乳腺癌循环肿瘤细胞研究及其检测进展

乳腺癌是女性发生率最高的恶性肿瘤之一,其引起死亡的主要原因是乳腺癌的复发转移.转移灶的形成是一个连续复杂的过程,从原发灶生长的早期即已开始.肿瘤细胞进入血液循环是乳腺癌发生转移的基础.经过多种复杂机制最终形成转移灶.因此,检测循环肿瘤细胞(CTC)对预测转移有重要作用.由于外周血中CTC数量稀少,增加了其检测的难度.目前对CTC的检测主要分为基于抗体的技术、核酸技术以及联合检测、系统检测等方式,随着试验方法的改进和新技术的不断出现,CTC检测的敏感度和特异性有较大提高.然而缺乏高特异性的标志物仍是CTC检测面临的最大问题.乳腺癌患者CTC水平与肿瘤的分期相关,其水平的升高提示有更高转移的可能.预示患者的预后较差.与传统的组织学及影像学检查相比,CTC检测有更高的可重复性和敏感度,并能更早地提供预后信息.与骨髓微转移肿瘤细胞检测相比,CTC检测具有创伤小、可连续多次检测等特点.CTC检测更重要的意义是能够帮助指导临床个体化治疗方案的制定.通过对CTC连续的检测可以及早地评估治疗效果,据此及时转变为更有效的治疗方案,提高患者的生存率.本文对乳腺癌循环肿瘤细胞的研究、检测技术和临床应用的新进展进行综述.     

乳腺癌循环肿瘤细胞研究进展与展望

目的:总结国内外乳腺癌循环肿瘤细胞(CTC)在检测手段及相关临床意义方面的研究进展,以及CTC检测项目在研究肿瘤转移过程中的地位、必要性及未来的发展方向.方法:应用Medline、PubMed及CNKI期刊全文数据库系统,以“乳腺癌、循环肿瘤细胞、转移、上皮间质转化”等为关键词,检索2006-06-2011-06的相关文献,共选择文献48篇加以分析总结.文献纳入标准:1)乳腺癌CTC的起源及其特点;2)乳腺癌CTC的检测方法;3)检测乳腺癌CTC的临床意义;4)乳腺癌CTC的特性.结果:CTC的检测在预测乳腺癌患者无病生存期和总生存期方面有重要价值;同时,它在预测和判断疗效方面优于一些传统方法.除此之外,CTC的分子学和生物学特性,如ER、PR、HER-2表达和上皮间质转化等,以及这些特性与乳腺癌转移的关系已成为新的研究热点.结论:对CTC的深入研究能够使我们进一步了解它,使其成为乳腺癌患者监测转移和预后的常规的检测指标,并有助于了解血行转移的机制,更有望发现乳腺癌治疗的新靶点.     

循环肿瘤细胞检测技术

循环肿瘤细胞(CTC)检测在肿瘤患者的实时个体化医疗中具有独特的优势.外周血中CTC稀少而又异质,这成为CTC研究的主要技术挑战.CTC检测技术往往首先利用细胞的物理性质或生物学特性等分离富集CTC,再进行基因型或表型分析等以计数CTC或鉴定其分子特征.单细胞分子分析的进展使CTC检测能够提供更准确全面的信息.CTC侵袭转移时上皮间质转化和聚集成簇等生物进程是促进CTC检测的临床应用需要考虑的问题.     

循环肿瘤细胞检测及其临床应用

循环肿瘤细胞(CTC)与恶性肿瘤的发生发展密切相关.目前检测CTC的方法有逆转录聚合酶链反应、免疫磁珠富集检测法等.检测CTC有助于发现早期肿瘤患者的微转移、重新确定临床分期,监测术后或者放化疗后患者肿瘤复发与转移,评估预后,选择个体化的治疗策略.     

