肿瘤的分子生物学检验课件

1肿瘤的分子诊断

Moleculardiagnosisoftumor

李江滨副教授2第一节肿瘤诊断的生物标志物染色体异常、基因异常、单核苷酸多态性、表观遗传异常、miRNA第二节肿瘤的分子生物学检验技术第三节肿瘤分子生物学检验的临床应用乳腺癌肿瘤分子诊断与个体化医疗3什么是肿瘤？肿瘤（Tumor）是机体在各种致癌因素作用下，局部组织的某一个细胞在基因水平上失去对其生长的正常调控，导致其克隆性异常增生而形成的异常病变。学界一般将肿瘤分为良性和恶性两大类。5AbnormalcellulargrowthTumorsandcancersaredifferent.Atumordevelopswhenalesionorlump病变或肿块isformedinyourbodyduetoabnormalcellulargrowth.Inthecaseofcancer,thiscellulargrowthisuncontrollableanditspreadsinthebody.6Treatment

CancerSurgery,chemotherapyandradiotherapy.TumorRemovingabenigntumorisrelativelyeasythroughsurgery,andtheconditiondoesnotrecur.7第一节肿瘤诊断的生物标志物最早的肿瘤标志物：本-周蛋白，1846年目前已发现一百多种肿瘤标志物蛋白与核酸两大类肿瘤相关染色体异常、基因异常、单核苷酸多态性、表观遗传异常、miRNA8一、肿瘤相关的染色体异常多数肿瘤细胞存在染色体异常。1.染色体数目异常例：某个癌细胞的染色体共104条，包括许多异常的染色体10二、肿瘤相关基因表达异常

原癌基因:sis、VEGF、EGFR、c-myc抑癌基因:APC、BRCA、p53、Rb细胞周期调节基因:cyclins、CDKs、CKIs细胞凋亡相关基因:Bcl-1、p53、bcr-abl基因组稳定相关基因(DNA修复基因):APE1肿瘤转移相关基因:nm23、WDNM、sis、p53肿瘤血管生成相关基因:VEGF

、EGFR、p531214MostSNPshavenoeffectonhealth.ResearchershavefoundSNPsthatmayhelppredictanindividual’sresponsetocertaindrugs,susceptibilitytoenvironmentalfactorssuchastoxins,andriskofdevelopingparticulardiseases.SNPscanalsobeusedtotracktheinheritanceofdiseasegeneswithinfamilies.FuturestudieswillworktoidentifySNPsassociatedwithcomplexdiseasessuchasheartdisease,diabetes,andcancer.15三、肿瘤相关单核苷酸多态性SNP:Singlenucleotidepolymorphisms1.SNP与肿瘤肿瘤易感基因、肿瘤药物治疗相关基因肿瘤个体化诊疗2.SNP的研究思路研究对象差异性研究技术：PCR、芯片研究难点：样本采集16四、肿瘤相关表观遗传异常表观遗传：表观遗传（epigenetics）是指DNA序列不发生变化，但基因表达却发生了可遗传的改变。这种改变是细胞内除了遗传信息以外的其他可遗传物质发生的改变，且这种改变在发育和细胞增殖过程中能稳定传递。几乎所有类型的人类肿瘤都存在表观遗传异常抑癌基因的高甲基化和癌基因的去甲基化。异常甲基化常发生在肿瘤细胞形成的早期。17五、肿瘤相关miRNAMicroRNAformationandfunction.flv

18肿瘤相关长链非编码RNA长链非编码RNA(longnoncodingRNA,lncRNA)长度在200-100000nt之间的RNA分子不编码蛋白lncRNA参与细胞内多种过程调控种类、数量、功能都不明确长链非编码RNA在肿瘤发生发展中的位置/rbmiRNA2012/article/i18822.html21第二节肿瘤分子生物学检验技术肿瘤分子诊断常用的检测标本外周血细胞病灶局部受损组织↓提取DNA或mRNA↓

