CN111830249A - 用于纯化分离及分析非典型循环肿瘤细胞的方法的用途及非典型循环肿瘤细胞的用途 - Google Patents
用于纯化分离及分析非典型循环肿瘤细胞的方法的用途及非典型循环肿瘤细胞的用途 Download PDFInfo
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Abstract
本发明涉及检测领域,尤其是有关于一种用于纯化分离及分析非典型循环肿瘤细胞的方法的用途及非典型循环肿瘤细胞的用途。所述纯化分离及分析非典型循环肿瘤细胞的方法,以及非典型循环肿瘤细胞可用于制备诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒。本发明具有高通量、高纯度及高回收率、无筛选偏倚的优点,可成功纯化分离及分析非典型循环肿瘤细胞。并且通过非典型循环肿瘤细胞的分析,可评估癌症风险、癌症预后及疗效等,提高检测的灵敏度及准确度。
Description
技术领域
本发明涉及检测领域,尤其是关于用于纯化分离及分析非典型循环肿瘤细胞的方法的用途及非典型循环肿瘤细胞的用途。
背景技术
癌症转移(cancer metastasis)是癌症引起死亡的主要原因。循环肿瘤细胞(circulating tumor cells,CTCs),自1869年即被证实,是从原发性肿瘤部位逃逸至邻近脉管系统并随后存在于血液循环中的细胞。已有证据表明,血液循环中循环肿瘤细胞的存在与癌症转移有关。因此,本领域的技术人员已致力于研究循环肿瘤细胞以理解癌症转移的机制。此研究方向可刺激本领域的技术人员开发出新的癌症治疗策略。
此外,在临床应用方面,循环肿瘤细胞的分析(被视为液体肿瘤活检(liquidtumor biopsy))可被使用作为诊断或预后工具,以供监测癌症转移或治疗反应,及指导个体化治疗。为了实现这些目标,有必要自血液样品中分离出高纯度的循环肿瘤细胞以尽可能避免周边血液细胞(主要是白血球)造成的分析干扰。
然而,循环肿瘤细胞在血液样品中非常稀少,其浓度大约为每105~107个血液单核细胞有1个循环肿瘤细胞。此现象使循环肿瘤细胞难以分离及纯化,特别是纯化分离出高纯度的循环肿瘤细胞。目前已有各种不同的循环肿瘤细胞分离及纯化方法,大致可分类为物理及生化方法。总体而言,以物理为基础的循环肿瘤细胞的分离方法(主要是过滤)易于实施且不需要标记收获的细胞,但细胞纯度低于生化方法所达到的纯度。在生化方法中,免疫式细胞分离法(例如免疫磁珠法)主要用于循环肿瘤细胞分离及纯化。在此方法中,与循环肿瘤细胞的表面标志蛋白(主要是上皮细胞黏附分子(epithelial cell adhesionmolecule,EpCAM)及细胞角质蛋白(cytokeratins,CKs))专一性抗体偶合的磁珠通常用于辨识及结合循环肿瘤细胞。透过施加的磁场将磁性标记的循环肿瘤细胞从周边细胞分离。根据此方法的循环肿瘤细胞分离主要用于目前主流的循环肿瘤细胞分离或检测系统(例如CellSearchTM系统、磁性活化的细胞筛选系统或DynabeadsTM)。
虽然已有上述循环肿瘤细胞分离方式,但仍有许多问题需要克服。其中一个问题为纯化分离得到的循环肿瘤细胞中白血球的污染往往是不可避免的,可能影响后续循环肿瘤细胞相关分析(特别是基因表现分析)的准确性,此事实突显了分离出高纯度循环肿瘤细胞以供后续高精度分析的重要性。除了循环肿瘤细胞的纯度问题,还有一些重要的生物学问题需要进一步考虑。如前所述,大部分循环肿瘤细胞的分离或纯化方式主要依赖使用EpCAM或CKs来鉴定循环肿瘤细胞。然而,循环肿瘤细胞(特别是具有高转移性的循环肿瘤细胞)可能经历上皮间质转化(epithelial-to-mesenchymal transition,EMT),使细胞获得转移所必需的特性,例如移行及侵犯能力、抗凋亡、癌干细胞特性等。这些经历EMT的循环肿瘤细胞可能会降低EpCAM及CKs这类上皮细胞标志基因的表现,在此情况下,若采用传统的循环肿瘤细胞分离方法(依赖EpCAM及CKs为基础的正向筛选法),可能会遗漏这些经历EMT且临床上与癌症转移有高度关联的循环肿瘤细胞。
