在結(jié)直腸癌循環(huán)腫瘤細(xì)胞中KRAS和PIK3CA的突變以及EGFR異質(zhì)性的研究

1、heterogeneity of epidermal growth factor receptor statusand mutations of kras / pik3ca in circulating tumorcells of patients with colorectal cancer clinical chemistry november 7， 2012study introduction美國(guó)國(guó)家癌癥綜合治療聯(lián)盟（nccn）結(jié)直腸癌臨床實(shí)踐指南(2011)明確指出： (1)所有轉(zhuǎn)移性結(jié)直腸癌患者都應(yīng)檢測(cè)kras基因狀態(tài)； (2)只有kras野生型患者才建議接受egfr抑制劑（如愛(ài)必妥

2、和帕尼單抗）治療。nccn非小細(xì)胞肺癌臨床實(shí)踐指南(2011)明確指出：當(dāng)kras基因發(fā)生突變時(shí)，不建議使用egfr-tkis靶向治療藥物。 衛(wèi)生部頒布的結(jié)直腸癌診療規(guī)范（2010年版）也明確指出：確定為復(fù)發(fā)或轉(zhuǎn)移性結(jié)直腸癌時(shí)，檢測(cè)腫瘤組織kras基因狀態(tài)，以確定合適的治療方案。1. amado r g， wolf m， peeters m，van cutsem e et al. journal of clinical oncology. april 2008. 2. van cutsem et al. asco annual meeting 2008: abstract 2. 3. boke

3、meyer et al. asco annual meeting 2008: abstract 4000. 4. livre et al. j clin oncol 2008; 26: 374-9. 5. 美國(guó)fda相關(guān)網(wǎng)站：/aboutfda/centersoffices/cder/ucm172905.htmegfr 是一種跨膜酪氨酸激酶受體，與配體結(jié)合后可引起下游信號(hào)傳導(dǎo)通路的活化， 如 ： kras braf(4%12%) ras-raf-mek-erk/mapk pik3ca(14%20%) p13k-akt-pkc-ikk 在許多實(shí)體瘤中 egfr

4、均有不同程度的表達(dá)，表達(dá)率最高為頭頸部腫瘤 ， 達(dá) 95% 100% 。 crc 次之 ， 為 72% 89% ， 胃 癌表達(dá)率為 41% 64% ，并且 egfr 表達(dá)率高的腫瘤惡性度高 ，侵襲力強(qiáng) ， 預(yù)后差 。而且在同一患者中不同的ctcs中，egfr的表達(dá)也存在著異質(zhì)性。1goldstein n s, armin m.epidermalgrowthfactorreceptorimmunohisto chemicalreactivityin patients with americanjointcommitteeon cancer stageiv colon adenocarcinoma

5、:implicationsfora standardized scoringsystemj. cancer ,2001,92(5):1331-1346. 2bergstrom j d, westermark b, heldin n e.epidermalgrowth factorre - ceptor signaling activates metin human anaplastic thyroid carcinomacellsj. experimentalcellresearch ,2000,259(1):292-299.3 denning m f, dlugosz a a, cheng

6、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-m

7、b-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 kitwga of dna from single tumor cells with t

8、he genomiphi kit identification of egfr gene amplifications by pcr on wga products (line1 reference,egfr target)sequencing of single-cell wga products materials and methodsresults applicability of wga from single-cell dna detection of egfr expression and gene amplificationson single ctcs mutational

9、analysis of single ctcs mutations in downstream genes of the egfr signaling pathway discussion genomiphi-amplified dna was more suitable the mean and median gene amplification rates(43.89-and 40.29- fold) determined on genomiphi wga products are similar to those determined by qpcr on dna extracts (a

10、pproximately 10 7 cells) and by fish analysis (30- to 40-fold) and are consistent with published data for mda-mb-468 cell populations. 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

11、 rates of 2 ctcs in 33%61% of patients with mcrc (17,30) and26% of patients with nmcrc .1cohen sj,punt cj,iannotti n,saidman bh,sabbath kd,gabrail ny,etal.relationship of circu lating tumor cells totumor response,progression-free survival,and overall survival in patients withmetastatic colorectal ca

12、ncer . j clin oncol 2008;26:321321. 2sastre j, maestro ml,puente j,veganzones s,alfonso r,rafael s,etal.circulating tumor cellsin colorectal cancer:correlation with clinical and pathological variables. ann oncol 2008;19:9358.58:15. although 30%90% of advanced primary crc cases were described to be p

13、ositive 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;15:15

14、767. 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 intratu

15、moral 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

16、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.e

17、gfr 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 2

18、003;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 the

19、rapies ,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 her

20、e 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 alan

21、alysis,and qpcr. genomeplex kit genomiphi kit manufacturesigma-aldrichge healthcare technique基于pcr技術(shù)-lmp不基于pcr技術(shù)-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

22、 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,egf

23、r 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 perform

24、ed 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 (27mc

25、rc,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

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 focu

27、sed 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

28、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 patient

29、s 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 investigat

30、e 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 o

31、f 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 detect

32、edpatient 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 (g1