Detection Of Circulating Tumor Cells From Peripheral Blood In Patients With Esophageal Squamous Cell Carcinoma And Its Clinical Significance

Background:Esophageal cancer, in which the most common pathological type is squamous cell carcinoma is a common malignant tumor in China and has the forth morbidity in human.The morbidity and mortality of esophageal cancer improve obviously by develeopment of medical and efforts of medical worker.Howerer,even after surgical resection,neoadjuvant and adjuvant chemotherapy, neoadjuvant and adjuvant radiotherapy, immunotherapy biotherapy and Chinese traditional treatment,the five-year survival rate remains30percent and which results from matestasis and recurrence of tumor.Many patients who has lower staging of tumor and no evidence of metastases die from matestasis and recurrence even undergo radical excision.This shows dissemination and unconspicuous metastasis of tumor cells cannot be found with routine clinical examinations and histopathology techniques.The favorite methods include image examinations, tumor marker detections in blood and biopsy.But,all of the techniques are incapale to identify the existence of tumors as early as possible,and may offer inaccurate informations because of false-negative or false positive results which could evaluate the persence of tumor.Recently,it is found that circulating tumor cells(CTCs)may be a promising approach for evaluating the recurrences and matastases in tumors in pace with intensive study in it. Nevertheless,there is no accepted standards in isolution,enrichment and characterazition of CTCs which hinders the study in CTCs. Whereas, large numbers of studies report the relationship between recurrence,metastasis and prognosis and CTCs. The progression in tumor evaluated by real-time detection of CTCs will be performed timely for preventing the appearance of symptom and deterioration of disease. Dynamic monitoring the changing of the CTCs will help to evaluate the effectiveness of treatment, select the best strategy for individual therapy in order to improve the effect of treatment,prolong the survival and improve the quality of life. At last, CTCs are given more and more attention. The various research of CTCs, especially in breast cancer, colorectal cancer, and prostate cancer, achieve many significant results,while there is few research in CTCs in patients with esophageal squamous cell carcinoma.Objective:This experiment establish a simple,fast and efficient method in detecting CTCs in patients with esophageal squamous carcinoma by immune fluorescence method based on negative strategy and evaluate the utility of it, while explore the clinical significance of the detection of CTCs by analyzing the correlation between CTCs and clinical pathological characteristics and different progression-free survival in various group of different level in CTCs both preoperative and postoperative by which assessing the relationship between CTCs and the development of tumor and providing the proof of CTCs serving as a prognostic factor in patients with esophageal squamous cell carcinoma.Methods:1.. Patienis and tissues amples. CTCs were detected in7.5ml venous blood preoperative and postoperative respectively in48patients with esophageal squamous cell carcinoma treated in Navy General Hospital from April,2009to May,2010..All tumors were confirmed to be esophageal squamous cell carcinoma.No any anti-cancer therapy was performed before operative. Informed consent was signed.All clinical pathologieal data and follow-up information were colleted. Time limited for the research is34months after surgery.Progressio-free survival was calculated as the period from surgery to recurrence. Gender, age, tumor location, diverse differentiation, size of tumor and invision situation, lymph node status, p-TNM stage,recurrence time point,CTCs counts both preoperative and postoperative were registered.The follow-up data was arranged carefuless.2、Experiment procedure.The CTCs was enrichment in24hours preoperative and postoperative respectively.Using negative selection system, red blood cells wsa depleted in red blood cell lysis buffer, white blood cells depleted with Miltenyi magnetic beads and enriched the rare cells from ESCC patients’peripheral blood.3、Explore the correlation between CTCs and clinical pathological characteristics.These was no accepted positive standards of CTCs counts.3was identified as a cut-off point in this experiment consulting studies of other solid tumors. The correlation between CTCs preoperative and clinical pathological characteristics including Gender, age, tumor location, diverse differentiation, size of tumor and invision situation, lymph node status, p-TNM stage and different progression-free survival in various group of different level in CTCs both preoperative and postoperative was analysed.4、Statistical analysis. Spearman’ correlation coefficient was used to assess the relationship between categorical variables.Chi-square test and Fish’s Exact Test were used to assess the relationship between ordinal variables.Progression-free survival was computed using the Kaplan-Meier method and comparison between groups was done by the log-rank test.Statistical analyses were performed using the SPSS(versionl3.0)statistical software, P<0.05was considered statistically significant.Results:1、Blood samples of both preoperative and postoperative had a CTCs detection rate of64.6%(31/48), the median was2(0-72),2(0-36) respectively. CTCs in patients with esophageal squamous cell carcinoma were significant correlation with cell differentiation grade (R=0.431, P=0.002),the invasion of primary cancer (R=0.403, P=0.005), lymph node status (R=0.339, P=0.019), and p-TNM stage (R=0.394, P=0.006). The higher CTCs detection was found in connection with worse cell differentiation grade (x2=9.636, P=0.008), and lymph node status (x2=4.218, P=0.040) and p-TNM stage (x2=8.392, P=0.004)2^The lower median progression-free survival was showed in CTCs positive group than negative group both preoperative (13months vs20months)and postoperative(13months vs20months)which displayed significant difference in preoperative(x2=9.403,P=0.002) and postoperative(x2=23.049, P=0.000) respectively and there was a significant difference between the groups.Conclusion:1、The method which based on negative strategy by immune fluorescence technique is effective for isolating,enriching and identifying CTCs in patients with esophageal squamous cell carcinoma.2、CTCs in patients with esophageal squamous cell carcinoma were significant correlation with cell differentiation grade,the invasion of primary cancer, lymph node status, and p-TNM stage.3、The lower median progression-free survival was showed in CTCs positive group than negative group both preoperative and postoperative.4、The results suggest that CTCs in the peripheral blood of patients with esophageal squamous cell carcinoma are closely related to the recurrence and metastasis status of tumor and potentially serve as a prognostic factor for survival.