原发性肝癌患者外周血循环肿瘤细胞的EMT分型及临床意义

发布时间：2018-04-16 05:39

  本文选题：原发性肝癌 + 肝良性病　； 参考：《南方医科大学》2017年博士论文

【摘要】：研究背景和目的原发性肝癌是世界上最常见的具有高发病率和死亡率的恶性肿瘤之一,手术切除是大多数原发性肝癌(primary liver cancer,PLC)的首选治疗方式,然而术后复发是影响PLC预后的主要原因。目前对于PLC的复发监测只要依靠B超、CT及血清AFP,这些传统监测手段是无法做到早期诊断复发。因此,发现具有早期诊断PLC复发的敏感性生物标志物至关重要。在过去的几十年中,肿瘤患者外周血中循环肿瘤细胞的检测已经获得越来越多的关注。因为外周血循环肿瘤细胞检测是一种简单、可重复和微创的操作,其可作为理想的取样检测手段。循环肿瘤细胞(circulating tumor cells,CTCs)是指存在于实体瘤外的肿瘤细胞。事实上,源自原发性肿瘤灶的具有高度侵袭性的肿瘤细胞不断增殖,它们在离开原发肿瘤灶并进入外周血后成为具有侵袭和转移潜能的循环肿瘤细胞。因此,近年来CTCs在肿瘤诊断,复发和转移中的作用得到越来越多的研究。已有研究证实,上皮-间质转化是一种细胞形态转化过程:细胞失去其上皮特征并获得间充质性质。同时,间充质细胞可能转化为上皮表型细胞,从而促使肿瘤细胞转移定值。许多研究已经建立了上皮-间质转化和各型CTCs形成之间的联系。然而,目前基于CTCs的检测技术有数十种,其每种检测技术的方法及原理各有不同,因而选取并深入研究肝癌CTCs检测技术对于实施本项目起着关键作用。在本研究中,我们首先选取并确立了CanPatrolrM肝癌CTCs检测技术,明确其检测技术的基本方法和原理,研究其检测技术的灵敏度、特异度及可重复性;在此检测技术的基础上分析了 EMT表型CTCs在PLC和非恶性肝脏疾病(nonmalignant liver diseases,NLD)之间的差异,试图提高PLC诊断的准确性。众所周知,肿瘤复发和转移是一个非常复杂的过程,而其血型转移过程通常需要CTCs从原发性肿瘤灶进入外周血.并且由于外周血CTCs检测是一种简单,可重复和微创过程,因此在过去几十年中,CTCs在肿瘤诊断、复发和转移中的功能一直在积极研究。但是对于各亚型循环肿瘤细胞与肿瘤复发的研究目前却甚少,本研究通过检测PLC患者外周血中的各亚型CTCs,分析其与PLC复发的关系,初步探讨各亚型CTCs检测在早期预测PLC复发中的临床价值。方法:采集PLC患者外周血,利用多重原位RNA杂交检测技术分离检测外周血中的CTCs,并通过细胞上皮型标志物(EpCAM、CK8,18,19)及间质型标志物(Vimentin、Twist)对CTCs进一步分型,第一组共采集73例入院诊断为肝占位患者的5-mL外周血用来检测CTCs,其中最后入组45位原发性肝癌患者(年龄中位数:55,范围:18至77)和13位肝良性病患者(年龄中位数:50,范围:35至74)。第二组选取2014年3月至2016年3月期间行根治性切除术的原发性肝癌患者62例(男:58例,女:4例;年龄:median:55,rang:30～79)行前瞻性研究,均抽取5-mL外周血用来检测CTCs。我们通过采用CanPatro1TM CTCs富集技术来检测外周血中的EMT表型CTCs。通过使用该技术,判断并计数不同表型肿瘤细胞,研究其在PLC中诊断及术后预测复发的临床价值。统计学处理:所有统计计算用SPSS v21.0进行,P值0.05被认为具有统计学意义。结果:我们应用CanPatrolrM系统将检测到的外周血中的EMT表型CTCs分成以下三类:上皮型CTCs(上皮标记染色),间质型CTCs(间质标记染色),混合型CTCs(上皮和间质标记染色)同时,为了检测该技术分离鉴定CTCs的回收效率及敏感性,我们将0,10,50,100,200个培养的肿瘤细胞加入健康人的5mL外周血样本中,与实际加入的肿瘤细胞数目进行对比分析,结果显示外周血中分别加入0,10,50,100,200个HepG2细胞的平均检出数为0,8±1.41,43.33±2.49,85±3.56,182.33±4.99,线性回归模型分析得到检出的CTCs与加入的肿瘤细胞数量具有明显线性相关性(r2=0.999)。通过应用该技术对PLC及NLD的诊断性研究,首先入组73例诊断为肝占位的患者中;3例患者未行进一步诊断,10名患者最终被诊断为其他恶性肿瘤肝转移,45名被诊断为原发性肝癌,13名被诊断为肝良性病;最终,我们发现除了上皮型CTCs,其它各类型EMT表型CTCs计数在PLC中显著高于NLD,差异具有统计学意义(P0.05)。在深入研究EMT表型的CTCs在PLC与复发的关系中,我们入组了62例经根治性手术治疗的PLC患者,通过随访,26例最终诊断为复发,36例诊断为未复发。复发组与未复发组CTCs总数(中位数:6vs2.5),上皮型CTCs(中位数:0.5vs0),混合型CTCs(中位数:3vs1),间质型CTCs(中位数:1vs0)。最终发现;复发组CTCs总数、间质型CTCs、混合型CTCs计数显着高于未复发患者。为了确定与复发相关的各型CTCs的截点值,行ROC曲线分析定义各型CTCs的截点值:CTCs总数≥4为阳性,间质型CTCs≥1为阳性,混合型CTCs≥3为阳性.经多因素cox回归发现,门脉癌栓(HR=2.905,P = 0.023)及间质型 CTCs(HR=3.453,P = 0.007)阳性为复发的独立危险因素。K-M检验进一步研究间质型CTCs与复发时间的关系,结果发现间质型CTCs阳性患者术后无瘤生存时间显著缩短(P0.001)。结论:1、研究证明了EMT现象存在于原发性肝癌患者外周血CTCs中;2、外周血中间质型CTCs和混合型CTCs计数将有望成为诊断原发性肝癌的新型生物标记物;3、原发性肝癌患者术后外周血中间质型CTCs阳性能增加复发的风险。间质型CTCs可以作为监测原发性肝癌预后的生物指标;4、门脉癌栓及间质型CTCs阳性为复发的独立危险因素,且间质型CTCs对复发结局的预测能力大于门脉癌栓。
