PIK3CATP53基因項目立項ppt課件

1、PIK3CA、TP53基因工程立項報告分子診斷部徐正紅20210302PIK3CA基因工程TP53基因工程技術(shù)方法風(fēng)險評價一. PIK3CA基因工程1. 工程背景吉非替尼厄洛替尼西妥昔單抗帕尼單抗FDA要求對擬采用易瑞沙、特羅凱等EGFR-TKI治療的患者,進展EGFR基因突變檢測FDA要求運用西妥昔單抗、帕尼單抗治療結(jié)直腸癌前，必需進展KRAS基因檢測耐藥PIK3CA 基因定位于3q26.3，長34kb，包含21個外顯子，編碼1068種氨基酸，該組氨基酸產(chǎn)生一組長124kD的蛋白。PIK3CA編碼I類磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinases,PI3K

2、s)的p110催化亞單位，即PI3Kp110a。 I類PI3K為異源二聚體，由一個調(diào)理亞基p85和一個催化亞基p110組成。1. 工程背景PIK3CA 的突變約4/5發(fā)生在螺旋區(qū)(exon9)和激酶區(qū)(exon20)這兩個熱點區(qū)域。PIK3CA基因突變引起p110a異常激活，進而活化PI3Ks，激活下游Akt等信號分子，激活或抑制其下游靶蛋白進而調(diào)理細胞的增殖、分化、凋亡以及遷移等生物學(xué)效應(yīng)，最終導(dǎo)致腫瘤的發(fā)生。1. 工程背景CFDA注冊產(chǎn)品：產(chǎn)品名稱生產(chǎn)單位批準(zhǔn)日期人PIK3CA基因突變檢測試劑盒（熒光PCR法）北京雅康博生物科技有限公司2014.06.17人類PIK3CA基因突變檢測試劑盒

3、（流式熒光雜交法）益善生物技術(shù)股份有限公司2014.07.07人類PIK3CA基因突變檢測試劑盒（熒光PCR法）廈門艾德生物醫(yī)藥科技有限公司2013.04.021. 工程背景其他的PIK3CA基因突變檢測試劑盒：產(chǎn)品名稱公司一種PIK3CA基因突變熒光定量PCR分型檢測試劑盒及檢測方法廣州達健一種檢測人PIK3CA基因突變的方法和試劑盒蘇州科貝用于檢測直腸癌PIK3CA基因熱點突變位點杭州艾迪康醫(yī)學(xué)檢驗PIK3CA基因突變檢測試劑盒(PCR-熒光探針法)北京鑫諾美迪基因檢測技術(shù)有限公司1. 工程背景國內(nèi)相關(guān)專利：專利名稱申請（專利權(quán)）人申請?zhí)柣蚴跈?quán)公告號PIK3CA基因驅(qū)動突變的檢測探針、引物

4、及試劑盒廈門艾德生物醫(yī)藥科技有限公司CN102453765BPIK3CA H1047R敲入非人動物乳腺癌模型霍夫曼-拉羅奇有限公司CN201280046858一種PIK3CA基因突變檢測液相芯片廣州益善生物技術(shù)股份有限公司CN201010160769PIK3CA基因突變的檢測探針、液相芯片及其檢測方法廣州益善生物技術(shù)股份有限公司CN101445832B一種PIK3CA基因突變熒光定量PCR檢測試劑盒及檢測方法寧波有成生物醫(yī)藥科技有限公司CN201310260412一種用于檢測PIK3CA基因突變的試劑盒武漢友芝友醫(yī)療科技有限公司CN201410734805引物及該引物質(zhì)譜檢測PIK3CA基因熱

5、點突變的方法武漢康圣達醫(yī)學(xué)檢驗所有限公司CN201210574584一種PIK3CA基因突變熒光定量PCR分型檢測試劑盒及其檢測方法廣州達健生物科技有限公司CN201210330990用于檢測結(jié)直腸癌PIK3CA基因熱點突變位點的試劑盒杭州艾迪康醫(yī)學(xué)檢驗中心有限公司CN201210234133一種檢測人PIK3CA基因突變的方法和試劑盒蘇州科貝生物技術(shù)有限公司CN2010106085001. 工程背景cancer% PIK3CA mutationSample source (primary tissuevs cancer cell line)Exon mutatedFunctional dom