因此,本领域的技术人员亟需研发出新颖的纯化分离及分析非典型循环肿瘤细胞(亦即不表现EpCAM及CKs这类典型循环肿瘤细胞标志的循环肿瘤细胞)的方法及非典型循环肿瘤细胞的新用途,以克服习知技术的缺点并造福有此需求的广大族群。
发明内容
有鉴于此,本发明的目的为提供一种纯化分离及分析一血球表面蛋白阴性及循环肿瘤细胞标志蛋白阴性的非典型循环肿瘤细胞的方法用于制备诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒的用途,其中该方法包含以下步骤:(a)提供来自一个体的一全血;(b)对该全血进行一处理,以去除复数红血球及复数血小板,得到一经处理的样品;(c)利用一免疫式细胞分离法对该经处理的样品进行负向筛选,将至少一血球表面蛋白阳性的白血球细胞移除,得到一经负向筛选的细胞群;(d)对该经负向筛选的细胞群进行一免疫荧光染色,辨识该经负向筛选的细胞群中的复数细胞亚群,其中该些细胞亚群包括至少一白血球及至少一非白血球有核细胞,其中该至少一非白血球有核细胞包含一血球表面蛋白阴性及循环肿瘤细胞标志蛋白阳性的典型循环肿瘤细胞及该非典型循环肿瘤细胞;以及(e)利用一单细胞分析技术,对该些细胞亚群进行分析、鉴定、计量及纯化分离,并藉由该血球表面蛋白及该循环肿瘤细胞标志蛋白排除血球细胞及该典型循环肿瘤细胞,得到该非典型循环肿瘤细胞及其数量信息;其中当该个体的非典型循环肿瘤细胞的数量或基因信息高于一截止值(cut-off value)时,判定为罹患癌症、癌症复发、癌症治疗成效不彰、或癌症预后不良的高风险族群,其中该截止值为经过一临床试验后透过统计分析方法所得的一数值。
在本发明的一实施例中,该血球表面蛋白是选自于下列所构成的群组:CD3、CD4、CD8、CD11b、CD11c、CD14、CD19、CD20、CD33、CD34、CD41、CD45、CD56、CD61、CD62、CD66b、CD68、CD123、CD146、Gly A,及其任意组合。
在本发明的一实施例中,该循环肿瘤细胞标志蛋白是选自于下列所构成的群组:上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)、细胞角质蛋白(cytokeratins,CKs)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、CD44、CD24、中间丝蛋白(vimentin)、黏液蛋白1(mucin 1,Muc-1)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、Ras、人类表皮生长因子受体2(human epidermalgrowth factor receptor 2,Her2)、MET,及其任意组合。
在本发明的一实施例中,该癌症是一肝癌、一肺癌、一大肠直肠癌、一乳癌、一鼻咽癌、一摄护腺癌、一食道癌、一胰脏癌、一皮肤癌、一甲状腺癌、一胃癌、一肾脏癌、一胆囊癌、一卵巢癌、一子宫颈癌、一骨癌、一脑癌、或一头颈癌。
在本发明的一实施例中,该单细胞分析技术是选自于下列所构成的群组:免疫荧光染色、流式细胞技术、荧光显微术、光诱导介电泳力式微流体生物芯片系统,及其任意组合。
本发明的另一目的为提供一种血球表面蛋白阴性及循环肿瘤细胞标志蛋白阴性的非典型循环肿瘤细胞用于制备诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒的用途,其中该非典型循环肿瘤细胞是由一个体的一全血所纯化分离出,且当该个体的非典型循环肿瘤细胞的数量或基因信息高于一截止值(cut-off value)时,判定为罹患癌症、癌症复发、癌症治疗成效不彰、或癌症预后不良的高风险族群,其中该截止值为经过一临床试验后透过统计分析方法所得的一数值。
