PIK3CATP53基因項目立項

1、PIK3CAPIK3CA、TP53TP53基因項目立項報告基因項目立項報告分子診斷部分子診斷部徐正紅徐正紅20150302201503022PIK3CA基因項目基因項目TP53基因項目基因項目技術方法技術方法風險評估風險評估3一一. PIK3CA. PIK3CA基因項目基因項目41. 1. 項目背景項目背景吉非替尼吉非替尼厄洛替尼厄洛替尼西妥昔單抗西妥昔單抗帕尼單抗帕尼單抗FDA要求對擬采用易瑞沙、特羅凱等要求對擬采用易瑞沙、特羅凱等EGFR-TKI治療的患者治療的患者,進行進行EGFR基因突基因突變檢測變檢測FDA要求使用西妥昔單抗、帕尼單抗要求使用西妥昔單抗、帕尼單抗治療結直腸癌前，必須進

2、行治療結直腸癌前，必須進行KRAS基基因檢測因檢測耐耐藥藥5PIK3CA 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,PI3Ks)的p110催化亞單位，即PI3Kp110a。 I類PI3K為異源二聚體，由一個調(diào)節(jié)亞基（p85）和一個催化亞基（p110）組成。1. 1. 項目背景項目背景6PIK3CA 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，

3、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)節(jié)細胞的增殖、分化、凋亡以及遷移等生物學效應，最終導致腫瘤的發(fā)生。1. 1. 項目背景項目背景7CFDA注冊產(chǎn)品：注冊產(chǎn)品：產(chǎn)品名稱產(chǎn)品名稱生產(chǎn)單位生產(chǎn)單位批準日期批準日期人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（流式熒光雜交法）益善生物技術股份有限公司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. 1. 項目背景項目背景8其他其他的的PIK3CAPIK3CA

4、基因突變檢測試劑盒：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有限公司1. 1. 項目背景項目背景9國內(nèi)相關專利國內(nèi)相關專利：專利名稱專利名稱申請（專利權）人申請（專利權）人申請?zhí)柣蚴跈喙嫣柹暾執(zhí)柣蚴跈喙嫣朠IK3CA基因驅動突變的檢測探針、引物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

5、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基因熱點突變的方法武漢康圣達醫(yī)學檢驗所有限公司CN201210574584一種PIK3CA基因突變熒光定量

6、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用于檢測結直腸癌PIK3CA基因熱點突變位點的試劑盒杭州艾迪康醫(yī)學檢驗中心有限公司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有限公司CN2010106085001. 1. 項目背景項目背景10cancer% PIK3CA mutationSample source (primary tissue vs cancer cell line)Exon mutatedFunctional domainReferenceLiver35.6 (26/73)Primary9 an

7、d 20Helical and kinaseLee et al (2004)Total liver 36% (26/73) Breast33.3 (4/12)Cell lines9 and 20Helical and kinaseBachman et al(2004)Breast21.4 (9/42)Primary1, 9 and 20p85, helical and kinaseBachman et al(2004)Breast18.1 (13/72)Primary9 and 20Helical and kinaseLevine et al(2005)Breast40.0 (28/70)Pr

8、imary6, 7, 9 and 20C2, helical and kinaseCampbell et al(2004)Breast20.7 (19/92)Primary9 and 20Helical and kinaseWu et al (2005a)Breast8.3 (1/12)Primary20kinaseSamuels et al(2004)Breast33.3 (5/15)Cell lines9 and 20Helical and kinaseWu et al (2005a)Breast26.9 (25/93)Primary9 and 20Helical and kinaseLe

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

10、l(2004)Colon13.6 (14/103)Primary9 and 20Helical and kinaseVelho et al(2005)Colon18.8 (6/32)Primary9 and 20Helical and kinaseCampbell et al(2004)Total colon 25% (94/369)2. 2. 臨床意義臨床意義-突變率突變率11Ovarian12.1 (24/198)Primary9 and 20Helical and kinaseLevine et al(2005)Ovarian6.0 (11/182)Primary9 and 20Heli

11、cal and kinaseCampbell et al(2004)Total ovarian 9% (35/380)Gastric25.0 (3/12)Primary18 and 20KinaseSamuels et al(2004)Gastric10.6 (5/47)Primary9 and 20Helical and kinaseVelho et al(2005)Gastric6.5 (12/185)Primary9 and 20Helical and kinaseLee et al (2004)Gastric4.3 (4/94)Primary9 and 20Helical and ki

12、naseLi et al (2005)Total gastric 7% (24/338) Brain26.7 (4/15)Primary4, 5 and 13C2 and helicalSamuels et al(2004)Brain4.6 (13/285)Primary9 and 20Helical and kinaseBroderick et al(2004)Total brain 6% (17/300) Lung1.3 (3/229)Primary9 and 20Helical and kinaseLee et al (2004)Lung4.2 (1/24)Primary9Helical

