1、PIK3CATP53基因项目基因项目立项立项2022-10-1PIK3CATP53基因项目立项一一PIK3CA基因项目基因项目二二TP53基因项目基因项目三三技术方法技术方法四四风险评估风险评估PIK3CATP53基因项目立项一一.PIK3CA.PIK3CA基因项目基因项目PIK3CATP53基因项目立项1.1.项目背景项目背景吉非替尼吉非替尼厄洛替尼厄洛替尼西妥昔单抗西妥昔单抗帕尼单抗帕尼单抗FDA要求对拟采用易瑞沙、特罗凯等要求对拟采用易瑞沙、特罗凯等EGFR-TKI治疗的患者治疗的患者,进行进行EGFR基因突基因突变检测变检测FDA要求使用西妥昔单抗、帕尼单要求使用西妥昔单抗、帕尼单抗治
2、疗结直肠癌前，必须进行抗治疗结直肠癌前，必须进行KRAS基因检测基因检测耐耐药药PIK3CATP53基因项目立项 PIK3CA 基因定位于3q26.3，长34kb，包含21个外显子，编码1068种氨基酸，该组氨基酸产生一组长124kD的蛋白。PIK3CA编码I类磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinases,PI3Ks)的p110催化亚单位，即PI3Kp110a。I类PI3K为异源二聚体，由一个调节亚基（p85）和一个催化亚基（p110）组成。1.1.项目背景项目背景PIK3CATP53基因项目立项 PIK3CA 的突变约4/5发生在螺旋区(exon9)和激
3、酶区(exon20)这两个热点区域。PIK3CA基因突变引起p110a异常激活，进而活化PI3Ks，激活下游Akt等信号分子，激活或抑制其下游靶蛋白进而调节细胞的增殖、分化、凋亡以及迁移等生物学效应，最终导致肿瘤的发生。1.1.项目背景项目背景PIK3CATP53基因项目立项 CFDA注册产品：注册产品：产品名称产品名称生产单位生产单位批准日期批准日期人PIK3CA基因突变检测试剂盒（荧光PCR法）北京雅康博生物科技有限公司2014.06.17人类PIK3CA基因突变检测试剂盒（流式荧光杂交法）益善生物技术股份有限公司2014.07.07人类PIK3CA基因突变检测试剂盒（荧光PCR法）厦门艾
4、德生物医药科技有限公司2013.04.021.1.项目背景项目背景PIK3CATP53基因项目立项 其他其他的的PIK3CAPIK3CA基因突变检测试剂盒：基因突变检测试剂盒：产品名称公司一种PIK3CA基因突变荧光定量PCR分型检测试剂盒及检测方法广州达健一种检测人PIK3CA基因突变的方法和试剂盒苏州科贝用于检测直肠癌PIK3CA基因热点突变位点杭州艾迪康医学检验PIK3CA 基因突变检测试剂盒(PCR-荧光探针法)北京鑫诺美迪基因检测技术有限公司1.1.项目背景项目背景PIK3CATP53基因项目立项 国内相关专利国内相关专利：专利名称专利名称申请（专利权）人申请（专利权）人申请号或授权
5、公告号申请号或授权公告号PIK3CA基因驱动突变的检测探针、引物及试剂盒厦门艾德生物医药科技有限公司CN102453765BPIK3CA H1047R敲入非人动物乳腺癌模型霍夫曼-拉罗奇有限公司CN201280046858一种PIK3CA基因突变检测液相芯片广州益善生物技术股份有限公司CN201010160769PIK3CA基因突变的检测探针、液相芯片及其检测方法广州益善生物技术股份有限公司CN101445832B一种PIK3CA基因突变荧光定量PCR检测试剂盒及检测方法宁波有成生物医药科技有限公司CN201310260412一种用于检测PIK3CA基因突变的试剂盒武汉友芝友医疗科技有限公司C
6、N201410734805引物及该引物质谱检测PIK3CA基因热点突变的方法武汉康圣达医学检验所有限公司CN201210574584一种PIK3CA基因突变荧光定量PCR分型检测试剂盒及其检测方法广州达健生物科技有限公司CN201210330990用于检测结直肠癌PIK3CA基因热点突变位点的试剂盒杭州艾迪康医学检验中心有限公司CN201210234133一种检测人PIK3CA基因突变的方法和试剂盒苏州科贝生物技术有限公司CN2010106085001.1.项目背景项目背景PIK3CATP53基因项目立项cancer%PIK3CA mutationSample source(primary t
7、issue vs cancer cell line)Exon mutatedFunctional domainReferenceLiver35.6(26/73)Primary9 and 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)Breas
8、t18.1(13/72)Primary9 and 20Helical and kinaseLevine et al(2005)Breast40.0(28/70)Primary6,7,9 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
9、kinaseWu et al(2005a)Breast26.9(25/93)Primary9 and 20Helical and kinaseLee 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,
