应用循环肿瘤细胞解析胰腺癌上皮—间质转化全程及其临床意义探究

发布时间：2018-08-03 16:25

【摘要】：[背景]胰腺癌早期即具有极强的侵袭转移能力,导致临床上确诊胰腺癌的患者手术可切除率低,临床预后极差。上皮-间质转化(Epithelial-Mesenchymal Transition, EMT)被认为是肿瘤的侵袭转移的关键机制之一,但通常只能对原位肿瘤及转移灶进行研究,无法对肿瘤细胞血行转移过程中的EMT进行观察,因此少有针对胰腺癌EMT全程的研究。而循环肿瘤细胞(Circulating tumor cell, CTC)除可作为“液体活检”之外,因其产生与EMT直接相关也被认为是研究循环血中EMT的重要工具,使得针对胰腺癌原位肿瘤-外周血肿瘤细胞-转移灶的EMT全程研究成为可能。[目的]1.评估胰腺癌CTC作为“液体活检”的临床应用前景。2.通过比较胰腺癌原位肿瘤、外周血CTC和转移淋巴结中上皮和间质标志物的表达对胰腺癌EMT的全程进行解析。[方法]1.采用SET-iFISH法对胰腺癌患者外周血CTC进行分类计数及动态监测,分析不同分类计数与手术、化疗、复发转移、临床病理及预后的相关性。2.采用芯片法对胰腺癌患者外周血CTC进行上皮标志物E-cadherin和间质标志物Vimentin的染色,分析不同EMT分类情况与手术、化疗、临床病理的相关性。3.应用免疫组化对确诊胰腺导管腺癌患者的原位肿瘤及转移淋巴结中上皮标志物E-cadherin和间质标志物Vimentin进行染色及评分,分析EMT水平与临床病理及预后的相关性；并通过与芯片法中CTC染色情况比较对胰腺癌EMT全程进行解析。[结果]1. SET-iFISH法：研究共对27名研究对象(20名胰腺导管腺癌患者、5名胰腺良性占位及2名健康受试者)的63例血标本进行检测。CTC分类计数与手术、化疗、临床病理特征呈现一定变化趋势,而术前检测到CTM的患者具有更短的OS(P=0.027)和PFS(p=0.015)。在原位肿瘤及胰腺癌腹水转移细胞中以上皮型细胞为主,而外周血CTC中绝大多数为非上皮型细胞。2.芯片法：研究共对24名研究对象(22名胰腺导管腺癌患者及2名健康受试者)的46例血标本进行检测,并对CTC中EMT情况(E、EM、E=M、EM和M型)进行分类计数。手术可导致CTC总数(p=0.005)及M型CTC(p=0.000)计数下降,而化疗则导致E型CTC (p=0.004)计数下降,进而导致间质为主型CTC比例则呈上升趋势。CTC总数与临床分期、T分期及远处转移相关(p0.05),但CTC中上皮和间质比例与侵袭转移等临床病理特征间相关性不具有统计学意义。3.研究共纳入27名确诊胰腺导管腺癌的患者,其癌组织中E-cadherin的IHC评分与分化程度(p=0.016)呈正相关,而Vimentin与分化程度负相关(p=0.016)。EM组和EM组平均无病生存期分别为627.5±49.0天和337.1±80.9天,差异具有统计学意义(χ2=4.867,p=0.027)。胰腺癌癌组织中E-cadherin表达下降而Vimentin表达升高。胰腺癌原位肿瘤及转移淋巴结中以E-cadherin表达为主,而外周血CTC上上皮及间质比例约各占50%。[结论]1.液体活检：CTM与胰腺癌不良预后密切相关；胰腺癌CTC分类计数与临床分期、T分期及远处转移相关,并随手术、化疗、复发转移成一定变化趋势。2.胰腺癌EMT全程：胰腺癌原位肿瘤-外周血CTC-转移淋巴结呈现EMT/MET变化趋势,EMT与化疗及不良预后相关,但其与侵袭转移的关系仍需进一步研究。
[Abstract]:[background] pancreatic cancer has a very strong invasion and metastasis ability in the early stage, resulting in low surgical excision rate and poor clinical prognosis in patients with clinically confirmed pancreatic cancer. Epithelial-Mesenchymal Transition (EMT) is considered to be one of the key mechanisms of tumor invasion and metastasis, but usually only in situ tumors and metastases. The study can not be used to observe the EMT in the metastasis of tumor cells. Therefore, there are few studies on the whole process of pancreatic cancer EMT, and the Circulating tumor cell (CTC) can be used as a "liquid biopsy", and the direct correlation with EMT is also recognized as an important tool to study EMT in circulating blood. EMT in situ tumor of pancreatic cancer - peripheral blood tumor cell metastasis is possible. [Objective]1. to evaluate the clinical application of pancreatic cancer CTC as "liquid biopsy".2. through comparison of pancreatic cancer in situ tumors, the expression of epithelial and interstitial markers in peripheral blood CTC and metastatic lymph nodes for the whole process of pancreatic cancer EMT. Methods]1. was used to count and monitor the peripheral blood CTC of the patients with pancreatic cancer by SET-iFISH method. The correlation between different classification counts and surgery, chemotherapy, recurrence and metastasis, clinicopathology and prognosis of.2. were analyzed by chip method for the staining of the