6、ainReferenceLiver35.6 (26/73)Primary9 and 20Helical and kinaseLeeet al(2004)Total liver 36% (26/73)Breast33.3 (4/12)Cell lines9 and 20Helical and kinaseBachmanet al(2004)Breast21.4 (9/42)Primary1, 9 and 20p85, helical and kinaseBachmanet al(2004)Breast18.1 (13/72)Primary9 and 20Helical and kinaseLev

7、ineet al(2005)Breast40.0 (28/70)Primary6, 7, 9 and 20C2, helical and kinaseCampbellet al(2004)Breast20.7 (19/92)Primary9 and 20Helical and kinaseWuet al(2005a)Breast8.3 (1/12)Primary20kinaseSamuelset al(2004)Breast33.3 (5/15)Cell lines9 and 20Helical and kinaseWuet al(2005a)Breast26.9 (25/93)Primary

8、9 and 20Helical and kinaseLeeet al(2004)Breast28.0 (14/50)Cell lines1, 9 and 20p85, helical and kinaseSaalet al(2005)Breast26.4 (77/292)Primary1, 4, 7, 9, 13, 18, 20p85, C2, helical and kinaseSaalet al(2005)Total breast 26% (195/750)Colon31.6 (74/234)Primary1, 2, 4, 7, 9, 18 and 20P85, C2, helical a

9、nd KinaseSamuelset al(2004)Colon13.6 (14/103)Primary9 and 20Helical and kinaseVelhoet al(2005)Colon18.8 (6/32)Primary9 and 20Helical and kinaseCampbellet al(2004)Total colon 25% (94/369)2. 臨床意義-突變率Ovarian12.1 (24/198)Primary9 and 20Helical and kinaseLevineet al(2005)Ovarian6.0 (11/182)Primary9 and 2

10、0Helical and kinaseCampbellet al(2004)Total ovarian 9% (35/380)Gastric25.0 (3/12)Primary18 and 20KinaseSamuelset al(2004)Gastric10.6 (5/47)Primary9 and 20Helical and kinaseVelhoet al(2005)Gastric6.5 (12/185)Primary9 and 20Helical and kinaseLeeet al(2004)Gastric4.3 (4/94)Primary9 and 20Helical and ki

11、naseLiet al(2005)Total gastric 7% (24/338)Brain26.7 (4/15)Primary4, 5 and 13C2 and helicalSamuelset al(2004)Brain4.6 (13/285)Primary9 and 20Helical and kinaseBrodericket al(2004)Total brain 6% (17/300)Lung1.3 (3/229)Primary9 and 20Helical and kinaseLeeet al(2004)Lung4.2 (1/24)Primary9HelicalSamuelse

12、t al(2004)Total lung 2% (4/253)Leukaemia1.1 (1/88)Primary9HelicalLeeet al(2004)Total leukaemia 1% (1/88)Total cancers reported 15% (382/2551) *2. 臨床意義-突變率藥物檢測項目檢測意義易瑞沙特羅凱EGFR基因突變預(yù)測療效KRAS基因突變預(yù)測療效西妥昔單抗帕尼單抗KRAS基因突變預(yù)測療效BRAF基因突變預(yù)測療效威羅菲尼BRAF基因突變預(yù)測療效易瑞沙 特羅凱西妥昔單抗 帕尼單抗PIK3CA基因突變耐藥相關(guān)2. 臨床意義-耐藥PI3K作為EGFR下游信號分子

13、被激活，可導(dǎo)致腫瘤細胞對EGFR-TKI藥物的耐藥，例如PIK3CA基因的突變可導(dǎo)致西妥昔單抗愛必妥，帕尼單抗維克替比對轉(zhuǎn)移性結(jié)直腸癌治療的耐受，導(dǎo)致吉非替尼，厄洛替尼對非小細胞肺癌和食道癌晚期患者的治療耐受1-4。K-ras，BRAF和PIK3CA基因突變率約占結(jié)直腸癌總體患者的56。KRAS，BRAF，PIK3CA任何一個或多個突變對個體化靶向藥物西妥昔單抗愛必妥，帕尼單抗維克替比治療無效，全部為野生型治療有效4。PIK3CA突變對于乳腺癌個體化靶向藥物曲妥珠單抗赫賽汀治療無效，野生型治療有效5-6。2. 臨床意義-耐藥COSMIC IDNMAA Change突變率775c.3140AGp

