肿瘤患者循环肿瘤细胞(CTCs)的分离工具:细胞角蛋白包被磁珠

多种癌症患者的外周血中均可检测到循环肿瘤细胞(circulating tumourcells,CTCs),它在肿瘤转移过程中发挥关键作用.此外,伴随CTCs检测技术的逐步发展,其敏感度和可重复性逐步提高,进而可充分发挥其在肿瘤诊断中的潜能.最近,美国食品和药物管理局(food and drug administration,FDA)已批准CellSearch?检测系统用于检测外周血CTCs,该系统采用的是上皮细胞粘附分子(EpCAM)抗体包被的磁珠.尽管它能有效检测出CTCs,但同样可因为上皮—间质转化中上皮抗原的丢失而错失CTCs,数据显示只有30%~50%的晚期NSCLC外周血CTCs检测阳性.为了克服这些缺点,研究人员正探索新的检测方法.本研究建立了细胞角蛋白(CK)包被的磁珠检测系统来分离肺癌临床病例外周血中的CTCs,并评估该系统所分离的CTCs是否可作为基因检测的有效材料来源.纳入研究的30例患者中,17例检测到CTCs (57%),细胞数从1~7个,其中2例患者中,仅存在EMT形式的CTCs.CTCs检测阳性率与恶性肿瘤的临床病史相关.在III-IV期肺癌患者首诊时或术后血标本中,超过80%可检测到CTCs.对化疗或TKIs治疗有效的患者,其检测阳性率下降到13%,肿瘤进展后又上升至57%.最后,我们对其中8例患者的CTCs进行EGFR和K-ras突变检测,但只有3例CTCs≥4个的患者基因扩增成功.     

原发性肝癌患者CTCs与CD4~+/CD8~+、NLR、TTV及肿瘤分期的相关性研究

目的:探讨原发性肝癌患者外周血循环肿瘤细胞(CTCs)与T细胞亚群CD4~+/CD8~+比值、中性粒细胞与淋巴细胞比值(NLR)、肿瘤总体积(TTV)及肿瘤分期的关系。方法:收集80例原发性肝癌患者外周血,采用益善生物技术公司使用的"免疫去除结合纳米过滤法"Can PatrolTM行CTCs检测。同时使用流式细胞术检测外周血中CD4+T细胞和CD8~+T细胞数量;收集患者外周血中性粒细胞数、淋巴细胞数、肿瘤最大直径及肿瘤分期等临床病理参数。计算出外周血CD4~+/CD8~+比值、NLR及TTV。根据CD4~+/CD8~+平均值及TTV、NLR中位数将CD4+/CD8+比值、TTV和NLR分为高、低2组;根据肝癌第7版TMN分期标准将患者分为I+II期和III+IV期2组。分别比较CTCs在2组中的差别。结果:高CD4~+/CD8~+比值组外周血CTCs数及间质型CTCs数明显低于低CD4~+/CD8~+比值组(P0.05)。高TTV组外周血的CTCs数、间质型CTCs数及混合型CTCs数明显高于低TTV组(P0.05);肿瘤分期I、II期组外周血的CTCs数、间质型CTCs数及混合型CTCs数明显低于III、IV期组(P0.05)。高NLR组与低NLR组外周血的CTCs数、间质型CTCs数、混合型CTCs数和上皮型CTCs数间的差异无统计学显著性。结论:原发性肝癌患者外周血中存在CTCs;外周血CTCs与T细胞免疫、TTV及肿瘤分期有显著相关:T细胞免疫越差、TTV越大及肿瘤分期越晚,外周血的CTCs越多。     

非小细胞肺癌中的循环肿瘤细胞

肺癌是世界上最致命的癌症类型。仅仅在美国，每年就有超过225000人被确诊为肺癌，2012年预计死于肺癌的患者大约有16万。与癌胚抗原（carcinoembryonic antigen，CEA）在结肠癌以及前列腺特异抗原（prostate specific antigen，PSA）在前列腺癌中的作用不同，在肺癌患者诊治过程中，肿瘤标志物的应用并不广泛。肺癌的诊断依赖于影像学检查，而不是体液中的肿瘤标志物。然而在最近的研究中，循环肿瘤细胞（circulating tumor cells，CTCs）的潜在价值被发现。基于CTCs的分析已经被视作“液相活组织检查”，它将有助于我们对恶性肿瘤进行定性和定量的分析。     