epithelial markers E-cadherin and the interstitial marker Vimentin of the peripheral blood CTC of the pancreatic cancer patients. The correlation of different EMT classification, operation, chemotherapy, and clinicopathological correlation.3. applied immunohistochemistry to stain and score the epithelial markers E-cadherin and interstitial marker Vimentin in the patients with pancreatic ductal adenocarcinoma in situ, and to analyze the correlation between the level of EMT and the clinicopathology and prognosis. The whole process of EMT in pancreatic cancer was analyzed by CTC staining. [results]1. SET-iFISH method: a total of 63 blood specimens of 27 subjects (20 patients with pancreatic duct adenocarcinoma, 5 benign pancreatic occupying sites and 2 healthy subjects) were examined for the.CTC classification count and operation, chemotherapy and clinicopathological features. Patients with CTM before operation had shorter OS (P=0.027) and PFS (p=0.015). In situ tumors and pancreatic carcinoma ascites metastasis cells were dominant, while most of the peripheral blood CTC was non epithelial cell.2. chip method: a total of 24 subjects (22 patients with pancreatic ductal adenocarcinoma and 2 healthy subjects) were studied. The blood samples were detected and the EMT status in CTC (E, EM, E=M, EM and M type) was counted. The operation could lead to the decrease of the total number of CTC (p=0.005) and M CTC (p=0.000). Shift correlation (P0.05), but the correlation between the proportion of epithelial and interstitial tissue in CTC and invasion and metastasis was not statistically significant. The.3. study was included in 27 patients with pancreatic ductal adenocarcinoma. The IHC score of E-cadherin was positively correlated with the degree of differentiation (p=0.016) in the cancer tissue, while Vimentin was negatively correlated with the degree of differentiation (p=0.016).EM. The average disease-free survival period of group and EM group was 627.5 + 49 days and 337.1 + 80.9 days respectively. The difference was statistically significant (x 2=4.867, p=0.027). The expression of E-cadherin in pancreatic cancer tissues was decreased, and Vimentin expression was increased. The majority of the pancreatic carcinoma in situ tumor and metastatic lymph node were E-cadherin table, while the proportion of epithelium and interstitial in peripheral blood was about CTC 50%.[conclusion]1. liquid biopsy: CTM is closely related to poor prognosis of pancreatic cancer; CTC classification of pancreatic cancer is associated with clinical staging, T staging and distant metastasis, and with surgery, chemotherapy, recurrence and metastasis to a certain trend of.2. pancreatic cancer EMT: pancreatic cancer in situ tumor - peripheral blood CTC- transfer lymph nodes present EMT/MET trend. EMT is associated with chemotherapy and poor prognosis, but its relationship with invasion and metastasis still needs further study.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

【相似文献】