14、.H1047R80%Common mutations776c.3140ATp.H1047L760c.1624GAp.E542K763c.1633GAp.E545K765c.1635GTp.E545D3.工程內(nèi)容PIK3CA mutation or gene amplification was detected in 30.5% of all ovarian cancers and 45% of the endometrioid and clear cell subtypes, and E542K, E545K, E545D, H1047R and H1047L mutations were d

15、etected 7.PIK3CA E542K, E545K, and E545D mutation in exon 9, H1047R and H1047L mutation in exon 20 are the common mutations, and may be associated with the efficacy of EGFR-TKI therapy 8-9.Most of the mutations occur at two hot spots, namely E545K in the helical domain and H1047R in the catalytic do

16、main, and E545K H1047R are associated with invasion and metastasis10-12.continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alphaE545K, was sufficient to abrogategefitinib-induced apoptosis.13PIK3CAE542K may affect the efficacy of EGFR-TKI therapy in lung cancer14. 3.工程內(nèi)容引文1 S

17、artore-Bianchi A, Martini M, Molinari F, et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodiesJ. Cancer research, 2021, 69(5): 1851-18572 Paez J G, Jnne P A, Lee J C, et al. EGFR mutations in lung cancer: correlation with clinical

18、 response to gefitinib therapyJ. Science, 2004, 304(5676): 1497-1500.3 Janmaat M L, Gallegos-Ruiz M I, Rodriguez J A, et al. Predictive factors for outcome in a phase II study of gefitinib in second-line treatment of advanced esophageal cancer patientsJ. Journal of Clinical Oncology, 2006, 24(10): 1

19、612-1619.4 Fidler M J, Morrison L E, Basu S, et al. PTEN and PIK3CA gene copy numbers and poor outcomes in non-small cell lung cancer patients with gefitinib therapyJ. British journal of cancer, 2021, 105(12): 1920-1926.5 Sueta A,Yamamoto Y,Yamamoto-Ibusuki M. An integrative analysis ofPIK3CAmutatio

20、n, PTEN, and INPP4B expression in terms oftrastuzumabefficacy in HER2-positive breast cancer. PLoS One.2021 Dec 26;9(12):e116054. doi: 10.1/journal.pone.0116054. eCollection 2021.6 Yuan K, Wu H, Wang Y, et al. Phospho-PRAS40Thr246 predicts trastuzumab response in patients with HER2-positive metastat

21、ic breast cancerJ. Oncology Letters, 2021, 9(2): 785-789.7 Campbell I G, Russell S E, Choong D Y H, et al. J. Cancer research, 2004, 64(21): 7678-7681. Mutation of the PIK3CA gene in ovarian and breast cancer8 Broderick D K, Di C, Parrett T J, et al. Mutations of PIK3CA in anaplastic oligodendroglio

22、mas, high-grade astrocytomas, and medulloblastomasJ. Cancer research, 2004, 64(15): 5048-5050.9 Zhang L, Shi L, Zhao X, et al. PIK3CA gene mutation associated with poor prognosis of lung adenocarcinomaJ. OncoTargets and therapy, 2021, 6: 497.10 Yamaguchi H, Yoshida S, Muroi E, et al. Phosphoinositid

23、e 3-kinase signaling pathway mediated by p110 regulates invadopodia formationJ. The Journal of cell biology, 2021, 193(7): 1275-1288.11 Isakoff,S.J.,J.A.Engelman,H.Y.Irie,J.Luo,S.M.Brachmann,R.V.Pearline,L.C.Cantley,J.S.Brugge.2005.Breast cancer-associated PIK3CA mutations are oncogenic in mammary e

24、pithelial cells.Cancer Res.65:1099211000.doi:10.1158/0008-5472.CAN-05-261212 Kang,S.,A.G.Bader,P.K.Vogt.2005.Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic.Proc. Natl. Acad. Sci. USA.102:802 807.doi:10.1073/pnas.040886410213 Engelman J A, Mukohara T, Zejnullahu K, e

25、t al. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancerJ. Journal of Clinical Investigation, 2006, 116(10): 2695.14 Han J Y, Kim S H, Lee Y S, et al. Comparison of targeted next-generation sequencing with conventional sequencing for p