循环肿瘤细胞与肺癌

循环肿瘤细胞(CTC)与肿瘤转移有明显的相关性,CTC检测有助于指导肿瘤治疗,为判断预后、预测疗效提供可靠依据.CTC与非小细胞肺癌的分期及远处转移相关.CTC数量变化与非小细胞肺癌患者化放疗疗效及预后有关.小细胞肺癌中CTC检出率和数量均明显高于其他肿瘤,与其分期及化疗疗效有关.CTC有望用于指导肺癌个体化治疗.初步研究结果提示可用CTC来动态了解肺癌患者分子靶向药物治疗过程中耐药肿瘤细胞的出现.     

循环肿瘤细胞纳米检测技术用于肿瘤早期诊断的探索

循环肿瘤细胞(circulating tumor cell,CTC)在外周血中的存在和循环被认为是引起肿瘤远处转移的主要原因.与传统的影像学检测手段相比,CTC检测技术作为一种非侵袭性的检测手段,其操作更简便、更安全和精准.此外,CTC技术也助于研究者深入探讨肿瘤转移复发和耐药的机制.因此,CTC作为一种生物标志物,在肿瘤的早筛、早诊、预后评估和疗效检测等过程中都有着极其重要的临床意义.中科院国家纳米科学中心研发的“肿瘤捕手”技术利用多肽纳米磁珠来捕获外周血中的CTC.目前,CTC检测技术已在国内多家顶级公立医院开展临床研究和试验,大量临床数据表明,其对于肿瘤早期诊断、预后判断和耐药性监测方面具有极大的指导意义.本文就CTC的研究进展,“肿瘤捕手”技术的研发和应用作一阐述.     

乳腺癌患者循环肿瘤细胞在预后和疾病进展中的作用

检测外周血循环肿瘤细胞(circulating tumor cell,CTC)已经成为在癌症诊断中热门领域之一。目前已有证据表明CTC和无病生存期,无进展生存期和总生存期相关。在乳腺癌患者骨髓或循环血检测出CTC是微转移的早期提示。检测CTC预测癌转移的方法证明优于一些传统的方法,如患者的临床表现、影像学检查及血清肿瘤标志的检查等。且具有早期、高效、可靠及检测过程低侵袭性的特点。本文总结国内外乳腺癌循环肿瘤细胞(CTC)在检测手段及相关临床意义方面的研究进展,以及CTC检测项目在研究肿瘤转移过程中的地位、必要性及未来的发展方向。     

直肠癌循环肿瘤细胞与原发肿瘤组织基因表达谱的差异分析

目的:通过直肠癌循环肿瘤细胞(circulating tumor cell,CTC)与原发肿瘤组织基因表达谱的差异分析,筛选直肠癌CTC的特异性基因.方法:选取2015年9月至2015年12月福建省肿瘤医院收治的直肠癌患者4例,取肿瘤组织和相应的癌旁组织并采集外周血20 ml.应用流式细胞术检测血液CTC,抽提癌组织和CTC的总RNA,采用人全转录组表达谱芯片检测CTC及相应肿瘤组织mRNA表达谱,经聚类分析筛选CTC特异性基因,并采用生物分子注释系统MAS软件对其进行基因功能及其相关的信号通路分析.结果:4例直肠癌患者检测到CTC数目分别是67、78、53和120个.筛选出肿瘤与癌旁组织差异基因共7 755个、CTC与白细胞差异基因共567个,肿瘤组织及CTC差异基因交集后共同上调或下调的特异性基因共36个,其中上调16个、下调20个(P＜0.05或P＜0.01).CTC特异性基因与肿瘤转移和干细胞功能有关,涉及与细胞转移、干细胞功能和免疫功能相关的22条信号通路.结论:CTC与原发肿瘤基因相比,具有独特的表达谱,是肿瘤复发和转移的重要基础.     