肝癌患者循环肿瘤细胞表型与临床病理特征的相关性研究

目的探讨原发性肝癌及复发性肝癌患者外周血中不同表型循环肿瘤细胞(CTCs)与其临床病理特征的相关性及其临床应用价值。方法回顾性分析2016年11月至2018年8月陆军军医大学第一附属医院肝胆科收治的111例原发性肝癌患者及56例复发性肝癌患者的临床资料。所有患者采用Canpatrol CTCs分型检测系统检测外周血上皮-间质表型CTCs,比较原发性与复发性肝癌患者外周血中各表型CTCs阳性率差异;分析原发性及复发性肝癌患者各表型CTCs与其临床病理特征的相关性。结果根据上皮及间质标志物将CTCs分为上皮型、间质型及混合型三种表型。原发性肝癌患者外周血上皮型CTCs、混合型CTCs、间质型CTCs的阳性率分别为30. 63%(34/111)、86. 49%(96/111)、46. 85%(52/111);复发性肝癌患者外周血上皮型CTCs、混合型CTCs、间质型CTCs阳性率分别为46. 43%(26/56)、26. 79%(15/56)、85. 71%(48/56);原发性及复发性肝癌患者外周血中上皮型、混合型、间质型CTCs阳性率差异均有统计学意义(χ~2=4. 035、23. 406、59. 522,P 0. 05)。原发性肝癌患者的上皮型CTCs与门静脉癌栓及微血管癌栓均呈正相关(r=0. 309和0. 330,P 0. 05),与肝硬化程度呈负相关(r=-0. 466,P 0. 05);混合型CTCs与微血管癌栓及门静脉癌栓分别呈正相关和负相关(r=0. 236、-0. 383,P 0. 05);间质型CTCs与肿瘤大小、门静脉癌栓、微血管癌栓、及巴塞罗那分期呈正相关(r=0. 757、0. 210、0. 708、0. 401,P 0. 05),与肿瘤分化程度呈负相关(r=-0. 243,P 0. 05)。复发性肝癌患者的上皮型CTCs与AFP及肿瘤个数呈正相关(r=0. 620、0. 391,P 0. 05);间质型CTCs与肿瘤大小、肿瘤个数呈负相关(r=-0. 335、-0. 354,P 0. 05);混合型CTCs与肿瘤大小及AFP分别呈正相关和负相关(r=0. 643、-0. 349,P 0. 05)。结论肝癌患者CTCs不同表型的阳性率与其临床病理特征具有显著相关性,可作为预测肝癌术后复发和评估疗效的辅助指标。     

血循环肿瘤细胞检测在中晚期乳腺癌疗效评价及转移监测中的应用

目的 探讨血循环肿瘤细胞(CTCs)检测对中晚期乳腺癌疗效评价及转移监测的应用价值.方法 纳入中晚期乳腺癌患者100例,于第1、2、3周期化疗前3天内分别取外周血,采用免疫磁珠阴性富集联合免疫荧光染色法进行CTCs检测;于第3周期化疗前进行影像学疗效评价,比较化疗2周期后不同转归患者的CTCs情况;所有患者于第3周期化疗开始每个月随访,直至出现新增转移灶时终止;于全部化疗结束后3个月、6个月、9个月再次分别进行CTCs检测及影像学检查.结果 第1、2、3周期化疗前,中晚期乳腺癌患者的CTCs数目分别为(15.34±4.12)、(7.82±1.56)、(3.71±1.04),阳性率分别为73.00%、56.00%、29.00%,化疗1、2周期后的CTCs数目及阳性率均较初次化疗前显著降低(P<0.05);化疗2周期后,CTCs阴性患者及CTCs数目减少患者的总有效率(RR)(73.24%、66.20%)分别高于CTCs阳性患者及CTCs数目增加的患者,差异均有统计学意义(P<0.05);全部化疗结束后3个月、6个月及9个月,CTCs阴性患者的转移复发率(5.41%、10.91%、25.00%)均明显低于CTCs阳性患者.结论 外周血CTCs检测可用于中晚期乳腺癌的化疗疗效评价和转移监测,对乳腺癌具有一定的预后预测价值.     