[Abstract]:Background and objective: primary hepatocellular carcinoma is one of the most common with high morbidity and mortality of malignant tumors in the world, surgical resection is the most primary liver cancer (primary liver cancer, PLC) the preferred treatment, but recurrence is the main factor influencing the prognosis of PLC patients. The recurrence monitoring as long as PLC by B-ultrasound, CT and AFP in serum, the traditional monitoring methods is unable to achieve the early diagnosis of recurrence. Therefore, it is important to find sensitive biomarkers for early diagnosis of the recurrence of PLC. In the past few decades, the tumor in the peripheral blood of patients with the detection of circulating tumor cells has received more and more attention. Because the detection of circulating tumor cells the peripheral blood is a simple, repeatable and minimally invasive operation, which can be used as sampling detection means. The ideal of circulating tumor cells (circulating tumor cells, CTCs) is present in the Solid tumors of tumor cells. In fact, highly invasive tumor cells from the primary tumor to proliferate, they leave the primary tumor and enter the peripheral blood circulating tumor cells has become invasive and metastatic potential. Therefore, in recent years CTCs in tumor diagnosis, recurrence and metastasis the role of more and more research. Studies have demonstrated that epithelial mesenchymal transition is a cell transformation process: cells lose their epithelial and mesenchymal features. At the same time, mesenchymal cells may be transformed into cells from epithelial phenotype, and promote tumor cell metastasis value. Many studies have been established between epithelial mesenchymal transition and the formation of CTCs. However, at present, based on several detection techniques of CTCs ten, the method of each kind of detection technology and principle are different, thus the selection and study of liver cancer CTCs Detection technology plays a key role in the implementation of the project. In this study, we firstly selected and established CanPatrolrM hepatoma CTCs detection technology, the detection technology of the basic method and principle, the sensitivity of detection, specificity and repeatability; based on the analysis of the EMT detection technology in CTCs phenotype PLC and non malignant liver disease (nonmalignant liver, diseases, NLD) the difference between, trying to improve the diagnostic accuracy of PLC. As everyone knows, the recurrence and metastasis of tumor is a very complicated process, and the blood transfer process usually takes CTCs from the primary tumor into peripheral blood. And the detection of CTCs in peripheral blood is a simple, minimally invasive and repeatable process, so in the past few decades, CTCs in the diagnosis of tumor recurrence and metastasis, the function has been active in research. But for each subtype of circulating tumor Study on cell and tumor recurrence is little, the study of each CTCs subtype by detection of PLC in the peripheral blood of patients and analyze its relationship with the recurrence of PLC, preliminary study of various