乳腺癌患者外周血循环肿瘤细胞阴性富集法检测及其临床意义

目的 循环肿瘤细胞(circulating tumor cells,CTC)的检测作为一种新兴的检测方法,在临床实践中拥有广阔的应用前景.本研究通过阴性富集法检测乳腺癌患者外周血CTC,探讨CTC水平与其分子分型及临床特征的相关性,研究其临床意义.方法 选取连云港市第一人民医院2015-08-18-2016-02 20接受CTC检测的乳腺癌患者152例为研究对象,收集临床资料,选择同期该院29例乳腺良性疾病患者作为对照组.采用免疫磁珠阴性富集及免疫荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测CD45-/CEP+/DAPI+的CTC,并结合乳腺癌分子分型和临床特征进行统计学分析.结果 152例乳腺癌患者与29例乳腺良性疾病患者进行ROC曲线分析,比较不同切点的敏感度及特异度,经计算Youden指数最高为0.390,对应切点为≥2.5,故取CTC个数≥3为阳性指标.152例乳腺癌患者中CTC阳性率Ⅰ～Ⅱ期为41.5％,Ⅲ期为60.0％,Ⅳ期为73.9％,差异有统计学意义,x2=9.360,P=0.009.CTC与乳腺癌分子分型无相关性,P＞0.05,与肿瘤原发灶分期、区域淋巴结转移、激素受体状况及HER2表达均无相关性,P＞0.05.但与是否发生远处转移密切相关,x2=5.863,P=0.015.结论 CTC与乳腺癌分子分型无明显相关,但与患者临床分期、是否发生转移密切相关,检测CTC有利于更准确的评估病情.     

磁性纳米载体用于循环肿瘤细胞的检测

循环肿瘤细胞（circulating tumor cell,CTC）是从原发肿瘤分离进入外周血或淋巴循环的肿瘤细胞,与肿瘤转移密切相 关。CTC作为液体活检重要标志物之一,支持实时动态重复检测,对于肿瘤的早期筛查、转移抑制、预后评估、复发监测、个性化 治疗指导具有重要意义。然而CTC在血液中含量极低,寻找稳定性好、灵敏度高及特异性强的CTC检测方法是当前研究的热 点。近年来,磁性纳米载体由于生物相容性好、表面易修饰和磁响应速度快等优势在CTC检测中受到广泛关注。本文阐述了 CTC检测的意义,系统归纳了磁性纳米载体用于CTC检测的设计方案,通过巧妙设计构建理想的磁性纳米载体,包括CTC的特 异性捕获,在特定刺激下实现CTC的释放,并对其捕获与释放过程进行监测,从而实现CTC的高效检测。     

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.     

Molecular mechanisms of metastasis in breast cancer--clinical applications

The metastatic cascade is a series of biological processes that enable the movement of tumor cells from the primary site to a distant location and the establishment of a new cancer growth. Circulating tumor cells (CTCs) have a crucial role in tumor dissemination. The role of CTCs in treatment failure and disease progression can be explained by their relation to biological processes, including the epithelial-to-mesenchymal transition and 'self seeding', defined as reinfiltration of the primary tumor or established metastasis by more aggressive CTCs. CTCs are a unique and heterogeneous cell population with established prognostic and predictive value in certain clinical situations. The possibility of collecting sequential blood samples for real-time monitoring of systemic-therapy efficacy presents new possibilities to evaluate targeted therapies based on the genomic profiling of CTCs and to improve the clinical management of patients by personalized therapy. Interruption of the metastatic cascade via the targeting of CTCs might be a promising therapeutic strategy.     

