在结直肠癌循环肿瘤细胞中KRAS和PIKCA的突变以及EGFR异质性的研究.pptVIP

Heterogeneity of Epidermal Growth Factor Receptor Statusand Mutations of KRAS / PIK3CA in Circulating TumorCells of Patients with Colorectal Cancer ;;Study introduction;EGFR 是一种跨膜酪氨酸激酶受体，与配体结合后可引起下游信号传导通路的活化， 如 ： KRAS BRAF(4%–12%) RAS-RAF-MEK-Erk/MAPK PIK3CA(14%–20%) P13K-AKT-PKC-IKK 在很多实体瘤中 EGFR 均有不同程度的表达，表达率最高为头颈部肿瘤 ， 达 95% — 100% 。 CRC 次之 ， 为 72% — 89% ， 胃 癌表达率为 41% — 64% ，并且 EGFR 表达率高的肿瘤恶性度高 ，侵袭力强 ， 预后差 。而且在同一患者中不同的CTCs中，EGFR的表达也存在着异质性。 [1]Goldstein N S, Armin M.Epidermalgrowthfactorreceptorimmunohisto chemicalreactivityin patients with AmericanJointCommitteeon Cancer StageIV colon adenocarcinoma:Implicationsfora standardized scoringsystem[J]. Cancer ,2001,92(5):1331-1346. [2]Bergstrom J D, Westermark B, Heldin N E.Epidermalgrowth factorre - ceptor signaling activates metin human anaplastic thyroid carcinomacells[J]. ExperimentalCellResearch ,2000,259(1):292-299.[3] Denning M F, Dlugosz A A, Cheng C, et al. Cross-talk betweenepidermalgrowth factorreceptorand protein kinase C during calciumnduced differentiation of keratinoytes [J]. Experimental Dermatology ,2000,9(3):192-199. ;Patients 4 Cell lines , culture conditions , and fluorescence in Situ hybridization analysis (MCF,MDA-MB-468,BT-20,MDA-MB-468A) Enumeration of CTCs by CS(FITC- the fourth channel of the CS) Sample preparation for molecular analysis after Cell Search detection Isolation of CTCs by micromanipulation Whole-Genome Amplification of DNA from single tumor cells with the GenomePlex kit WGA of DNA from single tumor cells with the GenomiPhi kit Identification of EGFR gene amplifications by PCR on WGA products (LINE1 reference,EGFR target) Sequencing of single-cell WGA products ;Results ;Discussion ;We detected ≥2 CTCs in 49% of patients withmCRC (24 of 49) and 22% of patients with nmCRC (7 of 32).These results are similar to previous findings revealing detection rates of ≥2 CTCs in 33%–61% of patients with mCRC (17,30) and26% of patients with nmCRC . [1]C