26、redicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinomaJ. Lung Cancer, 2021, 85(2): 161-167.3.工程內(nèi)容二. TP53基因工程TP53(tumor protein 53)基因定位于17p13.1，約20kb長，由11個外顯子和10個內(nèi)含子組成。編碼393個氨基酸蛋白，分子量53kD，是一種半衰期短的核內(nèi)磷酸化蛋白，

27、能經(jīng)過本身修飾或和其他蛋白相互作用來調(diào)理細胞周期及細胞凋亡。1. 工程背景當(dāng)細胞在正常形狀時，TP53維持一個較低的蛋白程度，不干擾正常細胞的運轉(zhuǎn)，當(dāng)細胞在有損傷或在應(yīng)熱情況下，TP53蛋白的程度迅速升高，使之很快的激活并啟動對正常細胞的修復(fù)功能。而當(dāng)細胞DNA損傷嚴重而不能完全修復(fù)時，TP53蛋白將引發(fā)細胞凋亡，發(fā)揚其抑制細胞生長的作用?！盎蚪M衛(wèi)士1. 工程背景CFDA注冊產(chǎn)品：無產(chǎn)品名稱生產(chǎn)單位TP53基因6種突變檢測試劑盒（僅供科研使用）廈門艾德生物醫(yī)藥科技有限公司人腫瘤蛋白p53(TP53)檢測試劑盒上海信裕生物技術(shù)有限公司科研類產(chǎn)品：1. 工程背景相關(guān)專利：發(fā)明名稱申請（專利權(quán)）人

28、申請/公開號一種檢測P53基因SNP位點的試劑及其應(yīng)用中國醫(yī)學(xué)科學(xué)院腫瘤醫(yī)院CN104131101Ap53基因突變的快速檢測方法上海賽安生物醫(yī)藥科技有限公司CN103436593A一種檢測P53基因突變的引物和探針遼寧琦潤生物科技有限公司CN102533957B基于納米探針直接檢測肺癌樣品中P53基因突變的方法中國科學(xué)院上海微系統(tǒng)與信息技術(shù)研究所CN101392286A一種鑒定外周血DNA肝癌相關(guān)P53基因249位密碼子突變的多重PCR的試劑盒四川大學(xué)華西醫(yī)院CN101067153A1. 工程背景-相關(guān)產(chǎn)品專利TP53是迄今發(fā)現(xiàn)與人類腫瘤相關(guān)性最高的基因50%以上人類腫瘤中發(fā)生TP53突變，同

29、時TP53的突變率在一切的腫瘤中也是最高的，尤其是在上皮組織發(fā)生的腫瘤中，TP53 的突變率可以高達50%-90%。野生型TP53 可以抑制多藥耐藥蛋白基因轉(zhuǎn)錄，減少多藥耐藥蛋白生成。突變型的TP53 是一種腫瘤促進因子，可引起腫瘤構(gòu)成或細胞轉(zhuǎn)化，加強多藥耐藥基因表達。TP53 變異與腫瘤細胞對鉑類化療藥的耐藥性相關(guān)，但不影響紫杉醇類藥的敏感性，所以TP53 作為一個新的耐藥基因，其突變檢測可用于指點臨床腫瘤患者化療的個體化用藥。臨床意義鉑類藥的作用機理：作用于DNA，引起DNA復(fù)制妨礙，從而抑制癌細胞的分裂。與其他抗癌藥一樣，鉑類抗癌藥影響DNA合成的作用是非特異性的。但腫瘤細胞比正常細胞增

30、殖快，合成DNA迅速，并且DNA受損后的修復(fù)功能不完善，因此，腫瘤細胞對抗癌藥的細胞毒作用更為敏感，從而顯示出藥物的抗癌作用。紫杉醇類藥物作用機理：抗微管藥物,經(jīng)過促進微管蛋白聚合抑制解聚,堅持微管蛋白穩(wěn)定,抑制細胞有絲分裂。體外實驗證明紫杉醇具有顯著的放射增敏作用，能夠是使細胞中止于對放療敏感的G2和M期。臨床意義COSMIC IDNMAA ChangeIARC TP53 Database: Number of reports10648c.524GAp.R175H1210Hotspots; common mutations; the most frequent mutations10656c

