The Establishment And Improvement Of EpCAM Immunomagnetic Beads Techniqueand Its Preliminary Clinical Studies

The first partThe establishment of hMAM combined with CK19 detection method for circulating tumor cells of breast cancer based on EpCAM immune magnetic beads methodBackground and Objection:Breast cancer is a common malignant tumor in female with high mortality.Tumor recurrence and metastasis is still the leading cause of death of patients.In recent years,it is found that circulating tumor cells(CTCs)are the important reasons for the recurrence and metastasis of breast cancer patients.Therefore,the detection of CTCs and its significance has become a hot spot in the current clinical research.Circulating tumor cell(CTCs)is a tumor cell which is released into the peripheral blood circulation by means of spontaneous or treatment operation.Breast cancer CellSearch system is a CTCs detection technology with the highest degree of automation,using epithelial cell adhesion molecule(EpCAM)immune magnetic beads and CK19 to enrich less number of circulating tumor cells in peripheral blood concentration.It has high specificity,sensitivity and less affected by human factors researchers.But breast cancer cells often happens transformation of epithelial-mesenchymal transition(EMT)which often lead to false negative test results.EMT refers to the epithelial cells lost cell polarity and basement membrane getting migration and invasion ability,so tumor cells may no longer express EpCAM/CK.Therefore,Cellsearch system inevitably appear false negatives which based on the existence of the epithelial cells such as EpCAM and CK.Human breast globin human mammaglobin(hMAM)is epithelial tissue derived tissue-specific markers.The Abnormal expression is closely related to breast cancer micro metastasis.Quantum dot is a new type of fluorescent probe,its fluorescence intensity is higher than the commonly used organic fluorescent dyes.The stability is good.Its symmetry emission peak is narrow,continuous distribution and small overlap.This study is an improvement of cytokeratin CK19 single labeling method,Exploring using CK19 combined with human mammaglobin-quantum dot quantum dots(hMAM-QDs)double labeling method to detect circulating tumor cells of breast cancer.Compare the clinical value of two methods.Methods and materials:1.Preparation of EpCAM immune hpid magnetic beads,Measure the size of EpCAM immune lipid magnetic beads and saturation magnetization.2.Detect breast cancer 231 cells using single labeling method and double labeling method.3.15 ml blood collected respectively from 15 patients,Detect CTCs using single labeling method and double labeling method.4.Using fluorescence microscopy to observe two staining immunofluorescence results.5.Using single labeling method and double labeling method to detect circulating tumor cells of breast cancer and Compare the relationship between clinical pathological features.Result:1.After testing,the solid content was(2.6 ±0.15)mg/ml.The average particle diameter of EpCAM was 300 ±50nm,which was meet the requirements.The maximum specific saturation magnetization of Fe3O4 was 42.4 while the maximum saturation magnetization of EpCAM was 18.7 Am2/Kg.Therefore,the coating of Fe3O4 magnetic fluid can reduce the magnetic properties to a certain extent.2.The capture efficiency test of the five groups showed that the magnetic ball is good,the separation ability of two methods on breast cancer cell are more than 85%,CK19 combined with hMAM-PL(QDs)detection method is better than CK19 single method,indicating that the double labeling method for clinical examination will have high sensitivity.3.Results show that:11 CTCs were detected by single labeling method,19 CTCs were detected by double labeling method.In most specimens,double staining method detected more CTCs than single labeling method,In No.9 patients,double staining method detected less CTCs than single labeling method,which may be related to false positive of the single labeling method.4.Results of fluorescence microscopy.5.In CK19 single labeling method,the positive of CTCs is associate with weather menopause,TNM stage,lymph node metastasis,distant metastasis.And in hMAM and CK19 double labeling method,the positive of CTCs is not associate with age,menopausal status,the degree of differentiation,lymph node metastasis and pathological differentiation,HR,ER,HER(P>0.05);But it is closely related to distant metastasis,and serum CEA and TNM stage.The difference is statistically significant.Conclusion:CTCs detection is an important