| PART Ⅰ: THE ROLE OF CIRCULATING TUMOR CELLS IN EVALUATION OF PROGNOSIS AND TREATMENT RESPONSE IN ADVANCED NON-SMALL-CELL LUNG CANCER1.PurposeNon-small-cell lung cancer(NSCLC)lacks validated biomarkers to predict the prognosis and treatment response.This study investigated whether circulating tumor cells(CTCs)detectable could reminder high risk of distant metastasis,provide prognostic information and early indicate the response to conventional therapy in patients with advanced NSCLC.2.MethodsIn this single-center prospective study,blood samples for CTC analysis were obtained from 59 patients with previously untreated,stage Ⅲ or Ⅳ NSCLC both before and after administration of two cycles of chemotherapy.CTCs took in peripheral blood were measured by Cell Search detect technique.The clinical data such as demography,overall survival(OS)and progression free survival(PFS)should be registed.The relationship between the amount of CTCs and the clinical features and prognosis was analysed.3.ResultsCarcino-embryonic antigen(CEA)and count of metastatic sites werepositively related with CTC count analyzed by multiple linear regression(P< 0.05).The median overall survival(OS)was 11.2 months(95% CI:10.37-12.03 months)for the baseline CTC ≥ 2 group compared with 8.3months(95% CI: 7.72-8.88 months)for the CTC < 2 group(log-rank test P< 0.05).Similarly,patients with CTC ≥ 2 at baseline had a significantly shorter median PFS(4.3 months,95% CI: 3.7-4.9 months)compared with patients with CTC < 2(6.2 months,95% CI: 5.59-6.82 months)(log-rank test P < 0.05).For the disease control(stable disease,partial response or complete response)group CTC value before treatment didn’t present difference with that after therapy compared by pared-samples T test(t=1.46,P=0.15),similarly with the result of progressed group(progressive disease)(t=-0.99,P=0.34).The CTC value of progressed group was higher than that of disease control group either at baseline or post chemotherapy(P<0.05).4.ConclusionThese data provide the evidence of a positive correlation between CTC counts with CEA,as well as count of metastatic sites.Meanwhile,CTCs could be an effective predictor of distant metastasis and poor prognosis.In this study,CTCs are poorly related with treatment response.Whether CTCs could be a predictor of curative effect in advanced NSCLC should be validated by more researches in future.PART Ⅱ: ESTABLISHING DETECTION TECHNIQUE OF EPITHELIAL-MESENCHYMAL TRANSITION PHENOTYPES IN CIRCULATING TUMOR CELLS AND EXPLORING ITS APPLICATION IN ADVANCED NON SMALL CELL LUNG CANCER1.Purpose Circulating tumor cells(CTCs)has been considered as a biomarker of real time liquid biopsy in monitoring metastasis and evaluating prognosis and treatment response gradually in non-small-cell lung cancer(NSCLC).CTCs could lose some epithelial marker because of epithelial-mesenchymal transition(EMT),so the widely used technique based on the epithelial marker existed dramatic defect.EMT is considered to be one of the key mechanism for invasion and metastasis in lung cancer,therefore detection and analysis of mesenchymal phenotype of CTCs has dramatic clinical value.This study aimed to explore the different phenotypes of CTCs and the corresponding biomarkers,which could supplement and complete existing technique based on epithelial biomarkers.This study tended to explore the relationship between the different phenotypes of CTCs and clinical feature and prognosis,which could provide evidence for CTCs becoming biomarker of metastasis prediction and prognostic evaluation.2.Methods Detection system concerning epithelial CTCs and mesenchymal CTCs should be established via negative enrichment(leukocyte specific antien CD45)independent of Ep CAM combining immunocytochemistry(ICC)technique.25,50,100 and 200 A549 cells were added in 5ml of peripheral blood collected from healthy volunteers,and then these tumor cells were detected,and finally the recovery was calculated to evaluate the efficiency of our detection system.The detection technique was used to test the different phenotype of CTCs at different period in NSCLC patients.The clinical data such as demography,overall survival(OS)and progression free survival(PFS)were registed.The relationship among the amount of CTCs and its characteristics of distribution in different phenotypes and the clinical features and prognosis was analysed.3.Results(1)It presented a linear correlation between the initial A549 cells and recycled tumor cells(R2=0.998,P<0.001),and the average detection rate exceeded 75%(76.4%~78.1%).(2)The detection rate of total CTCs was 88.37% in enrolled 43 patients,in which epithelial CTCs were 68(25%),merged ones were 98个(36%),and mesenchymal CTCs were 105(39%),which accounted for the vast majority.The mean value of epithelial CTCs was lower than that of mesenchymal ones and merged ones analysed by one-way ANOVA analysis.(3)Sex,smoking status,histological types and size of tumor were independent of the recall factor of total CTCs,and the mean value of CTCs in every group(P>0.05).The more lymphatic metastasis presented,the more CTCs’ recall factor was found(P<0.05).It detected more CTCs in patients on stage Ⅳ than that on stage Ⅲ.(4)The epithelial CTCs,mesenchymal CTCs and merged CTCs expressed no significant difference in different sex(P>0.05).However,the mean value of epithelial CTCs was lower than mesenchymal and merged ones in male patients(P=0.02).The female didn’t present this difference.The mean value of epithelial CTCs didn’t appeare difference in different histologic types,meanwhile the mesenchymal and merged ones presented the similar results.The univariate linear regression analysis was used to analyse the relationship of different phenotypes of CTCs with the size of tumors,metastatic lymph nodes and distant metastasis,the results indicated that the size of tumor was not related with the total CTCs and different type of CTCs.The metastatic lymph nodes and distant metastasis had a positive correlation with total CTCs,mesenchymal and merged CTCs(P<0.05,β>0),while this relationship was not found in epithelial CTCs.The mean value of total CTCs,mesenchymal CTCs and merged types in stage Ⅳ was higher than that in stage Ⅲ compared by independent-samples T test.Total CTCs amount was the independent predictor for PFS(HR 1.56,95%CI: 1.05 to 2.29,P<0.05),and it also could predict OS(HR 2.79,95%CI: 1.608 to 4.85,P<0.05)via Cox proportional hazards regression analysis.Mesenchymal CTCs were the unique ones in all the phenotypes that coud predict OS(HR 2.37,95% CI 1.27 to 4.42,P<0.05).At the aspect of clinical features,the amount of metastatic organs were not only the independent predictor of PFS(HR 5.83,95% CI 1.54 to22.07,P<0.05),but also the predictor for OS(HR 94.65,95% CI 2.79 to3208.02,P<0.05).4.Conclusion(1)Detection system concerning epithelial CTCs and mesenchymal CTCs that was independent of Ep CAM and combined with immunocytochemistry(ICC)technique presented high detection efficiency.(2)The mesenchymal CTCs accounted for the majority in total CTCs,and the mean value of epithelial CTCs was lower than that of the mesenchymal and merged ones.(3)The account of CTCs was not related with age,sex,histology,and size of tumor,while the mesenchymal CTCs accounted for the majority in total CTCs.The number of CTCs,especially the mesenchymal phenotype,was related with the metastatic lymph nodes,number of metastatic organs and stage.(4)CTCs,especially the mesenchymal phenotype,were the independent predictor of OS and PFS.(5)The mesenchymal phenotype of CTCs played an important role in evaluating distant metastasis a prognosis. |
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