subtypes of CTCs testing to predict the clinical value of PLC in early recurrence. Methods: collected peripheral blood of PLC patients, the use of multiple in situ RNA hybridization detection technology of separation and detection of CTCs in peripheral blood, and the epithelial cell type markers (EpCAM, CK8,18,19) and mesenchymal markers (Vimentin, Twist) of CTCs furtheranalysis, the first group were collected from 73 patients diagnosed as liver lesions in patients with peripheral blood 5-mL to detect CTCs the last group of 45 patients with primary liver cancer (median age: 55, range: 18 to 77) and 13 patients with benign liver disease (median age: 50, range: 35 to 74). The second group from March 2014 to March 2016 during radical resection of primary liver cancer patients (62 cases Male: 58 cases, female: 4 cases; age: median:55, rang:30 ~ 79) study prospectively, all were extracted from 5-mL peripheral blood to detect CTCs. by adopting CTCs concentration technique CanPatro1TM to detect the phenotype of EMT CTCs. in the peripheral blood by using this technology, determine and count the different phenotypes of tumor cells, prediction to study the clinical value of PLC in diagnosis of recurrence and postoperative. Statistical analysis: all statistical calculation was carried out with SPSS v21.0, the P value of 0.05 was considered statistically significant. Results: EMT phenotype of peripheral blood CTCs we used CanPatrolrM detected in the system will be divided into the following three categories: epithelial type CTCs (epithelial staining (CTCs), mesenchymal stromal staining), mixed CTCs (epithelial and stromal staining) at the same time, in order to detect the recovery efficiency and sensitivity of the isolation and identification of CTCs technology, we will 0,10,50100200 cultured tumor cells with health Blood samples the 5mL, compared with the number of tumor cells with actual, the results showed that the addition of 0,10,50100200 HepG2 cells in the peripheral blood of the average detection number of 0,8 + 1.41,43.33 + 2.49,85 + 3.56182.33 + 4.99, linear regression analysis was CTCs and added the number of cells with obvious linear correlation get (r2=0.999). By studying the diagnostic application of the technology of PLC and NLD, into the first group of 73 cases diagnosed as liver lesions in patients; 3 patients underwent no further diagnosis, 10 patients were diagnosed as other malignant tumors of liver metastasis, 45 were diagnosed with primary liver cancer, 13 men who were diagnosed with benign liver diseases; finally, we found that in addition to epithelial type CTCs, other types of EMT phenotype CTCs count in PLC was significantly higher than that of NLD, the difference was statistically significant (P0.05). On the research of EMT in PLC and CTCs phenotype 澶嶅彂鐨勫叧绯讳腑,鎴戜滑鍏ョ粍浜，

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