检测23循环DNA定量检测方法早期检测方法：二苯胺法、溴化乙锭法、对流免疫电泳法、RNA-DNA杂交法新的检测技术：放射免疫法，实时荧光定量PCR法等24新型检测标志物：血浆DNA

何谓血浆DNA(plasmaDNA)？又称循环DNA(circulatingDNA)，是一种无细胞状态的细胞外DNA(extracellularDNA)，由长度不等的单链或双链DNA及其混合物组成，主要以DNA-蛋白质混合物形式存在，但也存在部分游离DNA。26Cell-freeDNAresuscitatedfortumortestingNatureMedicine14,914-915(2008)

/nm/journal/v14/n9/full/nm0908-914.html27肿瘤分子诊断的常用方法以分子杂交和PCR为核心技术以DNA为主要检测对象1.致病基因结构异常检测2.基因转录水平检测3.基因序列分析及表达检测28肿瘤分子诊断的常用方法染色体数目异常荧光原位杂交（Fluorescenceinsituhybridization,FISH）30致病基因表达异常检测（1）mRNA检测定量PCR（2）蛋白检测免疫组化：定性、半定量、定位Westernblot：定量31第三节肿瘤分子生物学检验的临床应用乳腺癌32一、乳腺癌女性发病率高，5%-10%为家族性。90%家族性乳腺癌涉及BRCA1和BRCA2基因突变。BRCA1突变可发生在所有细胞，若发生在精子或卵子则可以传给下一代。其他涉及基因：p53、PTEN、c-myc、端粒酶等。论文导读:

Astrongcandidateforthebreastandovarian卵巢cancersusceptibilitygeneBRCA1/cgi/rapidpdf/266/5182/66.pdf

33BRCA1BRCA1(breastcancer1,earlyonset早发)isahumantumorsuppressorgene肿瘤抑制基因,whichproducesaprotein,calledbreastcancertype1susceptibility易感性

protein.Itisfoundinthecellsofbreastandothertissue,whereithelpsrepairdamagedDNA,anddestroythecellwhenDNAcan‘tberepaired.IfBRCA1itselfisdamaged,thedamagedDNAcanletthecellduplicate复制withoutcontrol,andturnintoacancer.34MutationsandcancerriskCertainvariationsoftheBRCA1geneleadtoanincreasedriskforbreastcancer.ResearchershaveidentifiedhundredsofmutationsintheBRCA1gene,manyofwhichareassociatedwithanincreasedriskofcancer.WomenwhohaveanabnormalBRCA1orBRCA2genehaveuptoan60%riskofdevelopingbreastcancer;increasedriskofdevelopingovariancancerisabout55%forwomenwithBRCA1mutationsandabout25%forwomenwithBRCA2mutations.35ThesemutationscanbechangesinoneorasmallnumberofDNAbasepairs.ThosemutationscanbeidentifiedwithPCRandDNAsequencing.Othermethodsareproposed:Q-PCR定量

andQuantitativeMultiplexPCR多重定量ofShortsFluorescentsFragments(QMPSF).Newmethodshavebeenrecentlyproposed:heteroduplexanalysis异源双链分析(HDA)bymulti-capillaryelectrophoresis多毛细管电泳oralsooligonucleotidesarraybasedoncomparativegenomichybridization比较基因组杂交(array-CGH).36ResearchersbelievethatthedefectiveBRCA1proteinisunabletohelpfixmutationsthatoccurinothergenes.Thesedefectsaccumulateandmayallowcellstogrowanddivideuncontrollablytoformatumor.37女性排名第一的常见恶性肿瘤。美国每8个人就有1个人一生中会得乳腺癌。美国患乳腺癌的女性占新发恶性肿瘤的30%，而其中的大约10%的乳腺癌是遗传性的。乳癌“株连”，一旦家里有人患此疾病，一级亲属（母亲、姐妹或孩子）的女性都应该做检查。38乳腺癌高危人群