在本发明的一实施例中,该血球表面蛋白是选自于下列所构成的群组:CD3、CD4、CD8、CD11b、CD11c、CD14、CD19、CD20、CD33、CD34、CD41、CD45、CD56、CD61、CD62、CD66b、CD68、CD123、CD146、Gly A,及其任意组合。
在本发明的一实施例中,该循环肿瘤细胞标志蛋白是选自于下列所构成的群组:上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)、细胞角质蛋白(cytokeratins,CKs)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、CD44、CD24、中间丝蛋白(vimentin)、黏液蛋白1(mucin 1,Muc-1)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、Ras、人类表皮生长因子受体2(human epidermalgrowth factor receptor 2,Her2)、MET,及其任意组合。
在本发明的一实施例中,该癌症是一肝癌、一肺癌、一大肠直肠癌、一乳癌、一鼻咽癌、一摄护腺癌、一食道癌、一胰脏癌、一皮肤癌、一甲状腺癌、一胃癌、一肾脏癌、一胆囊癌、一卵巢癌、一子宫颈癌、一骨癌、一脑癌、或一头颈癌。
在本发明的一实施例中,该非典型循环肿瘤细胞为游离态。
综上所述,本发明用于纯化分离及分析非典型循环肿瘤细胞的方法及非典型循环肿瘤细胞的用途的功效在于:高通量、高纯度及高回收率、无筛选偏倚,可成功纯化分离及分析非典型循环肿瘤细胞。通过本发明,可由单一检体回收多项参数用于临床诊断癌症、评估癌症预后、追踪监控癌症及评估癌症治疗成效。多参数同步分析更可提高临床应用的灵敏度及准确度,成为落实精准医疗的重要依据。是故,本发明在临床医学与基础研究两方面皆具有重要的应用价值。
以下将进一步说明本发明的实施方式,下述所列举的实施例是用以阐明本发明,并非用以限定本发明的范围,任何熟习此技艺者,在不脱离本发明的精神和范围内,当可做些许更动与润饰,因此本发明的保护范围当视后附的申请专利范围所界定者为准。
附图说明
图1是CD45阴性及EpCAM阴性细胞辨识及分析的示意图;
图2是22位健康受试者及39位癌症患者(包括肝癌、肺癌、鼻咽癌、摄护腺癌、食道癌、胰脏癌、及头颈癌)的典型循环肿瘤细胞(A)及CD45阴性及EpCAM阴性细胞(B)计量分析的示意图;
图3是22位健康受试者及27位头颈癌患者的典型循环肿瘤细胞(A)及CD45阴性及EpCAM阴性细胞(B)计量分析的示意图;
图4是22位健康受试者及27位头颈癌患者的存活分析的数据图。
具体实施方式
定义
本文中所使用数值为近似值,所有实验数据皆表示在20%的范围内,较佳为在10%的范围内,最佳为在5%的范围内。
如本文中所使用的,用语“循环肿瘤细胞(circulating tumor cell,CTC)”意为包含存在于与癌症有关的生物样品中的任何稀少的肿瘤细胞。
如本文中所使用的,用语“免疫磁珠法(magnetic activated cell-sorting,又称为磁性活化的细胞筛选)”意指利用免疫磁珠进行细胞筛选的方法,其是在磁珠表面包覆具免疫反应性的抗体,其可与目标细胞上的抗原进行抗原抗体反应,这些结合了磁珠的细胞一旦置于磁场下,就会与其他未被结合的细胞分群,具磁场的磁珠脱离磁场后立即消失磁性,藉此可以筛选或去除所标记的细胞,从而达到正向或负向细胞的目的。
实施例1.全血检体处理及CD45阴性与EpCAM阴性细胞计量方法
在本实施例中,实验是由长庚纪念医院的人体试验伦理部门(InstitutionalReview Board of the Chang Gung Memorial Hospital)所核准。全部的血液样品捐赠者皆取得知情同意书(批准号:201601081B0),且全部方法皆按照临床试验的相关指南进行。