循环肿瘤细胞检测技术研究进展

循环肿瘤细胞(circulating tumor cell,CTCs)与癌症转移密切相关,在癌症诊断中具有重要的意义。为了解CTCs的功能和分子特征以发展针对癌症有效的监测和治疗策略,从癌症患者外周血中捕获CTCs及提高捕获效率技术的研究是首要环节。本文总结了现有的CTCs富集技术、检测手段和它们用于离体培养的方法以及存在的问题。     

结直肠癌术后循环肿瘤细胞检测的临床意义

目的 探讨结直肠癌(CRC)患者术后外周血循环肿瘤细胞(CTCs)阳性率及其计数与临床病理特征、肿瘤复发转移及预后之间的关系及其临床意义。方法 回顾性分析2016年1月—2017年7月上海交通大学医学院附属第九人民医院首诊并经病理确诊的99例CRC患者的临床资料,其中男66例、女33例,年龄37~79岁。检测患者术后4~8周且辅助治疗前CTCs计数和血清癌胚抗原(CEA)水平,随访患者生存情况,比较患者不同临床病理学特征患者间CTCs表达及计数值的统计学差异,包括性别、年龄、原发灶部位、肿瘤分化程度、原发灶浸润深度(T分期)、区域淋巴结侵袭程度(N分期)、远处转移(M分期)、TNM总分期;观察患者生存情况;采用COX比例风险回归模型进行无进展生存期、总生存期的单因素和多因素分析。结果 99例CRC患者CTCs阳性率为60.6%(60/99),CTCs计数值为0~24(4.909±5.518)。患者的CTCs阳性率和CTCs计数值在T1+T2+T3期、N0期、M0期、Ⅰ~Ⅱ期、CEA＜5 ng/mL和中高分化组患者低于T4期、N1+N2期、M1期、Ⅲ~Ⅳ期、CEA≥5 ng/mL、低分化组患者,差异均有统计学意义(P值均＜0.05)。患者随访3~20个月,死亡22例(22/99,22.2%),其中CTCs阳性21例;疾病进展32例(32/99,32.3%),其中CTCs阳性29例。单因素分析显示:CTCs表达、N分期、M分期、肿瘤分期、和高CEA水平是无进展生存期的影响因素(P值均＜0.05);CTCs表达、N分期、M分期、肿瘤原发灶位于直肠、高CEA水平是总生存期的影响因素(P值均＜0.05)。CTCs和远处转移是CRC患者无进展生存期和总生存期的独立预后因素(HR= 5.418、2.254,95%可信区间:1.595~8.403、1.227~7.986,P值均＜0.05)。结论 在术后CRC患者中,CTCs表达与预后不良有关,对肿瘤复发转移评估具有一定的价值和临床意义。     

循环肿瘤细胞的生物学和临床意义

循环肿瘤细胞（circulating tumor cells,CTCs）评估为探索肿瘤生物学提供了一种新的方法,CTCs也有可能作为有临床意义的生物标志物。长期以来,人们一直在猜测CTCs的存在以及它们在转移中的作用。近年来,可靠分离CTCs技术的出现,使这个领域重新获得关注。CTCs多常见于恶性肿瘤患者的血液中,在健康志愿者和良性疾病患者的血液中比较罕见[1]。     

循环肿瘤细胞与肿瘤恶性行为间的关系

转移和复发是恶性肿瘤最主要的死因。近年研究发现,外周血循环肿瘤细胞（circulating tumor cells,CTCs）可成为判断肿瘤预后的重要指标。在提倡个体化治疗的当下,CTC检测可帮助指导临床设计更有效的治疗方案,从而延长患者寿命,提高患者生存率。本文综述CTCs与肿瘤复发转移的关系和CTCs分离富集检测技术研究进展。     

应用微流控芯片富集循环肿瘤细胞的发展现状及展望

循环肿瘤细胞(circulating tumor cell,CTCs)存在于肿瘤患者的外周血中,与恶性肿瘤的转移、预后评价以及临床的个体化治疗均密切相关。相比于组织活检,CTCs的检测仅需抽取静脉血即可进行,因而具有取样简单、可重复性、无创等优点。CTCs的富集方法有很多,近年来兴起的微流控芯片分选技术具有高通量、低消耗、可集成等特点。基于CTCs的物理性质和生物性质所设计的不同芯片在分选速度、效率、纯度以及细胞活性保持方面各有优势,本文将对此加以重点阐述。     

Detection and molecular analysis of circulating tumor cells for early diagnosis of pancreatic cancer

Circulating tumor cells (CTCs) have the potential to provide a surrogate for “real-time biopsy” of tumor biological activity. Enumeration and molecular characterization of CTCs in pancreatic cancer could play an important role in diagnosis, predicting the risk for tumor recurrence, and providing novel target therapy biomarkers. CTCs can disseminate into peripheral blood in the preinvasive and early stages of pancreatic cancer. In view of these facts, we propose that identification and molecular analysis of the malignant characteristics of CTCs may serve as a “liquid biopsy” in pancreatic cancer for early diagnosis.     