for the formation of distant metastasis of breast cancer.Early detection of CTC is of great significance for guiding clinical judgment of the patient.Clinical dynamic monitoring of CTCs changes is important to understand the patient’s condition timely or to guide the individualized comprehensive treatment.This study showed that CK19 and hMAM double labeling method can be used as biomarkers for the diagnosis of circulating tumor cells in patients with breast cancer.It is of great significance for preventing the early micro metastasis and estimating prognosis of breast cancer patients.The second partDetection of Circulating Tumor Cells using density-gradient centrifugation and immunofluorescence stain and its correlation with tumor recurrence and metastasisBackground and Objection:Ovarian cancer is one of the malignant tumors with high mortality in China.The main cause of death is metastasis and recurrence after treatment.A lot of patients with ovarian carcinoma received tumor and lymph node resection,and then accept chemotherapy and radiotherapy,tumor markers and imaging are timely detected.But finally found a sudden recurrence and distant metastasis.Therefore,early effective prediction of tumor recurrence is of great significance.Circulating tumor cells(CTCs)are tumor cells that are released into the peripheral blood circulation from the solid tumor or metastasis tumor.It has been proved that circulating tumor cells are helpful for the early diagnosis and prognosis of ovarian cancer.At present,there are few studies on ovarian cancer CTCs.In order to explore correlation between ovarian cancer prognosis and CTCs,Our experiment detected circulating tumor cells(CTCs)in patients with ovarian cancer and evaluated the relationship between CTCs and distant metastasis and recurrence of ovarian cancer.Methods:Peripheral blood samples were obtained from 50 patients with ovarian cancer.High fat and sugar was forbidden before blood collection.In general,7.5ml blood was taken from the median elbow vein when patients are in a quiet state.The collection of peripheral blood is transferred to a 10 ml centrifuge tube,CTCs were detected using density-gradient centrifugation and immunofluorescence staining.Correlation between clinical features and positive rates of CTCs in patients with ovarian cancer were investigated using SPSS 17.0 statistical software.Results:CTC-positive cases were defined by the presence of DAPI(+)、CK(+)、CD45(-).CTCs were found in 32 of the 50 patients with ovarian cancer.The presence of CTCs was significantly correlated with distant metastasis and recurrence(P=0.003,0.005respectively).However,CTCs were not significantly correlated with age,tumor location,TNM staging,node metastasis and tumor differentiation(P>0.05).Conclusion:CTCs are closely related to metastasis and prognosis of tunor.High levels of CTCs numbers suggest a higher risk of distant metastasis and recurrence.Monitoring CTCs can predict distant metastasis and recurrence of ovarian cancer.The Third part Meta-analysis of the prognostic value of Circulating and disseminated tumor Background and Objection:Ovarian cancer is one of the most common gynecologic malignancy with highest mortality rate.The etiology remains unknown.Early clinical symptoms were not obvious and Cancer metastasis is fast.In recent years,the age of the patients with ovarian cancer trends to become young.Because of lack of early diagnosis methods,the mortality of ovarian cancer is highest in gynecologic malignancy.Now,Imaging and CA125 are the common diagnosis and prognosis methods of ovarian cancer.Carbohydrate antigen125(CA125)has played a great role in the past clinical screening and treatment.But The CA125 is negative in half of early-stage ovarian cancer patients,meanwhile,CA125 is not a specific marker for ovarian cancer,it can be detected in other benign diseases for example breast cancer,endometrial cancer,lung cancer.So CA125 was not an effective marker for ovarian cancer.Thus it is necessary to find a sensitive and specific index that can help diagnose ovarian cancer earlier and predict distant metastasis timely.In recent years,the circulating tumor cell has become a hot research topic.Circulating tumor cells(CTCs)are tumor cells released from the primary tumors,recurrences,or metastases,then circulated in the peripheral blood or lymph node,Causing new tumor metastasis.It is an important element in the process of cancer netastases.Disseminated tumor cells(DTCs)are tumor cells in bone marrow of patients with cancer.It can be used as a ’liquid biopsy,to