13、Samuels et al(2004)Total lung 2% (4/253) Leukaemia1.1 (1/88)Primary9HelicalLee et al (2004)Total leukaemia 1% (1/88)Total cancers reported 15% (382/2551) *2. 2. 臨床意義臨床意義-突變率突變率12藥物檢測項目檢測意義易瑞沙特羅凱EGFR基因突變預測療效KRAS基因突變預測療效西妥昔單抗帕尼單抗KRAS基因突變預測療效BRAF基因突變預測療效威羅菲尼BRAF基因突變預測療效易瑞沙 特羅凱西妥昔單抗 帕尼單抗PIK3CA基因突變耐藥相關2.

14、 2. 臨床意義臨床意義-耐藥耐藥13PI3K作為EGFR下游信號分子被激活，可導致腫瘤細胞對EGFR-TKI藥物的耐藥，例如PIK3CA基因的突變可導致西妥昔單抗（愛必妥），帕尼單抗（維克替比）對轉移性結直腸癌治療的耐受，導致吉非替尼，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。

15、2. 2. 臨床意義臨床意義-耐藥耐藥14COSMICIDNMAAChange突變率突變率775c.3140AGp.H1047R80%Common mutations776c.3140ATp.H1047L760c.1624GAp.E542K763c.1633GAp.E545K765c.1635GTp.E545D3.3.項目內(nèi)容項目內(nèi)容15u PIK3CA mutation or gene amplification was detected in 30.5% of all ovarian cancers and 45% of the endometrioid and clear cell su

16、btypes, and E542K, E545K, E545D, H1047R and H1047L mutations were detected 7.u 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.u Most of the mutations occur at two hot s

17、pots, namely E545K in the helical domain and H1047R in the catalytic domain, and E545K H1047R are associated with invasion and metastasis10-12.u continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. 13u PIK3CA

18、E542K may affect the efficacy of EGFR-TKI therapy in lung cancer14. 3.3.項目內(nèi)容項目內(nèi)容16引文引文1 Sartore-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, 2009, 69(5): 1851-18572 Pae

19、z J G, Jnne P A, Lee J C, et al. EGFR mutations in lung cancer: correlation with clinical 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 treatme

20、nt of advanced esophageal cancer patientsJ. Journal of Clinical Oncology, 2006, 24(10): 1612-1619.4 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, 2011, 105(12): 1

21、920-1926.5 Sueta A, Yamamoto Y, Yamamoto-Ibusuki M. An integrative analysis of PIK3CA mutation, PTEN, and INPP4B expression in terms of trastuzumab efficacy in HER2-positive breast cancer. PLoS One. 2014 Dec 26;9(12):e116054. doi: 10.1371/journal.pone.0116054. eCollection 2014.6 Yuan K, Wu H, Wang Y

22、, et al. Phospho-PRAS40Thr246 predicts trastuzumab response in patients with HER2-positive metastatic breast cancerJ. Oncology Letters, 2015, 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

23、 cancer8 Broderick D K, Di C, Parrett T J, et al. Mutations of PIK3CA in anaplastic oligodendrogliomas, 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 adenocarcinoma

24、J. OncoTargets and therapy, 2013, 6: 497.10 Yamaguchi H, Yoshida S, Muroi E, et al. Phosphoinositide 3-kinase signaling pathway mediated by p110 regulates invadopodia formationJ. The Journal of cell biology, 2011, 193(7): 1275-1288.11 Isakoff, S.J., J.A. Engelman, H.Y. Irie, J. Luo, S.M. Brachmann,

25、R.V. Pearline, L.C. Cantley,J.S. Brugge. 2005. Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial 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

26、 are oncogenic. Proc. Natl. Acad. Sci. USA. 102:802 807.doi:10.1073/pnas.040886410213 Engelman J A, Mukohara T, Zejnullahu K, et al. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancerJ. Journal of Clinical Investigation, 2006, 116(10):

27、 2695.14 Han J Y, Kim S H, Lee Y S, et al. Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinomaJ. Lung Cancer, 2014,

28、85(2): 161-167.3.3.項目內(nèi)容項目內(nèi)容17二二. TP53. TP53基因項目基因項目18TP53(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，能通過自身修飾或和其他蛋白相互作用來調(diào)節(jié)細胞周期及細胞凋亡。1. 1. 項目背景項目背景19當細胞在正常狀態(tài)時，TP53維持一個較低的蛋白水平，不干擾正常細胞的運行，當細胞在有損傷或在應激情況下，TP53蛋白的水平迅速升高，使之很快的激活并啟動對正常細胞的修復功能。而當細胞DNA損傷嚴重而不能完全修復時

