| Background and Purpose:Esophageal cancer is a common malignant tumor in China and the fourth mortality rate of the malignancies which seriously harms to human health. The most histological type is esophageal squamous cell carcinoma (ESCC), which is about90%. The prognosis of esophageal cancer is poor, and the5-year survival rate is only15%. Even underwent surgical resection, the2-year survival rate is less than30%. Because of lack of obvious symptoms and precise tumor molecular markers, the diagnosis of ESCC was often late. Like the vast majority of tumor, for ESCC, better monitoring of early diagnosis and more discoveries of new molecular targets for diagnosis are needed for treatment and prognosis to improve the detection rate and the level of diagnosis and treatment of ESCCAt present, the main methods of detection of metastasis are biopsy to detect the lymph nodes and bone marrow or imaging methods. However, the acquisition histological specimens is a trauma and brings pain to patients which is difficult to be accepted; imaging are often unable to detect tumor lesions less than2mm; CTCs in peripheral blood drawn is convenient and small traumatic, easy detection, which is thought highly of people. It is the ideal clinical source samples for routine tests.Circulating tumor cells exist in blood of malignant patients, which are also known as a rare blood cells, tumor micrometastasis lesions, latent tumor cells and circulating epithelial cells (circulating epithelial cell, CEC), and CTCs are of epithelial origin malignant tumor recurrence and metastasis of early events, the theory of circulating tumor cells has been known for a long time. Tumor metastasis is a complex process involving multi-step series amplification cascade and the growth of tumor cells disseminated planting. Discovery and diagnosis of the tumor is still highly dependent on medical imaging and tumor-specific serum markers and tumor metastasis and recurrence can not be detected in time. Based on the theory, the tumor cells’ spreading through the blood circulatory system to distant organs and formation of obvious metastases of malignant tumors is main causes of death, then detecting circulating tumor cells may detect the cancer metastasis and recurrence early and effectively.Studies have shown that, the changes of CTCs emumeration and phenotypic can be used as a window to understand the malignant biological characteristics and tumor progression. CTCs detection by a non-invasive methods for early cancer screening, cancer treatment evaluation, metastasis and monitoring recurrence, provides the necessary information. However, these cells are difficult to detect by conventional methods. The majority of cancer researchers are particularly concerned about how to determine CTCs in peripheral blood, and how to find more CTCs’ specific markers to improve sensitivity and specificity of the detection means. The subject using series analysis, which is based on immunomagnetic bead negative selection CTCs in ESCC patients in peripheral blood,to summarize the relationship between the identification of CTCs with esophageal cancer clinical features and the clinical implication of cancer staging, tumor size, lymph node metastasis, and other clinical characteristics. The survival time of CTCs groups were different. The higher TNM stage and the worse the cell differentiation, the more peripheral blood CTCs were detected. The higher level of CTCs may indicate poor prognosis. There were statistical differences in CTCs detection of lymphocyte transfer, the depth of tumor invasion, TNM stage, differentiation. Before the operation, the survival rate of CTCs negative group was significantly higher than CTCs positive group, two groups had statistical differences (χ2=3.888, P=0.049). Before the operation, the survival rate of CTCs negative group was significantly higher than CTCs positive group, the survival rate of CTCs<5group was significantly higher than CTCs≥5groups. Using Cox regression to analysis influence factors of survival time, CTCs group (5was cut-off) and lymphatic metastasis were significant factors which affected the prognosis. During pre-operation, CTCs group (5was cut-off) and lymphatic transfer were the significant factors affecting the prognosis, the risk of the patient death of CTC≥5group was3.078times higher than CTC<5group, risk of lymph node metastasis group was3.31times higher than no lymph transfer group.Following-up a case of a patient with esophageal cancer, we analysis the disease progression by monitoring the dynamic changes in cell morphology and cell emumerations during the different stages of treatment in peripheral blood and combination of imaging and serological markers to explore the relationship between therapy and tumor progression. It is further confirmed that the relationship between CTCs and tumor progression, and its value for tumor detection and diagnosis. Because of heterogeneity of tumor, the tumor cells performed the process of epithelial mesenchymal transformation (EMT) and mesenchymal epithelial transition (MET) during the tumor development process, the various cellular signs changed. Out of the primary tumor, a part of CTCs acquire high metastatic potential, which lead to the phenotype of CTCs changed, we need to find the specific markers of CTCs. Current identification techniques based on immunology and molecular biology and a variety of CTCs physical and chemical character were not specific and the enrichment was not efficient. Antibodies are the basis and highlights in this area of the tumor molecular targeted therapy and tumor detection..The method of capture CTCs is mainly based on separation and enrichment cycle with epithelial cells in the blood of the characteristics of the cancer cells. In2004, the FDA approved CellSearch Procedure, which is the symbol of epithelial cells EpCAM, CK8, CK18and CK19combined with immune magnetic beads in the peripheral blood, however, because