循环肿瘤细胞检测的方法学建立和改良及在实体瘤中的监测应用研究
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摘要
循环肿瘤细胞(Circulating tumor cell, CTC)是由原发或转移病灶脱落进入外周血的肿瘤细胞,其作为一种实时“液体活检”手段反映了肿瘤是否发生侵袭转移。肺癌、乳腺癌、前列腺癌、结直肠癌等多种恶性肿瘤中的研究均提示,肿瘤诊断初期检测到CTC是疾病不良预后的独立预测因素,CTC检测阳性患者的无进展生存期和总生存期均明显短于CTC检测阴性的患者；而在有效地放化疗、激素治疗或靶向抑制剂治疗后CTC数目明显下降,提示CTC可用于监测辅助放化疗的疗效。同时,CTC中除含有类似于原发组织的遗传物质外,在播散过程中还会获得其他的额外遗传信息,因而,CTC分子水平变化可以为个体化疾病诊疗提供依据,CTC的分子基因分析有望监测疾病治疗过程中肿瘤基因型变化,如EGFR酪氨酸激酶TKIs治疗过程中耐药基因T790M突变出现,从而为非小细胞肺癌病人药物调整提供依据。总之,检测肿瘤患者外周血中的CTC具有十分重要的意义。
但是,相比较外周血中存在的大量白细胞,CTC仅为外周血中的稀有细胞,这就对其检测分析的方法提出了很高的要求,为了尽可能反应外周血中CTC的情况,目前CTC检测一般分为两个连续的步骤：分离和鉴定。其中又以依赖肿瘤细胞表面上皮特异性粘附分子(EpCAM)表达而富集和鉴定CTC的方法为多,如美国食品药监局审批通过的CellSeach系统。但是,随着研究的深入,人们发现,CTC从原发病灶脱落进入外周血的过程中会发生上皮间质转化(EMT),上皮特异性抗原表达弱化或者消失。循环肿瘤细胞的异质性使得传统的依赖上皮特异性抗原表达来鉴定CTC的方法应用受到限制,只能检测到EpCAM阳性的细胞,而无法检测EpCAM阴性的细胞。因此,我们旨在建立一种不依赖EpCAM的CTC分离和鉴定方法,以期提高实体瘤CTC的检出率,同时对单个CTC肿瘤细胞进行基因分子突变检测。
本研究第一部分在优化免疫磁珠包被CD45抗体差减富集CTC的基础上,通过引入不依赖EpCAM表达的荧光原位杂交(FISH)8号染色体着丝粒探针(CEP-8),并联合免疫荧光化学染色CD45建立免疫荧光化学染色-原位杂交法(CD45-FISH法)鉴定CTC。我们发现,优化后的免疫磁珠包被CD45抗体差减富集CTC法能够稳定的检测到掺入的一个肿瘤细胞。与传统的单纯免疫荧光化学染色上皮角蛋白CK(ICC-CK)法相比,我们所建立的CD45-FISH法检测肺癌患者中CTC的敏感性和特异性分别为83.3%和98.6%；卵巢癌患者中CD45-FISH法检测CTC的敏感性达76.2%,且卵巢癌患者术后7天CTC和血清肿瘤标记物CA125均明显下降。肺癌和卵巢癌患者不同CTC之间FISH杂交信号不同,提示肿瘤细胞的异质性,但是异质性的肿瘤细胞与CTC播散转移是否有关尚需进一步的验证。总之,我们所建立的CD45-FISH法鉴定CTC不依赖EpCAM的表达,与传统的ICC-CK法相比,能较好的检测肺癌、卵巢癌患者的外周血CTC; CD45-FISH法检测CTC有助于监测评估卵巢癌患者手术治疗疗效。
本研究第二部分内容是在第一部分研究的基础上继续优化CTC的鉴定方法,在CD45-FISH的基础上同时联合免疫组化染色上皮角蛋白(CK),建立CK/CD45-FISH的方法。CD45-FISH法是一种不依赖上皮细胞抗原表达的CTC鉴定方法,只能鉴别二倍体和多倍体细胞,而联合CK的CK/CD45-FISH法可以同时鉴别CK表达弱化或消失的细胞及FISH二体及多体的细胞。通过在正常人外周血掺入胰腺癌肿瘤细胞系,富集并CK/CD45-FISH法鉴定后,激光显微切割二次分离纯化单个细胞并全基因组扩增(WGA)、酶切富集联合DHPLC技术,我们检测到单个肿瘤细胞中的K-RAS突变。胰腺癌患者外周血经过富集并CK/CD45-FISH法杂交染色鉴定后,根据细胞杂交信号或抗体染色不同,胰腺癌患者外周血异常细胞可分为CK+CD45-CEP8=2、CK+CD45-CEP8>2、CK-CD45-CEP8>2、 CK-CD45-CEP8=2、CK+/-CD45+CEP8=2/>2五个亚群。其中,CK+CD45-CEP8=2、 CK+CD45-CEP8>2、CK-CD45-CEP8>2被定义为CTCs,CK-CD45-CEP8=2被定义为可疑细胞。22例胰腺癌患者中,有2例检测到CK阳性,其中检测到CK+CD45-CEP8=2亚群细胞和CK+CD45-CEP8>2亚群细胞的个数分别为6个、12个/3.75ml和2个、44个/3.75ml；16例检测到CK-CD45-CEP8>2亚群细胞,检测范围为1-14个/3.75ml,中位数为3个/3.75ml；21例胰腺癌患者检测到可疑CK-CD45-CEP8=2亚群细胞,检测范围为1-25个/3.75ml,中位数为5个/3.75ml。4例胰腺癌患者检测到CK+/-CD45+CEP8=2/>2亚群的造血细胞,检测范围为1-4个/3.75ml,中位数为2个/3.75ml。22例胰腺癌患者CTC检测cutoff值为2个/3.75ml时,CTC阳性率为68.18%。综上所述,我们所建立的联合免疫荧光化学染色CK、CD45和FISH CEP-8的方法具备兼顾上皮细胞特异性抗原表达和表达弱化或消失的特点,能够鉴定CTC亚群细胞,亚群细胞的发现再次提示外周血CTC的异质性。并且通过显微切割及酶切富集联合DHPLC技术,我们检测到单个CTC中K-ras基因的突变,这为以后临床单细胞亚群基因分析打下了良好的基础。
Circulating tumor cells (CTC) are the cells in peripheral blood that detached from the primary or metastasis cancer lesions. And as a real-time "liquid biopsy", CTC have long been considered a reflection of tumor aggressiveness. The efficacy of CTC detection among patients with solid malignancy, such as lung cancer, breast cancer, prostate cancer, colorectal cancer, has been investigated which shows CTC is an independent predictor of poor prognosis