31、.742CTp.R248W73410662c.743GA p.R248Q93310659c.817CTp.R273C70610660c.818GAp.R273H851工程內(nèi)容Codons 248, 273, and 175 are theTP53 mutation hot spots found in most human cancers. Most mutations at hot spots, including R248W, R248Q, R273C, R273H, R175H, are nonfunctional 1.p53 mutants such as R248W and R273

32、H can bind the Mre11-Rad5-NBS1 (MRN) complex and interfere with its ability to recruit the ataxiatelangiectasia-mutated (ATM) kinase to DNA double-strand breaks, ultimately causing genetic instability 2.p53His175 and p53His273 exerted very similar effects on the cellular response to cisplatin; both

33、conferred increased resistance to low concentrations of the drug 3.The expression of all forms of mutant p53 protein except p53His273 enhanced sensitivity to cisplatin and doxorubicin 4. R175H and R273H, exhibit significantly greater resistance to a number of antitumor drugs, including doxorubicin,

34、cisplatin, etoposide, and 5-FU5.Reconstitution of a mutant p53(R248W) in these cells inhibited the sensitivity to cisplatin treatment 6.The transformant of R248Q mutation gained higher activity of invasion, while its anti-cancer drug sensitivity also increased 7.R273H andR273Cincrease resistance toc

35、isplatintreatment 8.工程內(nèi)容Q : 廈門艾德的TP53基因檢測試劑盒可以6種突變R175H, R175C, R248W, R248Q, R273H, R273C)，為什么我們只檢測5種突變，不檢測R175C突變點？A：R175C突變?yōu)榉菬狳c突變，其突變蛋白具有部分功能，且無文獻支持該突變點與藥物的相關(guān)作用關(guān)系。工程內(nèi)容The mutant Cys175 exhibits wild-type properties. It is only recognized by PAb 1620 and does not bind to hsp7O. Indeed, it also beh

36、aves as a wild-type in transactivation assay 9.In vitroevidence for such differences was provided by the systematic mutagenesis of the R175 hotspot codon in humanp53: the R175C mutant was wild-type in its phenotype1,10.Functional classification based on the overall transcriptional activity of R175C

37、is classified as partially functional in IARC TP53 database.R175C is a false-positive detection, and the p.R175C mutant (c.523CT) is not impaired for any TP53 function. Considering this body of evidence, we hypothesize that p.R175C may be a passenger mutation coselected during neoplastic transformat

38、ion11.工程內(nèi)容1 Xu-Monette Z Y, Wu L, Visco C, et al. Mutational profile and prognostic significance of TP53 in diffuse large B-cell lymphoma patients treated with R-CHOP: report from an International DLBCL Rituximab-CHOP Consortium Program StudyJ. Blood, 2021, 120(19): 3986-3996. 2 Song H,Hollstein M,X

39、u Y.p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM.Nature Cell Biol2007;9:5735803 Blandino G, Levine A J, Oren M. Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapyJ. Oncogene, 1999, 18(2): 47

40、7-485.4 CHANG F U L I N, LAI M D. Various forms of mutant p53 confer sensitivity to cisplatin and doxorubicin in bladder cancer cellsJ. The Journal of urology, 2001, 166(1): 304-310.5 Martinez-Rivera M, Siddik Z H. Resistance and gain-of-resistance phenotypes in cancers harboring wild-type p53J. Bio

41、chemical pharmacology, 2021, 83(8): 1049-1062.6 Liu K, Ling S, Lin W C. TopBP1 mediates mutant p53 gain of function through NF-Y and p63/p73J. Molecular and cellular biology, 2021, 31(22): 4464-4481.7. Kamiya Y, Ohshima T. The individual cell properties of oral squamous cell carcinoma and p53 tumor

42、suppressor gene mutationJ. Oral Science International, 2005, 2(2): 104-117.8 Li J, Yang L, Gaur S, et al. Mutants TP53 p. R273H and p. R273C but not p. R273G Enhance Cancer Cell MalignancyJ. Human mutation, 2021, 35(5): 575-584.9 Goh A M, Coffill C R, Lane D P. The role of mutant p53 in human cancerJ. The Journal of pathology, 2021, 223(2): 116-126.10 Leroy B, Anderson M, Soussi T. TP53 mutations in human cancer: database reassessm