10、9,18 and 20P85,C2,helical and KinaseSamuels et al(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.临床意义临床意义-突变突变率率PIK3CATP53基因项目立项Ovarian12.1(24/198)Primary9 and 20Helical and kinase
11、Levine et al(2005)Ovarian6.0(11/182)Primary9 and 20Helical 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(2
12、004)Gastric4.3(4/94)Primary9 and 20Helical and kinaseLi 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 e
13、t al(2004)Lung4.2(1/24)Primary9HelicalSamuels 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.临床意义临床意义-突变突变率率PIK3CATP53基因项目立项药物检测项目检测意义易瑞沙特罗凯EGFR基因突变预测疗效KRAS基因突变预测疗效西妥昔单抗帕尼单抗KRAS基因突变预测疗效BRAF基因突变预测疗效威罗菲尼BR
14、AF基因突变预测疗效易瑞沙 特罗凯西妥昔单抗 帕尼单抗PIK3CA基因突变耐药相关2.2.临床意义临床意义-耐药耐药PIK3CATP53基因项目立项PI3K作为EGFR下游信号分子被激活，可导致肿瘤细胞对EGFR-TKI药物的耐药，例如PIK3CA基因的突变可导致西妥昔单抗（爱必妥），帕尼单抗（维克替比）对转移性结直肠癌治疗的耐受，导致吉非替尼，厄洛替尼对非小细胞肺癌和食道癌晚期患者的治疗耐受1-4。K-ras，BRAF和PIK3CA基因突变率约占结直肠癌总体患者的56。KRAS，BRAF，PIK3CA任何一个或多个突变对个体化靶向药物西妥昔单抗（爱必妥），帕尼单抗（维克替比）治疗无效，全部为
15、野生型治疗有效4。PIK3CA突变对于乳腺癌个体化靶向药物曲妥珠单抗（赫赛汀）治疗无效，野生型治疗有效5-6。2.2.临床意义临床意义-耐药耐药PIK3CATP53基因项目立项COSMICIDNMAAChange突变率突变率775c.3140AGp.H1047R80%Common mutations776c.3140ATp.H1047L760c.1624GAp.E542K763c.1633GAp.E545K765c.1635GTp.E545D3.3.项目内容项目内容PIK3CATP53基因项目立项u PIK3CA mutation or gene amplification was detec
16、ted 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 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 effi
17、cacy of EGFR-TKI therapy 8-9.u Most of the mutations occur at two hot spots,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,
18、was sufficient to abrogate gefitinib-induced apoptosis.13u PIK3CA E542K may affect the efficacy of EGFR-TKI therapy in lung cancer14.3.3.项目内容项目内容PIK3CATP53基因项目立项引文引文1 Sartore-Bianchi A,Martini M,Molinari F,et al.PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-ta
19、rgeted monoclonal antibodiesJ.Cancer research,2009,69(5):1851-18572 Paez 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 outco
20、me in a phase II study of gefitinib in second-line treatment 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 the
21、rapyJ.British journal of cancer,2011,105(12):1920-1926.5 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.eCo
22、llection 2014.6 Yuan K,Wu H,Wang Y,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
23、 ovarian and breast 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 adenocarc
24、inomaJ.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,R.V.Pearline,L.C.