Clinical application of circulating tumor cells in breast cancer

Circulating tumor cells (CTCs) play a major role in the metastatic spread of breast cancer. CTC detection has proven to be an important parameter for predicting progression free and overall survival. Collection of CTCs is minimally invasive and can be performed more often than disseminated tumor cell (DTC) collection from bone marrow, thus providing a real-time “liquid biopsy”. In this review, the most important techniques for enrichment and detection of CTCs are discussed for clinical application in low and higher staged breast cancer, as well as the genetic and molecular characterization of CTCs. For CTCs, the use of immunology-based enrichment techniques with multiple antibodies is recommended in a clinical setting, as well as the use of cytometric detection techniques, combined with RT-PCR for confirmation. Special attention is given to the value of cancer stem cell (CSC) activity, which may be the main cause of ineffectiveness of the control over metastatic lesions due to intratumor heterogeneity. Accumulating information on CSCs offers new paradigms to generate effective targets for the treatment of metastatic disease. Genetic and molecular characterization of CTCs has potential to stratify patients for optimal personalized treatment regimens. CTCs can be used for monitoring patients during treatment schedules.     

Clinical implications and perspectives of portal venous circulating tumor cells in pancreatic cancer

Despite recent improvements in the diagnosis and treatment of pancreatic cancer(PC), clinical outcomes remain dismal. Moreover, there are no effective prognostic or predictive biomarkers or options beyond carbohydrate antigen 19-9 for personalized and precise treatment. Circulating tumor cells(CTCs), as a member of the liquid biopsy family, could be a promising biomarker; however, the rarity of CTCs in peripheral venous blood limits their clinical use. Because the first venous drainage of PC is ...     

Multi-center study evaluating circulating tumor cells as a surrogate for response to treatment and overall survival in metastatic breast cancer

Background

Evaluating circulating tumor cells (CTCs) is one way to predict outcome and monitor treatment in patients with MBC. In this prospective study, we evaluated CTCs in predicting treatment efficacy and overall survival (OS) using the CellSearch? System (Veridex, LLC, Raritan, NJ).

Methods

One hundred nineteen patients with MBC with measurable disease were enrolled. Samples of 7.5 ml of blood from 107 eligible patients were tested for CTCs before starting therapy (baseline), after one cycle of therapy (3–4 weeks) and at 12 weeks. We compared CTC levels and imaging at baseline and at 12 weeks. Next, we determined the hazard ratios (HR) by comparing cases with zero CTCs to those with one or more CTCs. Moreover, HR was calculated when comparing cases that had greater than or equal to a certain number of CTCs to those with less than the number of CTCs.

Results

This study shows the incidence of detection of CTCs in patients with metastatic breast cancers. Of the patients, 64.4% (76/118) had one or more CTCs, and 37.3% (44/118) had five or more CTCs. First we set the baseline number of CTCs as 100%. Of the seven cases whose level of CTCs decreased more than 90%, six (85.7%) demonstrated a positive response (complete response and partial response) by imaging after one cycle (3–4 weeks later). For the patients whose CTC levels increased above 100% after one cycle (3–4 weeks later), 7 of 11 (63.6%) had progressive disease (PD). The HR for cases with five to ten CTCs was greater than 1.00 [HR = 2.450; 95% confidence interval (CI) 0.727–8.248]. Statistical significance was observed when comparing patients who had ≥3 CTCs to those with <3 CTCs (P = 0.0273). When comparing cases with ≥5 CTCs to those with <5 CTCs, the hazard ratio was 3.069 (95% CI 1.496–6.295; P = 0.0022).

Conclusions

Because the change in the number of CTCs was highly correlated with results from imaging before and after therapy, CTCs can be considered a biomarker that may predict the effect of treatment earlier than imaging modalities.     

纳米技术在循环肿瘤细胞中的研究进展

循环肿瘤细胞(CTC)对于监测肿瘤复发及判断预后具有重要意义.纳米技术为检测CTC提供了良好的平台,使CTC的应用具有广阔的发展前景.同时,利用纳米技术设计杀灭CTC的纳米装置在清除CTC方面有广阔的应用前景,为肿瘤治疗提供了新的研究方向.     