and co μ Id serve as an early marker to assess the therapeutic response in overt cancers. Several studies have shown that CTC may gain additional phenotypic and genotypic characteristics over time and develop independently from the primary tumor. Thus, direct molecular genetic analysis of CTC coμld provide important additional information before patients are stratified to expensive therapies that might have considerable side effects. In short, CTC detection in patients is of great significance.
However, owing to the rarity of CTC in peripheral blood, detecting these cells requires methods combined with high sensitivity and specificity, which sets tremendous challenges for the implementation of these assays into clinical routine. Generally, CTCs detection methods are composed of two consecutive steps:isolation process and identification process. Among these, Epithelial cell surface adhesion molecμle (EpCAM) dependent method to enrichment and detection CTC has been the most common, such as CellSeach system, which has been approved by USA food and drug administration (FDA). But, as research continues, people found that expression of EpCAM on CTCs varies greatly, and epithelial CTCs co μ ld undergo epithelial-mesenchymal transition (EMT) during metastasis, causing epithelial specific antigen expression weakening or loss. CTC heterogeneity has limited the clinical application of traditional methods relying on EpCAM to detect CTC, which co μ ld fail to detect EpCAM negative cells. Therefore, we aim to establish an EpCAM independent method for CTC isolation and identification, and to further study site mutagenesis of K-RAS on those single enriched and identified tumor cells.
In the first part of this study, we combined immunocytochemistry (ICC) staining of CD45, DAPI and fluorescence in situ hybridization with the centromere of chromosome8(CEP8) probe method (CD45-FISH) to improve the identification for CTCs with weak or negative CK. We found that, for detecting CTC negatively enriched with EpCAM independent magnetic beads coated with anti-CD45antibodies from lung cancers, CD45-FISH had higher sensitivity and specificity compared to ICC-CK,83.3%and43.3%,98.6%and89.5%, respectively. For CTC detection in ovarian cancers, CD45-FISH showed76.2%sensitivity. Four of six ovarian cancers showed dramatic ally decrease in both CTCs and serum CA125on the7th day after surgery. The FISH hybridization signals were different between different CTC, which provide evidence for cell heterogeneity. In brief, we have established an EpCAM-independent CD45-FISH method to identify CTC for lung cancer and ovarian cancer patients, and compared to traditional ICC-CK, CD45-FISH method had improved sensitivity and specificity. This combined detection strategy may be usefμl in detecting or monitoring CTCs after ovarian cancer surgery.