Circulating tumor cells as a window on metastasis biology in lung cancer

Circulating tumor cell (CTC) number in metastatic cancer patients yields prognostic information consistent with enhanced cell migration and invasion via loss of adhesion, a feature of epithelial-to-mesenchymal transition (EMT). Tumor cells also invade via collective migration with maintained cell-cell contacts and consistent with this is the circulating tumor microemboli (CTM; contiguous groups of tumor cells) that are observed in metastatic cancer patients. Using a blood filtration approach, we examined markers of EMT (cytokeratins, E-cadherin, vimentin, neural cadherin) and prevalence of apoptosis in CTCs and CTM to explore likely mechanism(s) of invasion in lung cancer patients and address the hypothesis that cells within CTM have a survival advantage. Intra-patient and inter-patient heterogeneity was observed for EMT markers in CTCs and CTM. Vimentin was only expressed in some CTCs, but in the majority of cells within CTM; E-cadherin expression was lost, cytoplasmic or nuclear, and rarely expressed at the surface of the cells within CTM. A subpopulation of CTCs was apoptotic, but apoptosis was absent within CTM. This pilot study suggests that EMT is not prosecuted homogeneously in tumor cells within the circulation of lung cancer patients and that collective migration and enhanced survival of cells within CTM might contribute to lung cancer metastasis. Multiplex analysis and further detailed exploration of metastatic potential and EMT in CTCs/CTM is now warranted in a larger patient cohort.     

肝癌患者循环肿瘤细胞的检测及其临床应用研究进展

原发性肝癌发病率和死亡率均较高,其术后转移与复发已经成为影响患者预后的重要因素,临床上迫切需要能够有效预测和防治肝癌转移的检测手段。循环肿瘤细胞（circulating tumor cell,CTC）,作为近些年来新出现的肿瘤检测指标,因其能够实时、无创地监测肿瘤患者的病情状态,在早期诊断、早期治疗,判断预后和制定个体化治疗方案等方面具有重要意义而备受关注。CTC在乳腺癌、前列腺癌、结直肠癌、肺癌等疾病的应用已初步得到临床认可。最新的研究表明,肝癌患者病情状态与血液中CTC的数量也显示出极强的相关性。目前各种CTC检测体系主要包括富集纯化和鉴定两方面;但由于检测方法繁多,不同方法的敏感性、特异性不同限制了其临床应用。近年来,新的CTC检测方法层见叠出,涌现出了很多新技术用于肝癌CTC的临床研究。本文就其相关研究进展做一综述。     

Simultaneous isolation and detection of circulating tumor cells with a microfluidic silicon-nanowire-array integrated with magnetic upconversion nanoprobes

The development of sensitive and convenient methods for detection, enrichment, and analysis of circulating tumor cells (CTCs), which serve as an importance diagnostic indicator for metastatic progression of cancer, has received tremendous attention in recent years. In this work, a new approach characteristic of simultaneous CTC capture and detection is developed by integrating a microfluidic silicon nanowire (SiNW) array with multifunctional magnetic upconversion nanoparticles (MUNPs). The MUNPs were conjugated with anti-EpCAM antibody, thus capable to specifically recognize tumor cells in the blood samples and pull them down under an external magnetic field. The capture efficiency of CTCs was further improved by the integration with a microfluidic SiNW array. Due to the autofluorescence free nature in upconversion luminescence (UCL) imaging, our approach allows for highly sensitive detection of small numbers of tumor cells, which afterward could be collected for further analysis and re-culturing. We have further demonstrated that this approach can be applied to detect CTCs in clinical blood samples from lung cancer patients, and obtained consistent results by analyzing the UCL signals and the clinical outcomes of lung cancer metastasis. Therefore our approach represents a promising platform in CTC capture and detection with potential clinical utilization in cancer diagnosis and prognosis.     

