腫瘤的分子生物學(xué)檢驗課件

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、表觀遺傳異常、miRNA第二節(jié)腫瘤的分子生物學(xué)檢驗技術(shù)第三節(jié)腫瘤分子生物學(xué)檢驗的臨床應(yīng)用乳腺癌腫瘤分子診斷與個體化醫(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，局部組織的某一個細(xì)胞在基因水平上失去對其生長的正常調(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兩大類。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腫瘤相關(guān)染色體異常、基因異常、單核苷酸多態(tài)性、表觀遺傳異常、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)基因腫瘤個體化診療2.SNP的研究思路研究對象差異性研究技術(shù)：PCR、芯片研究難點：樣本采集16四、腫瘤相關(guān)表觀遺傳異常表觀遺傳：表觀遺傳（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，且這種改變在發(fā)育和細(xì)胞增殖過程中能穩(wěn)定傳遞。幾乎所有類型的人類腫瘤都存在表觀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)長鏈非編碼RNA長鏈非編碼RNA(longnoncodingRNA,lncRNA)長度在200-100000nt之間的RNA分子不編碼蛋白lncRNA參與細(xì)胞內(nèi)多種過程調(diào)控種類、數(shù)量、功能都不明確長鏈非編碼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)檢驗技術(shù)腫瘤分子診斷常用的檢測標(biāo)本外周血細(xì)胞病灶局部受損組織↓提取DNA或mRNA↓

檢測23循環(huán)DNA定量檢測方法早期檢測方法：二苯胺法、溴化乙錠法、對流免疫電泳法、RNA-DNA雜交法新的檢測技術(shù)：放射免疫法，實時熒光定量PCR法等24新型檢測標(biāo)志物：血漿DNA

何謂血漿DNA(plasmaDNA)？又稱循環(huán)DNA(circulatingDNA)，是一種無細(xì)胞狀態(tài)的細(xì)胞外DNA(extracellularDNA)，由長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為主要檢測對象1.致病基因結(jié)構(gòu)異常檢測2.基因轉(zhuǎn)錄水平檢測3.基因序列分析及表達(dá)檢測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（1）mRNA檢測定量PCR（2）蛋白檢測免疫組化：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惡性腫瘤。美國每8個人就有1個人一生中會得乳腺癌。美國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、基因檢測到BRAC1/2基因突變、曾有過胸部放療史、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，一般患乳腺癌危險性會比較高。3940安吉麗娜·朱莉在2013年5月《紐約時報》上刊文“MyMedicalChoice”，講述了決定手術(shù)的原因及經(jīng)過，最后呼吁所有女性注意預(yù)防乳腺癌?！璏ydoctorsestimatedthatIhadan87percentriskofbreastcanceranda50percentriskofovariancancer,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《紐約時報》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)卵巢有一個小的良性腫瘤，任何組織都沒有癌癥跡象。

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