obtain helpful information for individual patients.But the number of CTC in the blood is very small,about 1 circulating tumor cells in every 1 billion normal blood cells.It is urgent to find sensitive CTCs detection means.But there are still no unified testing standards and programs for ovarian cancer circulating tumor cell.Although each of these technologies has their advantages,reagent composition and detection conditions still do not achieve standardization.The subjectivity of the operator is also affect the result of the experiment.So the experimental results are still controversial.In this paper,we systematically searched three databases about circulating tumor cells and the prognosis of ovarian cancer,we found the value of CTCs/DTCs in ovarian cancer is still controversial.Some studies considered CTCs/DTCs were not related to poor prognosis in ovarian cancer patients.In contrast,other studies revealed CTCs/DTCs were associated with adverse poor clinical outcomes and clinicopathological characteristics in clinical treatment of ovarian cancer patients.After considering the conflicting results from previous studies,we performed a meta-analysis to investigate the prognostic value of CTCs/DTCs on OS and DFS/PFS in ovarian cancer patients.Methods and materials:We searched the relationship between CTCs/DTCs and prognosis of ovarian cancer in the electronic databases of PubMed,EMBASE and Web of Science Up to October 20]5.Quality of included studies was assessed by Newcastle-Ottawa Scale(NOS).Statistical analysis was performed using Stata software(version 12.0,Stata Corp,College Station,TX,USA).The estimated odds ratio(OR)was used to summarize the association between CTCs/DTCs detection and ovarian cancer clinicopathological characteristics.OS and DFS/PFS in relation to CTCs/DTCs level in ovarian cancer were estimated by the Hazard ratio(HR)with its 95%confidence interval(95%CI).Publication bias used the funnel plot and the Egger’s/Begg’s test.Results:A total of 15 published studies met the inclusion criteria.According to analysis correlation between CTCs/DTCs and clinicopathological parameters,we found a significantly higher incidence of CTCs/DTCs in the CA125 high-expression group relative to the CA125 low-expression group.Higher incidence of CTCs/DTCs was observed in the serous group compared to the non-serous group.Higher incidence of CTCs/DTCs was observed in the Platinum Resistant group compared to the Sensitive group.the incidence of CTCs/DTCs was not associated with Resection status,FIGO,node metastasis and race.The meta-analysis revealed that ovarian patients in the CTCs/DTCs-positive group were significantly associated with shorter Overall survival(HR=1.84;95%CI[1.53-2.21])and Progression-free survival/Disease-Free Survival(HR=1.67,95%Cl[1.41-1.98]),The OS Subgroup analyses showed that both CTCs and DTCs were significantly associated with OS.Both RT-PCR and other ICC detection methods were able to detect CTCs/DTCs in predicting patient’s survival.The prognostic value of CTCs/DTCs for OS was significant in the "Surgery" subgroup and the "Surgery and Chemotherapy" subgroup,it was not significant in the "Chemotherapy" subgrou.The PFS/DFS Subgroup showed patients with detected CTCs in PB group and DTCs in BM group that had shorter PFS/DFS.The prognostic value of CTCs/DTCs for PFS/DFS was significant in the "RT-PCR" subgroup and the "other ICC" subgroup,whereas it was not significant in the "CellSearch" subgroup.The prognostic value of CTCs/DTCs for PFS/DFS was significant in the "Surgery" subgroup and the "Surgery and Chemotherapy,subgroup,it was not significant in the”Chemotherapy,subgroup.There was no publication bias for all subsequent subgroup analyses.It indicates there is no publication bias,and the result of the study is credible.It can serve as a tool to guide treatment in cancer patients in the future.ConclusionOur present meta-analysis indicates that the CTCs/DTCs are associated with OS and PFS/DFS in ovarian cancer.There was no publication bias for all subsequent subgroup analyses.The number of research about CTCs/DTCs was relatively small.The detection technology of circulating tumor cells is not mature.The diversity,complexity and the subjectivity of the test methods of CTCs/DTCs also caused the heterogeneity.Therefore,exploring the relationship between circulating tumor cells and the prognosis of ovarian cancer not only need advanced detection technology but also need more clinical evidence.It might be a new biomarker in patients with early-stage and metastatic ovarian cancer.