首先,提供4mL的来自一个体的全血检体,然后将4mL的全血检体中的血球去除。使用红血球裂解液,1L的红血球裂解液含有8.26g的NH4Cl、1.19g的NaHCO3、200μL的0.5M、pH 8的EDTA(乙二胺四乙酸),最终pH值为7.3。其中,全血检体与红血球裂解液的体积比例为1:10,作用不超过10分钟,作用完离心去除上清液。接着,以100~200xg的转速离心去除血小板,得到一经处理的样品。之后,利用免疫式细胞分离法对经处理的样品进行负向筛选,将至少一血球表面蛋白(例如CD3、CD4、CD8、CD11b、CD11c、CD14、CD19、CD20、CD33、CD34、CD41、CD45、CD56、CD61、CD62、CD66b、CD68、CD123、CD146、Gly A,及其任意组合)阳性的血球细胞移除。在本实施例中,按照EasySepTM CD45移除套组(EasySepTM CD45 depletion kit)(StemCell Technologies,温哥华,BC,加拿大)的操作流程去除CD45阳性的白血球细胞,得到一经负向筛选的细胞群。
接着,对经负向筛选的细胞群进行免疫荧光染色,辨识经负向筛选的细胞群中的复数细胞亚群,其中细胞亚群包括至少一白血球及至少一非白血球有核细胞,其中非白血球有核细胞包含一血球表面蛋白阴性及循环肿瘤细胞标志蛋白(例如上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)、细胞角质蛋白(cytokeratins,CKs)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、CD44、CD24、中间丝蛋白(vimentin)、黏液蛋白1(mucin 1,Muc-1)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、Ras、人类表皮生长因子受体2(human epidermal growth factor receptor 2,Her2)、MET,及其任意组合)阳性的典型循环肿瘤细胞,及血球表面蛋白阴性及循环肿瘤细胞标志蛋白阴性的非典型循环肿瘤细胞。
在本实施例中,免疫荧光染色流程如下:以细胞核染剂染细胞核、荧光物结合的抗体分别标记白血球及典型循环肿瘤细胞,其标记目标蛋白分别为CD45及EpCAM,或其它可辨识此二细胞种的蛋白标志专一性抗体。染色完成后以PBS洗去多余抗体即完成染色步骤。之后,利用单细胞分析技术,例如流式细胞技术及光诱导介电泳(optically-induceddielectrophoresis,ODEP)力式微流体生物芯片系统,对完成染色的细胞亚群进行荧光辨识、分析、鉴定、计量及纯化分离,并藉由血球表面蛋白及循环肿瘤细胞标志蛋白排除血球细胞及典型循环肿瘤细胞,得到CD45阴性及EpCAM阴性的非典型循环肿瘤细胞及其数量信息,如图1所示。
实施例2.CD45阴性及EpCAM阴性细胞用于癌症患者鉴别的专一性及灵敏度测试
在本实施例中,实验是由长庚纪念医院的人体试验伦理部门所核准。全部的血液样品捐赠者皆取得知情同意书(批准号:201601081B0),且全部方法皆按照临床试验的相关指南进行。