Circulating tumour cells: their utility in cancer management and predicting outcomes

Recent advances in technology now permit robust and reproducible detection of circulating tumour cells (CTCs) from a simple blood test. Standardization in methodology has been instrumental in facilitating multicentre trials with the purpose of evaluating the clinical utility of CTCs. We review the current body of evidence supporting the prognostic value of CTC enumeration in breast, prostate and colorectal cancer, using standardized approaches, and studies evaluating the correlation of CTC number with radiological outcome. The exploitation of CTCs in cancer management, however, is now extending beyond prognostication. As technologies emerge to characterize CTCs at the molecular level, biological information can be obtained in real time, with the promise of serving as a 'surrogate tumour biopsy'. Current studies illuminate the potential of CTCs as pharmacodynamic and predictive biomarkers and potentially their use in revealing drug resistance in real time. Approaches for CTC characterization are summarized and the potential of CTCs in cancer patient management exemplified via the detection of epidermal growth factor receptor mutations from CTCs in patients with non-small cell lung cancer. The opportunity to learn more about the biology of metastasis through CTC analysis is now being realized with the horizon of CTC-guided development of novel anticancer therapies.     

Circulating tumor cell isolation: the assets of filtration methods with polycarbonate track-etched filters

Circulating tumor cells (CTCs) arise from primary or secondary tumors and enter the bloodstream by active or passive intravasation. Given the low number of CTCs, enrichment is necessary for detection. Filtration methods are based on selection of CTCs by size using a filter with 6.5 to 8 µm pores. After coloration, collected CTCs are evaluated according to morphological criteria. Immunophenotyping and fluorescence in situ hybridization techniques may be used. Selected CTCs can also be cultivated in vitro to provide more material. Analysis of genomic mutations is difficult because it requires adapted techniques due to limited DNA materials. Filtration-selected CTCs have shown prognostic value in many studies but multicentric validating trials are mandatory to strengthen this assessment. Other clinical applications are promising such as follow-up, therapy response prediction and diagnosis. Microfluidic emerging systems could optimize filtration-selected CTCs by increasing selection accuracy.     

Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer

Background: Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. Objective: To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Methods: Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). Results: KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Conclusion: Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available.     

Detecting and phenotyping of aneuploid circulating tumor cells in patients with various malignancies

Circulating tumor cells (CTCs) have been exclusively studied and served to assess the clinical outcomes of treatments and progression of cancer. Most CTC data have mainly been derived from distinct cohorts or selected tumor types. In the present study, a total of 594 blood samples from 479 cases with 19 different carcinomas and 30 healthy samples were collected and analyzed by Subtraction enrichment method combined with immunostaining-fluorescence in situ hybridization (iFISH). Non-hematopoietic cells with aneuploid chromosome 8 (more than 2 copies) were regarded as positive CTCs. The results showed that none of CTCs was found in all 30 healthy samples. The overall positive rate of CTCs was 89.0% in diagnosed cancer patients (ranging from 75.0% to 100.0%). Average number of 11, 5, 8 and 4 CTCs per 7.5 mL was observed in lung cancer, liver cancer, renal cancer and colorectal cancer, respectively. Among 19 different carcinomas, the total number of CTCs, tetraploid chromosome 8, polyploid chromosome 8, CTM (Circulating tumor microemboli) and large CTCs in patients with stage Ⅲ and Ⅳ were statistically higher than patients with stage Ⅰ and Ⅱ (P < 0.05). Furthermore, EpCAM expression was more frequently found in most CTCs than vimentin expression, confirming that these CTCs were of epithelial origin. In addition, small and large CTCs were also classified, and the expression of vimentin was mostly observed in small CTCs and CTM. Our results revealed that there are higher numbers of CTCs, tetraploid, polyploid and large CTCs in patients with stage Ⅲ and Ⅳ, indicating that the quantification of chromosome ploidy performed by SE-iFISH for CTCs might be a useful tool to predict and evaluate therapeutic efficacy as well as to monitoring disease progression.