about20%to30%malignant tumor cells do not express epithelial or expression level is low, so the screening method of sensitivity is not high. Use CTCs physical properties, such as the cell size, because the heterogeneity of the tumor cells, cells size is different, so there are specific enrichment is not high.But the antibody approved using for tumor detection and treatment is relatively poor, researches and explorations are underway. The large phage antibody library is an important technological platform to screen human antibodies. The variable region genes are assembled into the expression a vector; fragments of antibody molecules fused phage coat protein are expressed onto the surface of phage particles, resulting in the collection of the diversity of phage antibodies. Phage antibody libraries are able to unite genotype and phenotype in one particle and have the choose capacity coupled with expansion potential in vitro by simulation of antibody generation process in vivo, and phage antibody libraries have the powerful screening capabilities. We explored that by screening form a large capacity of human single-chain phage antibody library to obtained antibody to tumor cells and the screening methods and modified strategies. Obtained the specific antibodies combining with esophageal squamous cancer cell line for the detection of CTCs in esophageal cancer patients, respectively, provides more antibodies to treat cancer. In general, we use immunomagetic negative enrichment together with fluoresce method to identify CTCs in peripheral blood, and through CTCs window such as the CTCs number and the dynamic changes of cell morphology and tumor risk stratification to predict diagnosis, the efficacy of adjuvant treatment, real-time monitoring the tumor recurrence and metastasis; and further explore the molecular targets of tumor cells. We use esophageal cancer cells as targets to screen specific single-chain antibody against esophageal CTCs from a large phage antibody library for provide additional molecular targets to identify peripheral blood CTCs sensitively and specifically. This study on CTCs of ESCC not only has great significance to individualized treatment of patients, but also provides new drug candidates for biological therapy.Method:Part I:In our study,59patients of esophageal cancer had been enrolled for CTCs by immunomagnetic bead based negative enrichment combined with immunofluorescence antibody. First, the CD45antibody-coupled immunomagnetic beads had been used to remove leukocytes. The cells with CK8/18/19and DAPI positive, CD45negative in fluorescence results, larger cellular size, heterogeneity nucleus size and cellular shape were considered as CTCs. The relationship of CTCs and clinical implication had been studied.Part Ⅱ:A case of report of a patient with esophageal squamous cell carcinoma patient was evaluated the response to treatment over a3-year follow-up period by testing CTCs level and imaging study. Immunomagetic negative enrichment together with immunofluorescence were used to identify CTCs. Monitoring the dynamic CTCs emumeration and molecular characteristics in peripheral blood of the patient at different stages of treatment before operation, after operation and during follow-up. To evaluation the clinical significance of CTCs changes of biological features and the relationship with treatment efficacy and disease progression.Part III:From the large phage antibody library, three kinds of human esophageal cancer cell lines KYSE-170, KYSE-180and EC0156cells were equally mixed for use as the target. These intact cells were divided into two groups, PF and NF, according to the treatment method of the cells (fixed with2%PFA or not, respectively). Positive phage antibodies were identified following4panning cycles of adhesion, elution and amplification. Phages were recovery combined with acidic eluting (s) and XL-Blue bacteria infection (c). The relative affinities of the phage antibodies were determined and soluble antibodies were prepared. Further positive antibodies were compared the binding ability with white blood cells and other cells then were used to detect CTCs in peripheral blood of ESCC.Statistical Analysis:Data analysis was completed by SPSS16.0. The first part of paper,χ2test was used to compare CTCs detection at different stages, tumor size, tumor, lymph node metastasis between different groups. During Follow-up visit, the survival rates of patients with different CTCs level were calculated by Kaplan-Meier method, two groups of survival time compared with Log-rank inspection, single factor mortality by χ2test, multiplicity survival analysis by cox regression model of risk proportion (Forward LR law). The third part of the thesis, the rate of positive clones and diversity of positive rate were compared by χ2test. Bilateral test was used and P<0.05was considered significant.Results: Part I:In this study,59ESCC patients were enrolled in CTCs research. By immunomagnetic bead negative enrichment and immunofluorescence method, the detection rate of CTCs in the peripheral blood of patients was79.6%(47/59), the total isolated CTCs number were462and an average of7.83(0-72), respectively. Average detected CTCs number was7.83, approximately1.044CTCs per milliliter of peripheral blood. The higher TNM stage and the worse the differentiation, the more peripheral blood CTCs were detected. The higher level of CTCs may indicate poor prognosis. There were statistical differences in CTCs detection of lymphocyte transfer(χ2=6.531, P=0.011), the depth of tumor invasion(χ2=5.025, P=0.025), TNM stage(χ2=5.025, P=0.025), cell differentiation (χ2=4.883, P=0.021).CTCs with tumor remote were not statistically significant difference (χ2=1.506, P=0.304), but the comparison of CTCs detection ratio was quite different. There were no statistically significant differences in age, gender, the tumor size, tumor location, and the statue of smoking. Before the