PIKCATP基因项目立项

PIK3CA、TP53基因项目立项报告,分子诊断部徐正红20150302,PIK3CA基因项目TP53基因项目技术方法风险评估,一. PIK3CA基因项目,1. 项目背景,吉非替尼,厄洛替尼,西妥昔单抗,帕尼单抗,FDA要求对拟采用易瑞沙、特罗凯等EGFR-TKI治疗的患者,进行EGFR基因突变检测,FDA要求使用西妥昔单抗、帕尼单抗治疗结直肠癌前，必须进行KRAS基因检测,耐药,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. 项目背景,PIK3CA 的突变约4/5发生在螺旋区(exon9)和激酶区(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. 项目背景,CFDA注册产品：,1. 项目背景,其他的PIK3CA基因突变检测试剂盒：,1. 项目背景,国内相关专利：,1. 项目背景,2. 临床意义-突变率,2. 临床意义-突变率,2. 临床意义-耐药,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. 临床意义-耐药,3.项目内容,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 detected 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 domain, 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.项目内容,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 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 outcome 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 therapyJ. British journal of cancer, 2011, 105(12): 1920-1926.5 Sueta A,Yamamoto Y,Yamamoto-Ibusuki M. An integrative analysis ofPIK3CAmutation, PTEN, and INPP4B expression in terms oftrastuzumabefficacy 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, 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 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 adenocarcinomaJ. 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.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. 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. 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, 85(2): 161-167.,3.项目内容,二. TP53基因项目,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. 项目背景,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，发挥其抑制细胞生长的作用。,“基因组卫士”,1. 项目背景,CFDA注册产品：无,科研类产品：,1. 项目背景,相关专利：,1. 项目背景-相关产品专利,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化疗药的耐药性相关，但不影响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。,临床意义,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：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。,临床意义,项目内容,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 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.p53His175 and p53His273 exerted very similar effects on the cellular response to cisplatin; both 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, 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 tocisplatintreatment 8.,项目内容,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。,项目内容,The mutant Cys175 exhibits wild-type properties. It is only recognized by PAb 1620 and does not bind to hsp7O. Indeed, it also behaves 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 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 transformation11.,项目内容,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, 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 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): 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 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 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, 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 decadeJ. Human mutation, 2014, 35(6): 672-688.11 Ory K, Legros Y, Auguin C, et al. Analysis of the most representative tumour-de