In the second part of this study, we further optimized CD45-FISH method by simμltaneously immunohistochemical staining of CK and established CK/CD45-FISH method which allowed us not only to distinguish between CK positive CTCs and CTCs with reduced or absent CK expression, but also between diploid and hyperdiploid CTCs. To analysis CTC in single cell level, MiaPaCa-2cells were spiked into normal peripheral bold and then enriched and identified, which were finally collected by laser capture micro-dissection (LCM) followed by whole genome amplification, enzymatic digestion and degeneration high performance liquid chromatography (DHPLC) to further study their site mutagenesis of K-RAS. Our resμlts showed that LCM co μ1d accurately separate and purify single tumor cells. Enzymatic digestion and DHPLC can stablely detect K-ras mutant from10MiaPaCa-2cells, while fluorescent PCR kit can detect from even one tumor cell. For CTC enriched from pancreatic cancers and identified by combining CK, CD45, DAPI and CEP8method (CK/CD45/CEP8), We found that remaining cells coμld be classified into5patterns:CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2, CK-CD45-DAPI+CE P8>2, CK-CD45-DAPI+CEP8=2, and CK+/-CD45+DAPI+CEP8=2or>2. Patterns of CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2and CK-CD45-DAPI+CEP8>2were considered as CTCs, and CK-CD45-CEP8=2and CK+/-CD45+DAPI+CEP8=2or>2were considered as indeterminate cells. Among22pancreatic cancers, patterns of CK+CD45-DAPI+CEP8=2and CK+CD45-DAPI+CEP8>2were identified in2cases, and the number of CTCs was6,12cells/3.75mL and2,44cells/3.75mL, respectively. CK-CD45-CEP8>2pattern was identified in16cases with the range of1-14cells/3.75mL and the median of3cells/3.75mL. CK-CD45-CEP8=2patterns was identified in21cases with the range of1-25cells/3.75mL and the median of5cells/3.75mL. CK+/-CD45+CEP8=2/>2patterns was identified in4cases with the range of1-4cells/3.75mL and the median of2cells/3.75mL. Above all, CK/CD45-FISH method can efficiently identify various tumors CTC, and detection efficiency is not affected by either degradation of cytokeratin in tumor cells. However, because tumor cell lines were very different from clinical samples in some respects, there are still many aspects needed to be modified for CTC separation and purification by LCM for clinical application.
但是,相比较外周血中存在的大量白细胞,CTC仅为外周血中的稀有细胞,这就对其检测分析的方法提出了很高的要求,为了尽可能反应外周血中CTC的情况,目前CTC检测一般分为两个连续的步骤：分离和鉴定。其中又以依赖肿瘤细胞表面上皮特异性粘附分子(EpCAM)表达而富集和鉴定CTC的方法为多,如美国食品药监局审批通过的CellSeach系统。但是,随着研究的深入,人们发现,CTC从原发病灶脱落进入外周血的过程中会发生上皮间质转化(EMT),上皮特异性抗原表达弱化或者消失。循环肿瘤细胞的异质性使得传统的依赖上皮特异性抗原表达来鉴定CTC的方法应用受到限制,只能检测到EpCAM阳性的细胞,而无法检测EpCAM阴性的细胞。因此,我们旨在建立一种不依赖EpCAM的CTC分离和鉴定方法,以期提高实体瘤CTC的检出率,同时对单个CTC肿瘤细胞进行基因分子突变检测。
