最新在结直肠癌循环肿瘤细胞中KRAS和PIK3CA的突变以及EGFR异质性的研究PPT课件.ppt

Although 30%90% of advanced primary CRC Cases were described to be positive for EGFR expression , CTCs with increased EGFR expression levels could be detected in only 7 of 33 (21%) CTC-positive patients. 1Shankaran V,Obel J,Benson AB 3rd.Predictingresponse to EGFR inhibitors in metastatic colorectal cancer:current practice and future directions.Oncologist 2010;15:15767. Inaddition to the low frequency of EGFR positivity in our patient cohort,not all CTCs of individual cases could be classified as EGFR overexpressing ,revealing a substantial heterogeneity in EGFR levels among CTCs from the same patient. These varying expression levels presumably reflect intratumoral heterogeneity of EGFR expression. Unlike the overexpression of the immunotherapy target human epidermal growth factor receptor 2(HER2) in breast cancer patients, which is most often connected with an amplification of the HER2 gene, the correlation of EGFR protein levels and gene amplification and their meaning for EGFR immunotherapy response is still controversial . As already shown for EGFR protein expression,we also obtained a heterogeneous distribution of EGFR gene amplification rates between CTCs of the same patient as well as of different patients. 1Nicholson RI, Gee JM, Harper ME.EGFR and cancer prognosis.Eur J Cancer 2001;37(Suppl4):S915. 2Ciardiello F,Tortora G.Epidermal growth factor receptor(EGFR) as a target in cancer therapy:understanding the role of receptor expression and other molecular determinants that could influ-ence the response to anti-EGFR drugs.Eur J Cancer 2003;39:134854. 3Moroni M,Veronese S,Benvenuti S, Marrapese G,Sartore-Bianchi A, DiNicolantonio F,etal.Gene copy number for epidermal growth factor receptor(EGFR) and clinical response to antiEGFRtreatment in colorectal cancer:a cohort study. Lancet Oncol 2005;6:27986. For are sponse to anti-EGFR therapies ,a normal function of downstream elements of the signaling pathway(e.g.,KRAS,BRAF,and PIK3CA)is essential. To overcome these limitations, we successfully established a protocol for the mutation alanalysis of DNA from single CTCs detected by CS. The main novelty of the technology described here is that it allows molecular analysis of individual CTCs after they are captured and immunostained by CS. Nevertheless,we could show feasibility of several downstream applications to further characterize molecular features of single CTCs detected with CS,including immunocytochemistry, mutation alanalysis,and qPCR. GenomePlex kit GenomiPhi kit ManufactureSigma-AldrichGE Healthcare Technique基于PCR技术-LMP不基于PCR技术-MDADNA amounts of WGA products Mean 8.9ug Range 4.615.4ug Mean 1.54ug Range 0.32.2ug Adequate DNA quality(atl east 2 of 4 PCR products after multiplex PCR) 8 of 11 11 of 11 Reactions 6 of 11 9 of 11 The mean EGFR gene amplification status14.72Range 0.56-40.35Median 11.2243.89Range 7.2290.07Median 40.29returnEGFR gene expression detected by CS(left images)correlates with EGFR gene amplification rates determined by qPCR and FISH(right tables) with MCF-7(low EGFR expression=score01,EGFR amplification rate 0.55/0.7),MDA-MB-468A(moderate EGFR expression = score 2,EGFR amplification rate1.83/not analyzed),BT-20(strong EGFR expression=score 3,EGFR amplification rate6.43/8.2),and MDA-MB-468(strong EGFR expression=score3, EGFRamplificationrate38.65/ 30) cells. The EGFR qPCR was performed on DNA extracts from approximately 10 7 Cells as well as on WGA products from 10 single cellsafter CS. continue10ng PurifiedproductAt least 2 CTCs were detected in 24 of 49 (49%) patients with mCRC and 7 of 32 (22%) patients with nmCRC. We further assessed 741 CTCs from 33 patients with CRC (27mCRC,6nmCRC) for EGFR protein expression. Altogether, 1 EGFR-positive CTC could be observed in 7 of 33(21%) patients,with only 2 of 33 patients (6%) possessing strongly EGFR-positive CTCs. Whereas all CTCs detected in nmCRC patients were EGFR-negative, increased EGFR levels were observed in 7 of 27(26%) patients with mCRC.Furthermore, EGFR was differently expressed between CTCs from the same patients,rangingfor example from EGFR-negative to strongly EGFR-positive. Heterogeneity in EGFR expression inthe CTC population of patient 9. continuecontinueTo analyze CTC heterogeneity molecularly, we focused on blood samples(n =5) with more than 20morphologically intact CTCs per7.5 mL, which explains in part the low number of samples analyzed by single-cell PCR.The failure to analyze a higher number of detected CTCs is mainly due to the inability to transfer all CTCs undisturbed from the CellSearch cartridge onto slides and reidentify them for micromanipulation. Thus,from all 33 patients analyzed for EGFR expression of CTCs, only CTCs from 3 mCRC patients could also be analyzed for EGFR gene amplification by qPCR .EGFR gene amplification rate determined by qPCR in 26 analyzed CTCs from patients 6,9,and 26. Comp.;CK-PE,cytokeratinphycoerythrin;DAPI,4,6-diamidino-2 -phenylindole;APC,allophycocyanin;FITC,fluorescein isothiocyanate. return For the establishment of a technique to detect mutations on WGA products from single cells, we used MDA-MB-231 cells carrying a p53 mutation.To investigate the impact of contamination of a single CTC with surrounding leukocytes during micromanipulation,we performed a mutation alanalysis on GenomiPhiWGA products from a MDA-MB-231 single cell supplemented with12 leukocytes.Detection of the p53 R280K mutation in a single MDA-MB-231 cell (red).Addition of up to 2 leukocytes (green) or cell-free liquid from the CS cartridge (blue waves) to a single MDA-MB-231 cell by micromanipulation did not disturb the detection of the p53 mutation. returnGeneCodons CTCs analyzed KRAS12,13 59 Can detectedBRAF 600 44 Not be detected PIK3CA 542,554,104739 Can detectedPatient IDMutated gene Patient 6 =1 mutated gene Patient 9 =1 mutated gene Patient 18 =1 mutated gene Patient 22-deletePatient 26 =1 mutated gene continue第一步第二步PIK3CA mutationPIK3CA mutation E545A PIK3CA mutation E542KPatient 69 of 15 CTCs 60% 6 of 9 3 of 9 Patient 91 of 5 CTCs 20% -Patient 181 of 3 CTCs 33% -Patient 263 of 11 CTCs 27% -Others CTCs not mutated -A KRAS (G12V) mutation from CTCsA KRAS (G12V) mutation fromPrimary tumor Patient 65 of 15 (33%)CTCs Mutation Patient 9-Wild-type Patient 18-Wild-type Patient 26-Wild-type (B),CTCs carrying PIK3CA (n =9)and KRAS mutation(n = 5)obtained from patient 6 (total analyzed CTCs,n =15;wild-type form of both genes,n = 6) illustrate the geneticheterogeneity present in a CTC population.(C), PIK3CAgene status in analyzed CTCs from patients9,18,22,and 26. MUT, mutation; WT, wild type. returnPatients The local ethics committee approved this study, and written informed consent was obtained from all par- ticipants. Blood samples (7.5 mL) 32 patients nmCRC49 patients mCRC The University Medical Center Hamburg-Eppendorf TheMedicalUniversity ofGraz continuecontinuecontinue+continue We performed further molecular analysis on CTCs from 5 patients with mCRC having advanced stage (Dukes D) disease A期癌肿浸润深度限于直肠壁内，未穿出深肌层，且无淋巴结转移。B期癌肿侵犯浆膜层，亦可侵入浆膜外或肠外周围组织，但尚能整块切除， 无淋巴结转移。C期癌肿侵犯肠壁全层或未侵犯全层，但伴有淋巴结转移：C1期癌肿伴有 癌灶附近肠旁及系膜淋巴结转移；C2期癌肿伴有系膜根部淋巴结转移， 尚能根治切除。D期癌肿伴有远处器官转移、局部广泛浸润或淋巴结广泛转移不能根治性 切除。returnWGA方法return