首先,依照上面实施例1所述操作流程得到CD45阴性及EpCAM阴性的非典型循环肿瘤细胞及其数量信息。接着,利用分析族群的接收者操作特征曲线(receiver operatingcharacteristic curve,ROC curve)设定截止值(cut-off value),其中截止值为需要诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的复数个体的非典型循环肿瘤细胞透过统计分析方法计算出的平均值、中位数、或透过接收者操作特征曲线分析所得的一数值。之后,利用统计分析法计算专一性(specificity)及灵敏度(sensitivity),其中专一性是健康受试者被诊断为阴性的比例(真阴性(True negative)÷健康受试者总数(真阴性+伪阳性)),灵敏度为癌症患者诊断结果为阳性的比例(真阳性(True positive)÷癌症患者总数(真阳性+伪阴性))。另外,当个体的非典型循环肿瘤细胞的数量高于截止值时,判定为罹患癌症、癌症复发、癌症治疗成效不彰、或癌症预后不良的高风险族群。在本实施例中,癌症是以头颈癌例示说明,并以系统作为对照组,结果显示于下表1。
表1
注1:健康受试者22名、晚期(三至四期)头颈癌患者27名
注2:健康受试者209名、晚期(三至四期)头颈癌患者852名
实施例3.典型循环肿瘤细胞数目及CD45阴性及EpCAM阴性细胞数目的临床意义
在本实施例中,实验是由长庚纪念医院的人体试验伦理部门所核准。全部的血液样品捐赠者皆取得知情同意书(批准号:201601081B0),且全部方法皆按照临床试验的相关指南进行。
首先,依照上面实施例1所述操作流程得到CD45阴性及EpCAM阴性的非典型循环肿瘤细胞及其数量信息。接着,透过统计分析方法比较健康受试者及癌症患者两群细胞的平均数是否有差异,其中统计方法为曼-惠特尼U检定(Mann-Whitney U test),P值小于0.05视为具显著差异。
图2是22位健康受试者及39位癌症患者(包括肝癌、肺癌、鼻咽癌、摄护腺癌、食道癌、胰脏癌、及头颈癌)的典型循环肿瘤细胞(A)及CD45阴性及EpCAM阴性细胞(B)计量分析的示意图,其中H表示健康受试者,Pt表示癌症患者。由图2可见,癌症患者的前述两群细胞皆显著高于健康受试者。
图3是22位健康受试者及27位头颈癌患者的典型循环肿瘤细胞(A)及CD45阴性及EpCAM阴性细胞(B)计量分析的示意图,其中H表示健康受试者,Pt表示头颈癌患者。由图3可见,头颈癌患者的前述两群细胞皆显著高于健康受试者。因此,本发明方法确实可应用于制备鉴别癌症患者,即诊断癌症的试剂盒。
接着,头颈癌患者依据前述两群细胞计数结果各别区分为细胞数高及低两次族群。接着透过统计方法针对两群细胞各别的高、低次族群患者进行存活分析,即克普兰-麦尔分析(Kaplan-Meier analysis)。受试者的血液样品被收集后首次治疗经临床医师评估疾病是否恶化作为存活分析的事件(event)。结果显示于图4。图4是22位健康受试者及27位头颈癌患者的存活分析的数据图,其中PFS表示无恶化存活时间(progression-freesurvival),单位为月。由图4可见,无恶化存活时间在CD45阴性及EpCAM阴性细胞高(虚线)的收案族群相较于该群细胞低的族群(实线)有较差的趋势。本实施例的结果证实,本发明方法确实可应用于制备评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒。
实施例4.分离自头颈癌患者的血液样品中CD45阴性及EpCAM阴性细胞群并分析其基因表现的临床意义
在本实施例中,实验是由长庚纪念医院的人体试验伦理部门所核准。全部的血液样品捐赠者皆取得知情同意书(批准号:201601081B0),且全部方法皆按照临床试验的相关指南进行。
在传统循环肿瘤细胞相关研究中,细胞蛋白EpCAM及CKs主要用作鉴定循环肿瘤细胞的生物标志。然而,越来越多证据表明由于循环肿瘤细胞的异质特征,使用这些生物标志来鉴定循环肿瘤细胞是不够的。例如经历EMT的循环肿瘤细胞其EpCAM及CKs表现会下降,因此,在传统基于EpCAM及CKs表现的循环肿瘤细胞正向筛选方法中,这些历经EMT后与癌症转移高度相关的循环肿瘤细胞通常会被忽略。而透过负向筛选则可避免正向筛选面临的筛选偏倚的问题。此外,我们发现到癌症患者的血液样品经负向筛选后,其中所包含的CD45阴性及EpCAM阴性的有核细胞的数目显著高于健康捐赠者,且其数目与患者预后相关(如图2~4所示)。