循环肿瘤细胞的微流控芯片分离及其物理性质测量分析

目的建立一种使用微吸管测量具有良好生理活性的循环肿瘤细胞(circulating tumor cells, CTCs)弹性模量的方法。方法使用商品化的微流控芯片富集血液中具有良好生理活性的CTCs,使用EpCAM抗体确定CTCs,并使用微吸管测量其弹性模量,同时与癌细胞系弹性模量相对比。结果对于癌细胞系的弹性模量,不仅在不同细胞系间存在较大的差异,在同一细胞系间也存在较大的异质性。血液中CTCs相比于同种癌细胞系属于弹性模量较小的癌细胞。结论该方法能够获得生理活性较好的CTCs并测量其弹性模量,为进一步研究CTCs力学特性与癌症诊断和治疗预后之间的相关关系,推进癌细胞物理标志物的发展提供细胞力学数据支持。     

CTCs hemodialysis: Can it be a new therapy for breast cancer?

Circulating tumor cells (CTCs) play an important role in Blood-borne distant metastasis, which is the leading cause of cancer-related death in breast cancer. So far, the impacts of CTCs as a tool for predicting or monitoring the efficacy of systemic therapy and that it is a independent prognostic factor have been confirmed. However, CTCs cannot be generally removed at primary surgery or by systemic therapy. In some EMT (epithelial mesenchymal transition)-related treatment fail, CTCs can be accumulated in the postoperative course of the patient which lead to a bad prognosis. In view of these, considering mature hemodialysis technology, we further propose CTCs hemodialysis (CHD), which filtrate CTCs out of blood, as a new therapy for the breast cancer.     

Clinical significance of circulating tumor cells from lung cancer patients using microfluidic chip

Circulating tumor cells (CTCs) exist in the peripheral blood and have an important role in the disease development, tumor metastasis and clinical surveillance, especially in the process of metastasis. However, the technology of detecting CTCs still had a large challenge since they were rare in the peripheral blood. Here, we developed a size-based microfluidic chip, which contained array and filter channel array that could enrich CTCs from blood samples more quickly and conveniently. Combined with clinical specimen, we analyzed CTCs in 200 lung cancer patients by this microfluidic chip. The microfluidic device has high specificity and sensitivity in detecting CTCs (86.0% sensitivity and 98% specificity). Furthermore, the number of CTCs showed a increasing trend according to the stage of the disease (the mean number of I stage 5.0 ± 5.121 versus II stage 8.731 ± 6.36 versus III stage 16.81 ± 9.556 versus IV stage 28.72 ± 17.39 cells/mL, P < 0.05). The number of CTCs was concurrent with the condition of pathological type and metastasis patients. Compared to conventional markers like CEA, CY211, SCC, CTCs showed a higher positive rate in diagnosed patients. The advanced microfluidic device could capture tumor cells without reliance on cell surface expression markers and provide a fast, convenient, economical method in detecting CTCs, thereby offering potential to design effective and individualized cancer therapies.     

循环肿瘤标志物在甲状腺癌临床诊疗中应用的研究进展

目的 总结循环肿瘤标志物应用于甲状腺癌临床诊疗中的研究进展。方法 在中国知网、万方数据库以及PubMed等中英文数据库中,检索2022年7月之前发表的肿瘤标志物与甲状腺癌临床应用的相关文献共568篇,剔除与内容不符、无法获得全文、重复性研究以及较为陈旧的文献,最终纳入63篇文献进行总结和分析。结果 甲状腺癌是临床最常见的内分泌肿瘤,超声引导下细针穿刺细胞学检查（FNAB）是其诊断的金标准,但存在15%~20%的不准确性。近年来,作为一种新型的非侵入性诊断工具,对包括循环肿瘤细胞（CTCs）、细胞游离DNA（cfDNA）/循环肿瘤DNA（ctDNA）、外泌体以及游离的非编码RNAncRNA）等在内的循环肿瘤标志物的检测,在甲状腺乳头状癌和滤泡状癌患者的临床诊疗中发挥了一定的作用,能够在较早期监测到肿瘤进展或复发,从而帮助临床医生制定个体化的治疗方案。然而,关于循环肿瘤标志物与甲状腺髓样癌以及未分化癌的研究仍旧较少,同时ctDNA与非编码RNA（ncRNA）在甲状腺癌临床诊疗中的作用仍不明确,仍需对其分子机制进一步探讨。结论 循环肿瘤标志物检测具有侵袭性小、副作用少、肿瘤异质性代表性强等优势,在甲状腺癌的临床诊疗中具有广泛的应用前景。     

Comparative analysis of innate immune system function in metastatic breast,colorectal, and prostate cancer patients with circulating tumor cells

In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry.     

Case study of the morphologic variation of circulating tumor cells

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.