 amplicons, one dayABL1ATMBRD3CD74CIITADICER1ETV4ABL2AXIN2BRD4CD79ACLP1DNMT3AETV5ACSL3BAP1B

48、RIP1CDNBNNRASPCSK7PRDM16SDHDSSX1TFPTACSL6BARD1BTG1CDNCKIPSDNSD1PDE4DIPPRF1SEPT5SSX2TFRCAFF1BCAR3BUB1BCNCOA1NTRK1PDGFBPRKAR1ASEPT6SSX4THRAP3AFF3BCL10C15orf55CDNCOA2NTRK3PDGFRAFANCCFNBP1HIP1IKZF1KLF6MDM4MSH6AFF4BCL11ACANT1CNCOA4NUMA1PDGFRBFANCD2FOXL2HIST1H4IIL2KLK2MECOMMSI2AKAP9BCL11BCARD11CNDE1NUP214

49、PER1FANCEFOXO1HLFIL21RKRASMEN1MSNAKT1BCL2CARSCDNEK9NUP98PHOX2BFANCFFOXO3HMGA1IL6STKTN1METMTCP1AKT2BCL3CASC5CDNF1OGG1PICALMFANCGFOXO4HMGA2IRF4LASP1MITFMTORALKBCL6CBFA2T3CDNF2OLIG2PIK3CAFASFOXP1HNF1AITGA10LCKMKL1MUC1APCBCL7ACBFBCNFE2L2OMDPIK3R1FBXW7FSTL3NRNPA2BITGA9LCP1MLF1MUTYHARHGAP26BCL9CBLCENFIBPAFAH1B2PIM1FCGR2BFUSHOOK3ITKL