本研究第一部分在优化免疫磁珠包被CD45抗体差减富集CTC的基础上,通过引入不依赖EpCAM表达的荧光原位杂交(FISH)8号染色体着丝粒探针(CEP-8),并联合免疫荧光化学染色CD45建立免疫荧光化学染色-原位杂交法(CD45-FISH法)鉴定CTC。我们发现,优化后的免疫磁珠包被CD45抗体差减富集CTC法能够稳定的检测到掺入的一个肿瘤细胞。与传统的单纯免疫荧光化学染色上皮角蛋白CK(ICC-CK)法相比,我们所建立的CD45-FISH法检测肺癌患者中CTC的敏感性和特异性分别为83.3%和98.6%；卵巢癌患者中CD45-FISH法检测CTC的敏感性达76.2%,且卵巢癌患者术后7天CTC和血清肿瘤标记物CA125均明显下降。肺癌和卵巢癌患者不同CTC之间FISH杂交信号不同,提示肿瘤细胞的异质性,但是异质性的肿瘤细胞与CTC播散转移是否有关尚需进一步的验证。总之,我们所建立的CD45-FISH法鉴定CTC不依赖EpCAM的表达,与传统的ICC-CK法相比,能较好的检测肺癌、卵巢癌患者的外周血CTC; CD45-FISH法检测CTC有助于监测评估卵巢癌患者手术治疗疗效。
本研究第二部分内容是在第一部分研究的基础上继续优化CTC的鉴定方法,在CD45-FISH的基础上同时联合免疫组化染色上皮角蛋白(CK),建立CK/CD45-FISH的方法。CD45-FISH法是一种不依赖上皮细胞抗原表达的CTC鉴定方法,只能鉴别二倍体和多倍体细胞,而联合CK的CK/CD45-FISH法可以同时鉴别CK表达弱化或消失的细胞及FISH二体及多体的细胞。通过在正常人外周血掺入胰腺癌肿瘤细胞系,富集并CK/CD45-FISH法鉴定后,激光显微切割二次分离纯化单个细胞并全基因组扩增(WGA)、酶切富集联合DHPLC技术,我们检测到单个肿瘤细胞中的K-RAS突变。胰腺癌患者外周血经过富集并CK/CD45-FISH法杂交染色鉴定后,根据细胞杂交信号或抗体染色不同,胰腺癌患者外周血异常细胞可分为CK+CD45-CEP8=2、CK+CD45-CEP8>2、CK-CD45-CEP8>2、 CK-CD45-CEP8=2、CK+/-CD45+CEP8=2/>2五个亚群。其中,CK+CD45-CEP8=2、 CK+CD45-CEP8>2、CK-CD45-CEP8>2被定义为CTCs,CK-CD45-CEP8=2被定义为可疑细胞。22例胰腺癌患者中,有2例检测到CK阳性,其中检测到CK+CD45-CEP8=2亚群细胞和CK+CD45-CEP8>2亚群细胞的个数分别为6个、12个/3.75ml和2个、44个/3.75ml；16例检测到CK-CD45-CEP8>2亚群细胞,检测范围为1-14个/3.75ml,中位数为3个/3.75ml；21例胰腺癌患者检测到可疑CK-CD45-CEP8=2亚群细胞,检测范围为1-25个/3.75ml,中位数为5个/3.75ml。4例胰腺癌患者检测到CK+/-CD45+CEP8=2/>2亚群的造血细胞,检测范围为1-4个/3.75ml,中位数为2个/3.75ml。22例胰腺癌患者CTC检测cutoff值为2个/3.75ml时,CTC阳性率为68.18%。综上所述,我们所建立的联合免疫荧光化学染色CK、CD45和FISH CEP-8的方法具备兼顾上皮细胞特异性抗原表达和表达弱化或消失的特点,能够鉴定CTC亚群细胞,亚群细胞的发现再次提示外周血CTC的异质性。并且通过显微切割及酶切富集联合DHPLC技术,我们检测到单个CTC中K-ras基因的突变,这为以后临床单细胞亚群基因分析打下了良好的基础。
Circulating tumor cells (CTC) are the cells in peripheral blood that detached from the primary or metastasis cancer lesions. And as a real-time "liquid biopsy", CTC have long been considered a reflection of tumor aggressiveness. The efficacy of CTC detection among patients with solid malignancy, such as lung cancer, breast cancer, prostate cancer, colorectal cancer, has been investigated which shows CTC is an independent predictor of poor prognosis and co μ Id serve as an early marker to assess the therapeutic response in overt cancers. Several studies have shown that CTC may gain additional phenotypic and genotypic characteristics over time and develop independently from the primary tumor. Thus, direct molecular genetic analysis of CTC coμld provide important additional information before patients are stratified to expensive therapies that might have considerable side effects. In short, CTC detection in patients is of great significance.