有乳腺癌家族史、基因检测到BRAC1/2基因突变、曾有过胸部放疗史、乳腺活检为高危良性病变、乳腺密度高、初潮年龄早于12岁,以及绝经年龄推迟等的人群，一般患乳腺癌危险性会比较高。3940安吉丽娜·朱莉在2013年5月《纽约时报》上刊文“MyMedicalChoice”，讲述了决定手术的原因及经过，最后呼吁所有女性注意预防乳腺癌。……MydoctorsestimatedthatIhadan87percentriskofbreastcanceranda50percentriskofovariancancer,althoughtheriskisdifferentinthecaseofeachwoman。Onlyafractionofbreastcancersresultfromaninheritedgenemutation.ThosewithadefectinBRCA1havea65percentriskofgettingit,onaverage。41OnceIknewthatthiswasmyreality,IdecidedtobeproactiveandtominimizetheriskasmuchIcould.Imadeadecisiontohaveapreventivedoublemastectomy预防性的双乳切除手术

.Istartedwiththebreasts,asmyriskofbreastcancerishigherthanmyriskofovariancancer。

422015年3月《纽约时报》上刊文“diaryofasurgery”Iwantedotherwomenatrisktoknowabouttheoptions.Ipromisedtofollowupwithanyinformationthatcouldbeuseful,includingaboutmynextpreventivesurgery,theremovalofmyovariesandfallopiantubes卵巢和输卵管.Ihadbeenplanningthisforsometime.Itisalesscomplexsurgerythanthemastectomy,butitseffectsaremoresevere.Itputsawomanintoforcedmenopause更年期.

43ThentwoweeksagoIgotacallfrommydoctorwithblood-testresults.“YourCA-125isnormal,”hesaid.Ibreathedasighofrelief.ThattestmeasurestheamountoftheproteinCA-125intheblood,andisusedtomonitorovariancancer.Ihaveiteveryyearbecauseofmyfamilyhistory.Butthatwasn’tall.Hewenton.“Thereareanumberofinflammatorymarkersthatareelevated,andtakentogethertheycouldbeasignofearlycancer.”Itookapause.“CA-125hasa50to75percentchanceofmissingovariancanceratearlystages,”hesaid.Hewantedmetoseethesurgeonimmediatelytocheckmyovaries.

44Thedayoftheresultscame.ThePET/CTscanlookedclear,andthetumortestwasnegative.Iwasfullofhappiness,althoughtheradioactivetracermeantIcouldn’thugmychildren.Therewasstillachanceofearlystagecancer,butthatwasminorcomparedwithafull-blowntumor.Tomyrelief,IstillhadtheoptionofremovingmyovariesandfallopiantubesandIchosetodoit.

45Inmycase,theEasternandWesterndoctorsImetagreedthatsurgerytoremovemytubesandovarieswasthebestoption,becauseontopoftheBRCAgene,threewomeninmyfamilyhavediedfromcancer.MydoctorsindicatedIshouldhavepreventivesurgeryaboutadecadebeforetheearliestonsetofcancerinmyfemalerelatives.Mymother’sovariancancerwasdiagnosedwhenshewas49.I’m39.

46Lastweek,Ihadtheprocedure:alaparoscopicbilateralsalpingo-oophorectomy腹腔镜双侧输卵管卵巢切除术.Therewasasmallbenigntumorononeovary,butnosignsofcancerinanyofthetissues.在一侧卵巢有一个小的良性肿瘤，任何组织都没有癌症迹象。

47Ihavealittleclearpatch透明贴thatcontainsbio-identicalestrogen雌激素.AprogesteroneIUD黄体酮节育器wasinsertedinmyuterus子宫.Itwillhelpmemaintainahormonalbalance,butmoreimportantitwillhelppreventuterinecancer子宫癌.Ichosetokeepmyuterusbecausecancerinthatlocationisnotpartofmyfamilyhistory.

48RegardlessofthehormonereplacementsI’mtaking,Iamnowinmenopause更年期.Iwillnotbeabletohaveanymorechildren,andIexpectsomephysicalchanges.ButIfeelateasewithwhateverwillcome,notbecauseIamstrongbutbecausethisisapartoflife