25、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 are oncogenic.Proc.Natl.Acad.Sci
26、.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):2695.14 Han J Y,Kim S H,Lee Y S,et al.Comparis
27、on 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,2014,85(2):161-167.3.3.项目内容项目内容PIK3CATP53基因项目立项二二.TP53.TP53基因
28、项目基因项目PIK3CATP53基因项目立项 TP53(tumor protein 53)基因定位于17p13.1，约20kb长，由11个外显子和10个内含子组成。编码393个氨基酸蛋白，分子量53kD，是一种半衰期短的核内磷酸化蛋白，能通过自身修饰或和其他蛋白相互作用来调节细胞周期及细胞凋亡。1.1.项目背景项目背景PIK3CATP53基因项目立项当细胞在正常状态时，TP53维持一个较低的蛋白水平，不干扰正常细胞的运行，当细胞在有损伤或在应激情况下，TP53蛋白的水平迅速升高，使之很快的激活并启动对正常细胞的修复功能。而当细胞DNA损伤严重而不能完全修复时，TP53蛋白将引发细胞凋亡，发挥其
29、抑制细胞生长的作用。“基因组卫士”1.1.项目背景项目背景PIK3CATP53基因项目立项 CFDA注册产品：无产品名称产品名称生产单位生产单位TP53基因6种突变检测试剂盒（仅供科研使用）厦门艾德生物医药科技有限公司人肿瘤蛋白p53(TP53)检测试剂盒上海信裕生物技术有限公司 科研类产品：1.1.项目背景项目背景PIK3CATP53基因项目立项 相关专利：发明名称发明名称申请（专利权）人申请（专利权）人申请申请/公开号公开号一种检测P53基因SNP位点的试剂及其应用中国医学科学院肿瘤医院CN104131101Ap53基因突变的快速检测方法上海赛安生物医药科技有限公司CN103436593A
30、一种检测P53基因突变的引物和探针辽宁琦润生物科技有限公司CN102533957B基于纳米探针直接检测肺癌样品中P53基因突变的方法中国科学院上海微系统与信息技术研究所CN101392286A一种鉴定外周血DNA肝癌相关P53基因249位密码子突变的多重PCR的试剂盒四川大学华西医院CN101067153A1.1.项目背景项目背景-相关产品专利相关产品专利PIK3CATP53基因项目立项 TP53是迄今发现与人类肿瘤相关性最高的基因（50%以上人类肿瘤中发生TP53突变），同时TP53的突变率在所有的肿瘤中也是最高的，尤其是在上皮组织发生的肿瘤中，TP53 的突变率可以高达50%-90%。野生
31、型TP53 能够抑制多药耐药蛋白基因转录，减少多药耐药蛋白生成。突变型的TP53 是一种肿瘤促进因子，可引起肿瘤形成或细胞转化，增强多药耐药基因表达。TP53 变异与肿瘤细胞对铂类铂类化疗药的耐药性相关，但不影响紫杉醇类紫杉醇类药的敏感性，所以TP53 作为一个新的耐药基因，其突变检测可用于指导临床肿瘤患者化疗的个体化用药。2.2.临床意义临床意义PIK3CATP53基因项目立项铂类药的作用机理：铂类药的作用机理：作用于DNA，引起DNA复制障碍，从而抑制癌细胞的分裂。与其他抗癌药一样，铂类抗癌药影响DNA合成的作用是非特异性的。但肿瘤细胞比正常细胞增殖快，合成DNA迅速，并且DNA受损后的修
32、复功能不完善，因此，肿瘤细胞对抗癌药的细胞毒作用更为敏感，从而显示出药物的抗癌作用。紫杉醇类药物作用机理：紫杉醇类药物作用机理：抗微管药物,通过促进微管蛋白聚合抑制解聚,保持微管蛋白稳定,抑制细胞有丝分裂。体外实验证明紫杉醇具有显著的放射增敏作用，可能是使细胞中止于对放疗敏感的G2和M期。2.2.临床意义临床意义PIK3CATP53基因项目立项COSMIC IDNMAA ChangeIARC TP53 Database:Number of reports10648c.524GAp.R175H1210Hotspots;common mutations;the most frequent muta
33、tions10656c.742CTp.R248W73410662 c.743GA p.R248Q93310659c.817CTp.R273C70610660c.818GAp.R273H8513.3.项目内容项目内容PIK3CATP53基因项目立项u 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 p5
34、3 mutants such as R248W and R273H 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.u p53His175 and p53His273 exerted very similar effects on the cellular
35、 response to cisplatin;both conferred increased resistance 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
36、drugs,including doxorubicin,cisplatin,etoposide,and 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
37、and R273C increase resistance to cisplatin treatment 8.3.3.项目内容项目内容PIK3CATP53基因项目立项Q:厦门艾德的TP53基因检测试剂盒可以6种突变（R175H,R175C,R248W,R248Q,R273H,R273C)，为什么我们只检测5种突变，不检测R175C突变点？A：R175C突变为非热点突变，其突变蛋白具有部分功能，且无文献支持该突变点与药物的相关作用关系。3.3.项目内容项目内容PIK3CATP53基因项目立项u The mutant Cys175 exhibits wild-type properties.It