However, owing to the rarity of CTC in peripheral blood, detecting these cells requires methods combined with high sensitivity and specificity, which sets tremendous challenges for the implementation of these assays into clinical routine. Generally, CTCs detection methods are composed of two consecutive steps:isolation process and identification process. Among these, Epithelial cell surface adhesion molecμle (EpCAM) dependent method to enrichment and detection CTC has been the most common, such as CellSeach system, which has been approved by USA food and drug administration (FDA). But, as research continues, people found that expression of EpCAM on CTCs varies greatly, and epithelial CTCs co μ ld undergo epithelial-mesenchymal transition (EMT) during metastasis, causing epithelial specific antigen expression weakening or loss. CTC heterogeneity has limited the clinical application of traditional methods relying on EpCAM to detect CTC, which co μ ld fail to detect EpCAM negative cells. Therefore, we aim to establish an EpCAM independent method for CTC isolation and identification, and to further study site mutagenesis of K-RAS on those single enriched and identified tumor cells.
In the first part of this study, we combined immunocytochemistry (ICC) staining of CD45, DAPI and fluorescence in situ hybridization with the centromere of chromosome8(CEP8) probe method (CD45-FISH) to improve the identification for CTCs with weak or negative CK. We found that, for detecting CTC negatively enriched with EpCAM independent magnetic beads coated with anti-CD45antibodies from lung cancers, CD45-FISH had higher sensitivity and specificity compared to ICC-CK,83.3%and43.3%,98.6%and89.5%, respectively. For CTC detection in ovarian cancers, CD45-FISH showed76.2%sensitivity. Four of six ovarian cancers showed dramatic ally decrease in both CTCs and serum CA125on the7th day after surgery. The FISH hybridization signals were different between different CTC, which provide evidence for cell heterogeneity. In brief, we have established an EpCAM-independent CD45-FISH method to identify CTC for lung cancer and ovarian cancer patients, and compared to traditional ICC-CK, CD45-FISH method had improved sensitivity and specificity. This combined detection strategy may be usefμl in detecting or monitoring CTCs after ovarian cancer surgery.
In the second part of this study, we further optimized CD45-FISH method by simμltaneously immunohistochemical staining of CK and established CK/CD45-FISH method which allowed us not only to distinguish between CK positive CTCs and CTCs with reduced or absent CK expression, but also between diploid and hyperdiploid CTCs. To analysis CTC in single cell level, MiaPaCa-2cells were spiked into normal peripheral bold and then enriched and identified, which were finally collected by laser capture micro-dissection (LCM) followed by whole genome amplification, enzymatic digestion and degeneration high performance liquid chromatography (DHPLC) to further study their site mutagenesis of K-RAS. Our resμlts showed that LCM co μ1d accurately separate and purify single tumor cells. Enzymatic digestion and DHPLC can stablely detect K-ras mutant from10MiaPaCa-2cells, while fluorescent PCR kit can detect from even one tumor cell. For CTC enriched from pancreatic cancers and identified by combining CK, CD45, DAPI and CEP8method (CK/CD45/CEP8), We found that remaining cells coμld be classified into5patterns:CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2, CK-CD45-DAPI+CE P8>2, CK-CD45-DAPI+CEP8=2, and CK+/-CD45+DAPI+CEP8=2or>2. Patterns of CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2and CK-CD45-DAPI+CEP8>2were considered as CTCs, and CK-CD45-CEP8=2and CK+/-CD45+DAPI+CEP8=2or>2were considered as indeterminate cells. Among22pancreatic cancers, patterns of CK+CD45-DAPI+CEP8=2and CK+CD45-DAPI+CEP8>2were identified in2cases, and the number of CTCs was6,12cells/3.75mL and2,44cells/3.75mL, respectively. CK-CD45-CEP8>2pattern was identified in16cases with the range of1-14cells/3.75mL and the median of3cells/3.75mL. CK-CD45-CEP8=2patterns was identified in21cases with the range of1-25cells/3.75mL and the median of5cells/3.75mL. CK+/-CD45+CEP8=2/>2patterns was identified in4cases with the range of1-4cells/3.75mL and the median of2cells/3.75mL. Above all, CK/CD45-FISH method can efficiently identify various tumors CTC, and detection efficiency is not affected by either degradation of cytokeratin in tumor cells. However, because tumor cell lines were very different from clinical samples in some respects, there are still many aspects needed to be modified for CTC separation and purification by LCM for clinical application.
引文
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