operation, the survival rate of CTCs negative group was significantly higher than CTCs positive group, two groups had statistical differences (χ2=3.888, P=0.049). the survival rate of CTCs<5group was significantly higher than CTCs≥5groups, two groups had statistical differences (χ2=12.388, P<0.001). Using Cox regression to analysis influence factors of survival time, CTCs group (5was cut-off) and lymphatic metastasis were significant factors which affected the prognosis. Using Kaplan-Meier method, survival rate of CTCs<5group was significantly higher than CTCs≥5groups. Post-operation, survival time of different groups had not statistical significance, but the number had large differences. During preoperation, CTCs group (5was cut-off)(B=1.124, P=0.010) and lymphatic transfer (B=1.203, P=0.003) were the significant factors affecting the prognosis, the risk of the patient death of CTC≥5group was3.078times higher than CTC<5group, general relative risk of95%CI was1.313-7.231; risk of lymph node metastasis group was3.31times higher than no lymph transfer group, general relative risk of95%CI is1.493-7.430.Part Ⅱ:The case of ESCC patient was followed up about3years since the first hospitalization, T3N2M0, clinical stage IV. Collect peripheral blood total eight times, each time7.5ml.1CTCs and14CTCs were identified in peripheral blood before and after surgery. Postoperative chemotherapy course of treatment was performed, Postoperative chemotherapy course of treatment, serum CA19-9, AFP, and other markers during the course of treatment maintained in normal level; immunohistochemisty showed, tumor cells CKAE1/AE3(++), CK17(++) and p63(++), p53(++), EGFR,(+) positive; of CK7(-), CK20(-), CEA (-). The dynamic changes with CTCs of patients and cell morphology were associated with disease progression, and consistent with the imaging results.Part III:Using a cell ELISA assay, it was found that the additional procedure of directly infecting XL1-Blue bacteria with the cell pellet following washing with an acidic solution can effectively decrease the loss of positive phage, yielding a positive screening rate of11.6%(61/525). Most of the phage antibodies displayed binding activity with all three esophageal cancer cell lines. Moreover, other phages (such as NFc53a and NFc70a) appeared to be specific for certain cell lines. Regarding the method used to treat the target cells, positive screening rate of PFc, PFs, NFc or NFs four groups method, the differences between the groups are statistically significant (χ2=12.046, P=0.007), both the genetic diversity of the antibody variable region were significantly higher in the NF group (12.9and71.4%, respectively) than in the PF group (10.5and14.2%, respectively), two groups of diversity of differences have statistically significant (χ2=4.667, P=0.031). The majority of phage antibodies had binding activity with three esophageal cancer cells, and some antibodies showed specificity to the cell lines. Immunohistochemical analysis demonstrated that the scFv phages have good specificity for esophageal cancer located in the esophageal cancer cell membrane and low binding activity of white blood cells. This technology is helpful for developing small molecular tracers and targeting therapies for malignant tumors.Conclusion:Part I:Comparison clinical data of patients showed CTCs in peripheral blood were closely associated with the diagnosis, treatment and prognosis of patients with ESCC. Consequently, as shown in our studies, the detection of disseminated tumor cells in peripheral blood might be of clinical relevance with respect to individual patients’prognosis and stage or response evaluation, detection early metastasis.Part Ⅱ:Monitoring the dynamic CTCs and molecular characteristics in peripheral blood of ESCC before and after may predict disease progression and treatment efficacy. CTCs levels at all time points were significantly associated with disease progress. In general, the dynamic of CTCs was in according with the image examination for monitoring the efficacy of treatment and disease progression. CTCs could reflect the unfavorable prognosis of the patient, especially those whose serum tumor markers were negative. CTCs thus predicted outcomes at any time during treatment and could be used to guide the scheme of treatment to perform appropriate individualized treatment.Part III:For some complex antigens, intact cancer cells for antibody screening requires a higher quality antibody library and a tighter screening process because most cell surface antigens have a specific conformation with glycosylation and other post-translational modifications. Using large antibody library and allowed for the identification of complex and varied antigens in tumor tracing and targeted will have a broad application prospects. Single-chain antibodies from large phage antibody library had specific binding ability to esophageal cells and leukocyte-free combination, which can be used to identify CTCs in peripheral blood of patients and provide more antibody targets for tumor immunotherapy and detection.This research shows that, CTCs detection were associated with tumor stage, infiltration degree, lymph node metastasis and the number of preoperative CTCs,survival time, so CTCs can guide the cancer treatment, judge provide information and appraisal cancer treatment effect, monitor the tumor recurrence. This screened antibodies expected to improve CTCs detection sensitivity and specificity, and to be more widely used in cancer treatment areas.The main innovation1. By experimental date and statistical methods to analyze the evaluation of CTCs for diagnosis of ESCC and improvement the applicational value for disease diagnosis and progression.2. Using a large phage antibody library to obtain specific antibodies of esophageal cancer provides more targets for the diagnosis and identification of esophageal cancer cells. |
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