38、is only recognized by PAb 1620 and does not bind to hsp7O.Indeed,it also behaves 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
39、Functional classification based on the overall transcriptional activity of 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 tha
40、t p.R175C may be a passenger mutation coselected during neoplastic transformation11.3.3.项目内容项目内容PIK3CATP53基因项目立项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
41、Rituximab-CHOP Consortium Program StudyJ.Blood,2012,120(19):3986-3996.2 Song H,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 differen
42、t p53 mutants on resistance of cultured cells to chemotherapyJ.Oncogene,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.Resistan
43、ce and gain-of-resistance phenotypes in cancers harboring 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
44、 cell properties of oral squamous cell carcinoma 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,La
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46、epresentative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferationJ.The EMBO journal,1994,13(15):3496.3.3.项目内容项目内容引文PIK3CATP53基因项目立项三三.技术方法技术方法PIK3CATP53基因项目立项PCR技术技术：castPCRtechnologyTaqManMutatio
47、nDetectionAssays1.1.技术原理技术原理PIK3CATP53基因项目立项u Highspecificitymutant allele detection is based on an allele-specific primer,while wild type background is suppressed by the proprietary MGB blocker oligonucleotideu Highsensitivityassays can detect down to 0.1%mutation in a background of wild type DNA,a
48、s demonstrated in spiking experimentsu WidedynamicrangeandexcellentPCRefficiencyassays demonstrate at least 4 logs of dynamic range and an average PCR efficiency of 100%10%uFast,simpleworkflowlike other TaqMan Assays,typically requires 3 hours from sample to results,with minimum hands-on time 2.2.技术
49、优势技术优势PIK3CATP53基因项目立项样本类型：样本类型：gDNA,FFPE tissues,fresh frozen tissues,cell lines适合机型：适合机型：3.3.样本和机型样本和机型PIK3CATP53基因项目立项时间4月5-7月8-10月11-12月内容采购相关试剂，质粒，开始预实验反应体系优化，灵敏度，精密度，稳定性等试验临床试验，测序实验数据整理，相关材料的编写4.4.项目进程项目进程PIK3CATP53基因项目立项5.5.项目预算项目预算产品产品价格价格使用次数使用次数单次检测单次检测TaqMan mutation detection assay8130.0
50、075108.4TaqMan Genotyping Master Mix,28600.0010008.6IPC Mix2767.005005.5每孔合计122.5使用使用ABI试剂研发的预算：试剂研发的预算：PIK3CATP53基因项目立项5.5.项目预算项目预算项目项目价格（万元）价格（万元）试剂3.0耗材1.0测序0.6其他0.4合计5.0自己研发的预算：自己研发的预算：PIK3CATP53基因项目立项u 试剂和PCR技术的稳定性u 实验过程中的不确定性（实验室、试剂等污染情况）u 技术路线调整和时间u 临床样本的收集、样本的质量u 试剂盒生产u 市场推广四四.风险评估风险评估PIK3CA