29、，TP53蛋白將引發(fā)細胞凋亡，發(fā)揮其抑制細胞生長的作用?！盎蚪M衛(wèi)士”1. 1. 項目背景項目背景20CFDA注冊產(chǎn)品：無產(chǎn)品名稱產(chǎn)品名稱生產(chǎn)單位生產(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有限公司科研類產(chǎn)品：1. 1. 項目背景項目背景21相關專利：發(fā)明名稱發(fā)明名稱申請（專利權）人申請（專利權）人申請申請/公開號公開號一種檢測P53基因SNP位點的試劑及其應用中國醫(yī)學科學院腫瘤醫(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

30、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院上海微系統(tǒng)與信息技術研究所CN101392286A一種鑒定外周血DNA肝癌相關P53基因249位密碼子突變的多重PCR的試劑盒四川大學華西醫(yī)院CN101067153A1. 1. 項目背景項目背景- -相關產(chǎn)品專利相關產(chǎn)品專利22TP53是迄今發(fā)現(xiàn)與人類腫瘤相關性最高的基因（50%以上人類腫瘤中發(fā)生TP53突變），同時TP53的突變率在所有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 能夠抑制多藥耐藥蛋白基因轉錄

31、，減少多藥耐藥蛋白生成。突變型的TP53 是一種腫瘤促進因子，可引起腫瘤形成或細胞轉化，增強多藥耐藥基因表達。TP53 變異與腫瘤細胞對鉑類鉑類化療藥的耐藥性相關，但不影響紫杉醇類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導臨床腫瘤患者化療的個體化用藥。臨床意義臨床意義23鉑類藥的作用機理：鉑類藥的作用機理：作用于DNA，引起DNA復制障礙，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殖快，合成DNA迅速，并且DNA受損后的修復功能不完善，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

32、癌作用。紫杉醇類藥物作用機理：紫杉醇類藥物作用機理：抗微管藥物,通過促進微管蛋白聚合抑制解聚,保持微管蛋白穩(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，可能是使細胞中止于對放療敏感的G2和M期。臨床意義臨床意義24COSMIC IDNMAA ChangeIARC TP53 Database: Number of reports10648c.524GAp.R175H1210Hotspots; common mutations; the most frequent mutations10656c.742CTp.R248W73410662 c.743GA p.R248Q93310

33、659c.817CTp.R273C70610660c.818GAp.R273H851項目內(nèi)容項目內(nèi)容25u Codons 248, 273, and 175 are the TP53 mutation hot spots found in most human cancers. Most mutations at hot spots, including R248W, R248Q, R273C, R273H, R175H, are nonfunctional 1.u p53 mutants such as R248W and R273H can bind the Mre11-Rad5-NBS1

34、 (MRN) complex and interfere with its ability to recruit the ataxiatelangiectasia-mutated (ATM) kinase to DNA double-strand breaks, ultimately causing genetic instability 2.u p53His175 and p53His273 exerted very similar effects on the cellular response to cisplatin; both conferred increased resistan

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

36、d 5-FU 5.u Reconstitution of a mutant p53(R248W) in these cells inhibited the sensitivity to cisplatin treatment 6.u The transformant of R248Q mutation gained higher activity of invasion, while its anti-cancer drug sensitivity also increased 7.u R273H and R273C increase resistance to cisplatin treat

37、ment 8.項目內(nèi)容項目內(nèi)容26Q: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作用關系。項目內(nèi)容項目內(nèi)容27u The mutant Cys175 exhibits wild-type properties. It is only recognized by PAb 1620 and does not bind to hsp7O. Indeed, it also beh

38、aves as a wild-type in transactivation assay 9.u In vitro evidence for such differences was provided by the systematic mutagenesis of the R175 hotspot codon in human p53: the R175C mutant was wild-type in its phenotype1,10.u Functional classification based on the overall transcriptional activity of

39、R175C is classified as partially functional in IARC TP53 database.u 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 tra

40、nsformation11.項目內(nèi)容項目內(nèi)容281 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, 2012, 120(19): 3986-3996. 2 Song H

41、, Hollstein M, Xu Y. p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nature Cell Biol 2007; 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. Onco

42、gene, 1999, 18(2): 477-485.4 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 harborin

43、g wild-type p53J. Biochemical pharmacology, 2012, 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, 2011, 31(22): 4464-4481.7. Kamiya Y, Ohshima T. The individual cell properties of oral squamous cell car

44、cinoma and p53 tumor 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, 2014, 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, 2011, 223(2): 116-126.10 Leroy B, Anderson M, Soussi T. TP53 mutations in human cancer: database reassessment and prospects for the next decad