腫瘤的分子生物學(xué)檢驗(yàn)課件

1腫瘤的分子診斷

Moleculardiagnosisoftumor

李江濱副教授2第一節(jié)腫瘤診斷的生物標(biāo)志物染色體異常、基因異常、單核苷酸多態(tài)性、表觀(guān)遺傳異常、miRNA第二節(jié)腫瘤的分子生物學(xué)檢驗(yàn)技術(shù)第三節(jié)腫瘤分子生物學(xué)檢驗(yàn)的臨床應(yīng)用乳腺癌腫瘤分子診斷與個(gè)體化醫(yī)療3什么是腫瘤？腫瘤（Tumor）是機(jī)體在各種致癌因素作用下，局部組織的某一個(gè)細(xì)胞在基因水平上失去對(duì)其生長(zhǎng)的正常調(diào)控，導(dǎo)致其克隆性異常增生而形成的異常病變。學(xué)界一般將腫瘤分為良性和惡性?xún)纱箢?lèi)。5AbnormalcellulargrowthTumorsandcancersaredifferent.Atumordevelopswhenalesionorlump病變或腫塊isformedinyourbodyduetoabnormalcellulargrowth.Inthecaseofcancer,thiscellulargrowthisuncontrollableanditspreadsinthebody.6Treatment

CancerSurgery,chemotherapyandradiotherapy.TumorRemovingabenigntumorisrelativelyeasythroughsurgery,andtheconditiondoesnotrecur.7第一節(jié)腫瘤診斷的生物標(biāo)志物最早的腫瘤標(biāo)志物：本-周蛋白，1846年目前已發(fā)現(xiàn)一百多種腫瘤標(biāo)志物蛋白與核酸兩大類(lèi)腫瘤相關(guān)染色體異常、基因異常、單核苷酸多態(tài)性、表觀(guān)遺傳異常、miRNA8一、腫瘤相關(guān)的染色體異常多數(shù)腫瘤細(xì)胞存在染色體異常。1.染色體數(shù)目異常例：某個(gè)癌細(xì)胞的染色體共104條，包括許多異常的染色體10二、腫瘤相關(guān)基因表達(dá)異常

原癌基因:sis、VEGF、EGFR、c-myc抑癌基因:APC、BRCA、p53、Rb細(xì)胞周期調(diào)節(jié)基因:cyclins、CDKs、CKIs細(xì)胞凋亡相關(guān)基因:Bcl-1、p53、bcr-abl基因組穩(wěn)定相關(guān)基因(DNA修復(fù)基因):APE1腫瘤轉(zhuǎn)移相關(guān)基因:nm23、WDNM、sis、p53腫瘤血管生成相關(guān)基因:VEGF

、EGFR、p531214MostSNPshavenoeffectonhealth.ResearchershavefoundSNPsthatmayhelppredictanindividual’sresponsetocertaindrugs,susceptibilitytoenvironmentalfactorssuchastoxins,andriskofdevelopingparticulardiseases.SNPscanalsobeusedtotracktheinheritanceofdiseasegeneswithinfamilies.FuturestudieswillworktoidentifySNPsassociatedwithcomplexdiseasessuchasheartdisease,diabetes,andcancer.15三、腫瘤相關(guān)單核苷酸多態(tài)性SNP:Singlenucleotidepolymorphisms1.SNP與腫瘤腫瘤易感基因、腫瘤藥物治療相關(guān)基因腫瘤個(gè)體化診療2.SNP的研究思路研究對(duì)象差異性研究技術(shù)：PCR、芯片研究難點(diǎn)：樣本采集16四、腫瘤相關(guān)表觀(guān)遺傳異常表觀(guān)遺傳：表觀(guān)遺傳（epigenetics）是指DNA序列不發(fā)生變化，但基因表達(dá)卻發(fā)生了可遺傳的改變。這種改變是細(xì)胞內(nèi)除了遺傳信息以外的其他可遺傳物質(zhì)發(fā)生的改變，且這種改變?cè)诎l(fā)育和細(xì)胞增殖過(guò)程中能穩(wěn)定傳遞。幾乎所有類(lèi)型的人類(lèi)腫瘤都存在表觀(guān)遺傳異常抑癌基因的高甲基化和癌基因的去甲基化。異常甲基化常發(fā)生在腫瘤細(xì)胞形成的早期。17五、腫瘤相關(guān)miRNAMicroRNAformationandfunction.flv

18腫瘤相關(guān)長(zhǎng)鏈非編碼RNA長(zhǎng)鏈非編碼RNA(longnoncodingRNA,lncRNA)長(zhǎng)度在200-100000nt之間的RNA分子不編碼蛋白lncRNA參與細(xì)胞內(nèi)多種過(guò)程調(diào)控種類(lèi)、數(shù)量、功能都不明確長(zhǎng)鏈非編碼RNA在腫瘤發(fā)生發(fā)展中的位置/rbmiRNA2012/article/i18822.html21第二節(jié)腫瘤分子生物學(xué)檢驗(yàn)技術(shù)腫瘤分子診斷常用的檢測(cè)標(biāo)本外周血細(xì)胞病灶局部受損組織↓提取DNA或mRNA↓

檢測(cè)23循環(huán)DNA定量檢測(cè)方法早期檢測(cè)方法：二苯胺法、溴化乙錠法、對(duì)流免疫電泳法、RNA-DNA雜交法新的檢測(cè)技術(shù)：放射免疫法，實(shí)時(shí)熒光定量PCR法等24新型檢測(cè)標(biāo)志物：血漿DNA