为探讨癌症患者的血液样品中CD45阴性及EpCAM阴性细胞群的基因表现的临床意义,本发明自头颈癌患者(n=7)取得血液样品(8mL),然后依照前面实施例1所述方式处理血液样品以分离出CD45阳性白血球和CD45阴性及EpCAM阴性细胞群。然后,以次世代定序技术分析两群细胞的转录体差异,经分析后筛选出CD45阴性及EpCAM阴性细胞独特表现的基因,再以实时聚合酶链反应(real-time polymerase chain reaction)技术验证次世代定序结果。
简言之,纯化分离CD45阴性及EpCAM阴性细胞后,以Ovation Solo RNA-Seq系统(NuGEN Technologies,Inc.)处理细胞以制备定序所需基因库,接着利用Illumina HiSeq4000定序系统执行定序,定序结果经过质量检测分析(Quality Value≥20)、修剪(Trim)、与参考序列(Hg19)进行序列比对(mapping)、及计算转录体表现量(transcript PerMillion,TPM),然后透过比较分析,筛选出CD45阴性及EpCAM阴性细胞独特表现的基因。接着,纯化分离所得的CD45阴性及EpCAM阴性细胞。然后,进行基于TaqMan的检测以确定经分离的细胞的基因表现位准(level)。在本实施例中,硫醇氧化还原蛋白相关的穿膜蛋白2(thioredoxin related transmembrane protein 2,TMX2)基因的TaqMan套组(Hs01128573_g1)是购自于Thermo Fisher Scientific并依据厂商的操作指引进行分析。β-2-微球蛋白(β-2-microglobulin)使用作为内部对照基因。
本实施例的次世代定序结果显示,TMX2为其中一个独特表现于CD45阴性及EpCAM阴性非典型循环肿瘤细胞的基因(其TPM=7.78;而白血球的TPM=0),经过real-time PCR验证,TMX2基因在CD45阴性及EpCAM阴性非典型循环肿瘤细胞的表现(17.25±30.79)的确高于白血球的表现(2.86±2.52)。结果显示于表2。
表2
注1,此实施例为CD45阴性EpCAM阴性有核细胞;
注2,Transcript Per Million,TPM;
注3,相对表现量(ΔΔCq)。
接着,根据TMX2基因在CD45阴性及EpCAM阴性非典型循环肿瘤细胞的表现量将患者分为TMX2低表现及高表现两族群(截止值为中位数),然后比较两族群疾病无恶化存活时间的差异。结果显示于表3。
表3
注1,疾病无恶化存活时间,单位:月。
由表3可见,TMX2基因高表现患者的中位生存期(3.5个月)及半年存活率(33%)相较于TMX2基因低表现患者(中位生存期为11.4个月,半年存活率为80%)有较差的趋势。此结果呼应过去研究发现过量表现TMX2基因与肝癌及头颈癌的预后相关。此结果证实来自CD45阴性及EpCAM阴性非典型循环肿瘤细胞的基因信息,确实有潜力可应用于制备诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒中。
综上所述,本发明用于纯化分离及分析非典型循环肿瘤细胞的方法及非典型循环肿瘤细胞的应用确实可达成以下功效:高通量、高纯度及高回收率、无筛选偏倚,可成功纯化分离及分析非典型循环肿瘤细胞。透过本发明,可由单一检体回收多项参数用于临床诊断癌症、评估癌症预后、追踪监控癌症及评估癌症治疗成效。多参数同步分析更可提高临床应用的灵敏度及准确度,成为落实精准医疗的重要依据。是故,本发明在临床医学与基础研究两方面皆具有重要的应用价值。
以上所述仅为举例性,而非为限制性者。任何未脱离本发明的精神与范畴,而对其进行的等效修改或变更,均应包含于后附的申请专利范围中。
Claims (10)
