| Objective:Circulating tumor cells(CTCs)have been proved to be closely related to the cancer metastasis and patients prognosis.The counting and downstream gene analysis of CTCs have become a hot spot in individualized therapy.However,enumeration and phenotyping in most capture devices is challenging due to the rarity and heterogeneity of CTCs.The aim of this study is to develop a multi-aptamer combined with nanochip system to improve the isolation performance and phenotyping of CTCs,and to evaluate the consistency of breast cancer tumor-related gene mutations between CTCs and tissue samples.This study provides a more scientific basis for the clinical application of CTCs in the diagnosis,treatment and disease monitoring of breast cancer.Methods:1.A total of 235 patients with malignant tumors who were treated in Shenzhen Luohu Hospital Group People’s Hospital from June 2018 to November 2020 were retrospectively analyzed,involving 13 types of malignant tumor.The relationship between the positive rate and the number of CTCs detected by im FISH and the patients’disease stage,clinical stage and tumor marker levels was analyzed.The relationship between the changes of CTCs and clinical outcome was also studied.2.Aptamercocktail recognizing Ep CAM/Vimentin/EGFR/CD44 were bound to a nanopillar array on a nano-microfluidic chip.The recognition was validated with cancer cells by flow cytometry and the critical parameters were optimized with the nanomicrofluidic chip.The platform was used to capture CTCs in simulated human peripheral circulation blood samples,peripheral blood samples from 51 healthy volunteers,51 patients with benign breast diseases and 51 patients with breast cancer,and the target cells were identified by four-color immunofluorescence.Droplet digital PCR was used to evaluate the HER2 gene amplification status of single CTCs from 10randomly selected patients to verify whether the CTCs sorted by the nano-microfluidic chip in this study could be used for downstream gene detection.3.Breast cancer patients who were treated in the Department of Thyroid and Breast Surgery of Shenzhen Luohu Hospital Group People’s Hospital from April 2021to June 2022 were prospectively enrolled.A total of 42 blood samples,30 tissue samples,and 30 paired blood,and the remaining 12 blood samples were obtained from the follow-up samples of 10 patients.Genomic DNA was extracted from tissue samples and CTC samples,respectively.Gene mutation analysis of PIK3CA,TP53,EGFR,KRAS,BRAF,MET and HER2 genes in the genome was performed by digital PCR.Results:1.The total positive rate of CTCs was 65.53%.The positive rate of CTCs in patients who did not receive relevant treatment(54.17%)was not significantly different from that of patients with postoperative chemotherapy(61.79%,X~2=0.833,P=0.361).The positive detection rate of CTCs in patients with recurrence and metastasis was 81.25%,which was significantly different from the previous two groups(X~2=9.516,P=0.002;X~2=7.383,P=0.007).The positive rate of CTCs was related to clinical stage,maximum diameter of lesions,lymph node metastasis and distant metastasis(P<0.05).The results of continuous CTCs monitoring in 6 patients with malignant tumors showed that the changes of CTCs count in different stages of the disease were related to the prognosis of the disease.2.The proposed aptamer-cocktail showed the predominant affinity with MDA-MB-231 and SK-BR-3.When utilized to capture artificial clinical samples,it showed 71%to 83%capture efficiency.Three types of CTCs were isolated by this system:epithelial CTCs,mesenchymal CTCs and epithelial-mesenchymal CTCs.The nuclei of CTCs were multinucleated and heterogeneous in shape,and the cell volume of epithelial CTCs was more than 2 times larger than that of normal lymphocytes.CTCs were detected in all breast cancer clinical samples(100%,counting data:1-94/2m L).The total CTC detection rate of benign breast disease samples was 11.77%,among which the maximum number of CTC detected in two patients was 3/2m L peripheral blood,and the rest were 1/2m L peripheral blood.The total number of CTCs and the number of mesenchymal CTCs were correlated with the occurrence and metastasis of breast cancer(P<0.05).There were significant differences in the levels of total CTC,mesenchymal CTC and epithelial-mesenchymal CTC among different stages of breast cancer(P<0.05),and the number of interstitial CTC in stageⅣpatients was significantly higher than that in stageⅡpatients(P=0.01).Digital PCR results showed that the reference genes could be detected in the DNA samples of 10patients,which indicated that the CTC sorted by the nano microfluidic system could be used for downstream gene analysis.3.The gene mutation rate of tissue samples was 53.33%,and the number of gene mutations was 19,including PIK3CA H1047R,PIK3CA E545X,PIK3CA E542K.The rate of gene mutation in CTC samples was 53.33%,and the number of gene mutations was 24,mainly PIK3CA H1047R,PIK3CA E545X,PIK3CA E542K,and TP53 R175H.Among the 30 paired samples of blood and tissue,12 cases had the same mutation,and3 cases had two mutations.The three same gene mutations had a high degree of consistency in CTC and tissue samples(Kappa>0.600).Conclusion:1.The positive rate of CTCs can be used for clinical staging analysis,recurrence and metastasis prediction of patients with malignant tumors.The number of CTCs can be used as an indicator to evaluate recurrence and metastasis and predict the prognosis of malignant tumors.2.The multi-aptamer recognition unit combined with nano-microfluidic chip system established in this study achieved efficient detection and phenotypic characterization of circulating tumor cells,and single CTC could be obtained for genetic detection.The corresponding clinical application of breast cancer patients was executed.3.In this study,PIK3CA,TP53 and EGFR gene mutations in CTCs could be accurately detected by digital PCR counting,which verified a certain degree of consistency of gene mutations between paired tissue and CTC samples,which is expected to settle the problem sooner of obtaining tumor tissue in the clinical setting.Figure[19]Table[7]Reference[77]... |
PDF Full Text Request