何謂血漿DNA(plasmaDNA)？又稱(chēng)循環(huán)DNA(circulatingDNA)，是一種無(wú)細(xì)胞狀態(tài)的細(xì)胞外DNA(extracellularDNA)，由長(zhǎng)度不等的單鏈或雙鏈DNA及其混合物組成，主要以DNA-蛋白質(zhì)混合物形式存在，但也存在部分游離DNA。26Cell-freeDNAresuscitatedfortumortestingNatureMedicine14,914-915(2008)

/nm/journal/v14/n9/full/nm0908-914.html27腫瘤分子診斷的常用方法以分子雜交和PCR為核心技術(shù)以DNA為主要檢測(cè)對(duì)象1.致病基因結(jié)構(gòu)異常檢測(cè)2.基因轉(zhuǎn)錄水平檢測(cè)3.基因序列分析及表達(dá)檢測(cè)28腫瘤分子診斷的常用方法染色體數(shù)目異常熒光原位雜交（Fluorescenceinsituhybridization,FISH）30致病基因表達(dá)異常檢測(cè)（1）mRNA檢測(cè)定量PCR（2）蛋白檢測(cè)免疫組化：定性、半定量、定位Westernblot：定量31第三節(jié)腫瘤分子生物學(xué)檢驗(yàn)的臨床應(yīng)用乳腺癌32一、乳腺癌女性發(fā)病率高，5%-10%為家族性。90%家族性乳腺癌涉及BRCA1和BRCA2基因突變。BRCA1突變可發(fā)生在所有細(xì)胞，若發(fā)生在精子或卵子則可以傳給下一代。其他涉及基因：p53、PTEN、c-myc、端粒酶等。論文導(dǎo)讀:

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

33BRCA1BRCA1(breastcancer1,earlyonset早發(fā))isahumantumorsuppressorgene腫瘤抑制基因,whichproducesaprotein,calledbreastcancertype1susceptibility易感性

protein.Itisfoundinthecellsofbreastandothertissue,whereithelpsrepairdamagedDNA,anddestroythecellwhenDNAcan‘tberepaired.IfBRCA1itselfisdamaged,thedamagedDNAcanletthecellduplicate復(fù)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異源雙鏈分析(HDA)bymulti-capillaryelectrophoresis多毛細(xì)管電泳oralsooligonucleotidesarraybasedoncomparativegenomichybridization比較基因組雜交(array-CGH).36ResearchersbelievethatthedefectiveBRCA1proteinisunabletohelpfixmutationsthatoccurinothergenes.Thesedefectsaccumulateandmayallowcellstogrowanddivideuncontrollablytoformatumor.37女性排名第一的常見(jiàn)惡性腫瘤。美國(guó)每8個(gè)人就有1個(gè)人一生中會(huì)得乳腺癌。美國(guó)患乳腺癌的女性占新發(fā)惡性腫瘤的30%，而其中的大約10%的乳腺癌是遺傳性的。乳癌“株連”，一旦家里有人患此疾病，一級(jí)親屬（母親、姐妹或孩子）的女性都應(yīng)該做檢查。38乳腺癌高危人群

有乳腺癌家族史、基因檢測(cè)到BRAC1/2基因突變、曾有過(guò)胸部放療史、乳腺活檢為高危良性病變、乳腺密度高、初潮年齡早于12歲,以及絕經(jīng)年齡推遲等的人群，一般患乳腺癌危險(xiǎn)性會(huì)比較高。3940安吉麗娜·朱莉在2013年5月《紐約時(shí)報(bào)》上刊文“MyMedicalChoice”，講述了決定手術(shù)的原因及經(jīng)過(guò)，最后呼吁所有女性注意預(yù)防乳腺癌。……MydoctorsestimatedthatIhadan87percentriskofbreastcanceranda50percentriskofovariancancer,althoughtheriskisdifferentinthecaseofeachwoman。Onlyafractionofbreastcancersresultfromaninheritedgenemutation.ThosewithadefectinBRCA1havea65percentriskofgettingit,onaverage。41OnceIknewthatthiswasmyreality,IdecidedtobeproactiveandtominimizetheriskasmuchIcould.Imadeadecisiontohaveapreventivedoublemastectomy預(yù)防性的雙乳切除手術(shù)

.Istartedwiththebreasts,asmyriskofbreastcancerishigherthanmyriskofovariancancer。

422015年3月《紐約時(shí)報(bào)》上刊文“diaryofasurgery”Iwantedotherwomenatrisktoknowabouttheoptions.Ipromisedtofollowupwithanyinformationthatcouldbeuseful,includingaboutmynextpreventivesurgery,theremovalofmyovariesandfallopiantubes卵巢和輸卵管.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腹腔鏡雙側(cè)輸卵管卵巢切除術(shù).Therewasasmallbenigntumorononeovary,butnosignsofcancerinanyofthetissues.在一側(cè)卵巢有一個(gè)小的良性腫瘤，任何組織都沒(méi)有癌癥跡象。

47Ihavealittleclearpatch透明貼thatcontainsbio-identicalestrogen雌激素.AprogesteroneIUD黃體酮節(jié)育器wasinsertedinmyuterus子宮.Itwillhelpmemaintainahormonalbalance,butmoreimportantitwillhelppreventuterinecancer子宮癌.Ichosetokeepmyuterusbecausecancerinthatlocationisnotpartofmyfamilyhistory.

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