1.一种纯化分离及分析血球表面蛋白阴性及循环肿瘤细胞标志蛋白阴性的非典型循环肿瘤细胞的方法用于制备诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒的用途,其中所述方法包含以下步骤:
(a)提供来自一个体的全血;
(b)对所述全血进行处理,以去除复数红血球及复数血小板,得到经处理的样品;
(c)利用免疫式细胞分离法对所述经处理的样品进行负向筛选,将至少血球表面蛋白阳性的白血球细胞移除,得到经负向筛选的细胞群;
(d)对所述经负向筛选的细胞群进行免疫荧光染色,辨识所述经负向筛选的细胞群中的复数细胞亚群,其中所述细胞亚群包括至少一白血球及至少一非白血球有核细胞,其中所述至少一非白血球有核细胞包含血球表面蛋白阴性及循环肿瘤细胞标志蛋白阳性的典型循环肿瘤细胞及所述非典型循环肿瘤细胞;以及
(e)利用单细胞分析技术,对所述细胞亚群进行分析、鉴定、计量及纯化分离,并藉由所述血球表面蛋白及该循环肿瘤细胞标志蛋白排除血球细胞及该典型循环肿瘤细胞,得到所述非典型循环肿瘤细胞及其数量信息;
其中当该个体的非典型循环肿瘤细胞的数量或基因信息高于截止值时,判定为罹患癌症、癌症复发、癌症治疗成效不彰、或癌症预后不良的高风险族群,其中所述截止值为经过一临床试验后通过统计分析方法所得的数值。
2.根据权利要求1所述的用途,其特征在于,所述血球表面蛋白是选自于下列所构成的群组:CD3、CD4、CD8、CD11b、CD11c、CD14、CD19、CD20、CD33、CD34、CD41、CD45、CD56、CD61、CD62、CD66b、CD68、CD123、CD146、Gly A,及其任意组合。
3.根据权利要求1所述的用途,其特征在于,所述循环肿瘤细胞标志蛋白是选自于下列所构成的群组:上皮细胞黏附分子、细胞角质蛋白、表皮生长因子受体、CD44、CD24、中间丝蛋白、黏液蛋白1、E-钙黏蛋白、N-钙黏蛋白、Ras、人类表皮生长因子受体2、MET,及其任意组合。
4.根据权利要求1所述的用途,其特征在于,所述癌症是肝癌、肺癌、大肠直肠癌、乳癌、鼻咽癌、摄护腺癌、食道癌、胰脏癌、皮肤癌、甲状腺癌、胃癌、肾脏癌、胆囊癌、卵巢癌、子宫颈癌、骨癌、脑癌、或头颈癌。
5.根据权利要求1所述的用途,其特征在于,所述单细胞分析技术是选自于下列所构成的群组:免疫荧光染色、流式细胞技术、荧光显微术、光诱导介电泳力式微流体生物芯片系统,及其任意组合。
6.一种血球表面蛋白阴性及循环肿瘤细胞标志蛋白阴性的非典型循环肿瘤细胞用于制备诊断癌症、评估癌症预后、追踪监控癌症或评估癌症治疗成效的试剂盒用途,其中所述非典型循环肿瘤细胞是由一个体的全血所纯化分离出,且当该个体的非典型循环肿瘤细胞的数量或基因信息高于截止值时,判定为罹患癌症、癌症复发、癌症治疗成效不彰、或癌症预后不良的高风险族群,其中所述截止值为经过临床试验后透过统计分析方法所得的数值。
7.根据权利要求6所述的用途,其特征在于,所述血球表面蛋白是选自于下列所构成的群组:CD3、CD4、CD8、CD11b、CD11c、CD14、CD19、CD20、CD33、CD34、CD41、CD45、CD56、CD61、CD62、CD66b、CD68、CD123、CD146、Gly A,及其任意组合。
8.根据权利要求6所述的用途,其特征在于,所述循环肿瘤细胞标志蛋白是选自于下列所构成的群组:上皮细胞黏附分子、细胞角质蛋白、表皮生长因子受体、CD44、CD24、中间丝蛋白、黏液蛋白1、E-钙黏蛋白、N-钙黏蛋白、Ras、人类表皮生长因子受体2、MET,及其任意组合。
9.根据权利要求6所述的用途,其特征在于,所述癌症是肝癌、肺癌、大肠直肠癌、乳癌、鼻咽癌、摄护腺癌、食道癌、胰脏癌、皮肤癌、甲状腺癌、胃癌、肾脏癌、胆囊癌、卵巢癌、子宫颈癌、骨癌、脑癌、或头颈癌。
10.根据权利要求1或6所述的用途,其特征在于,所述非典型循环肿瘤细胞为游离态。
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