Circulating tumor cells: the 'leukemic phase' of solid cancers

It is well known that malignant cells circulate in the bloodstream of patients with solid tumors. However, the biological significance of circulating tumor cells (CTCs) and the clinical relevance of their detection are still debated. Besides technical issues regarding CTC-detection methods, discontinuous shedding of CTCs from established cancer deposits, genomic instability and metastatic inefficiency might underlie the conflicting results currently available. Nevertheless, technological advances and recent clinical findings are prompting researchers to dissect CTC biology further. Here, we review these recent findings, and discuss the prospects for the identification and molecular characterization of the CTC subset that is responsible for metastasis development. This would provide a formidable tool for prognosis evaluation, anticancer-drug development and, ultimately, cancer-therapy personalization.     

Quantification of Rare Cancer Cells in Patients With Gastrointestinal Cancer by Nanostructured Substrate

Detecting the cancer cells in the peripheral blood, i.e. circulating tumor cell (CTC), have been considered as the “liquid biopsy” and become a particular area of focus. A deep insight into CTC provides a potential alternative method for early diagnosis of solid tumor. Previous studies showed that CTC counts could be regarded as an indicator in tumor diagnosis, predicting clinical outcomes and monitoring treatment responses. In this report, we utilize our facile and efficient CTC detection device made of hydroxyapatite/chitosan (HA/CTS) for rare cancer cells isolation and enumeration in clinical use. A biocompatible and surface roughness controllable nanofilm was deposited onto a glass slide to achieve enhanced topographic interactions with nanoscale cellular surface components, anti-EpCAM (epithelial cell adhesion molecule, EpCAM) were then coated onto the surface of nanosubstrate for specific capture of CTCs. This device performed a considerable and stable capture yields. We evaluated the relationship performance between serial CTC changes and the changes of tumor volume/serum tumor marker in gastrointestinal cancer patients undergoing anti-cancer treatments. The present study results showed that changes in the number of CTC were associated with tumor burden and progression. Enumeration of CTCs in cancer patients may predict clinical response. Longitudinal monitoring of individual patients during the therapeutic process showed a close correlation between CTC quantity and clinical response to anti-cancer therapy. Effectively capture of this device is capable of CTCs isolation and quantification for monitoring of cancer and predicting treatment response.     

Circulating tumor cells in the diagnosis and management of pancreatic cancer

Pancreatic cancers are typically resistant to chemo and radiation therapy and are predisposed to distant metastases. Circulating tumor cells (CTCs) are tumor cells disseminated from primary and metastatic sites and can be isolated from peripheral blood. CTC may overcome the limitation of the current available tumor markers, CA19-9. As a surrogate for 'real-time biopsy', CTCs allow recurrent assessment of a tumor's biological activity. We review the current methodologies for CTC extraction and characterization including antibody-based immunological assays, PCR-based assays, and novel technologies based on the physical or biological characteristics of CTCs. CTCs also provide an accessible link to the existence of epithelial to mesenchymal transition, tumor stem cell markers, and ongoing clonal mutations and epigenetic changes in the tumor. We also explore the potential of using CTC profiling in diagnosis, selection of neoadjuvant and adjuvant therapy, detection of recurrent disease, examination of pharmacodynamic biomarkers, as well as in gene therapy and immunotherapy for pancreatic cancer. Ongoing CTC characterization not only has the potential to represent all cells shed from primary pancreatic tumor and each metastatic site, but also allows dynamic sampling at multiple time points during the clinical course to identify the subpopulations of CTCs and the specific molecules driving metastasis and chemo resistance. We predict that CTC genotyping and phenotyping will play an increasing role in personalized therapy and in identification of novel therapeutic targets as well as monitoring the course and status of the disease.     

Advanced technologies for studying circulating tumor cells at the protein level

Metastasis is the main cause of cancer death. As the tumor progresses, cells from the primary tumor site are shed into the bloodstream as circulating tumor cells (CTCs). Eventually, these cells colonize other organs and form distant metastases. It is therefore imperative that we gain a better understanding of the biological characteristics of CTCs for development of novel treatment modalities to minimize metastasis-associated cancer deaths. In recent years, rapid developments in technologies for the study of CTCs have taken place. We now have a variety of tools for the isolation and examination of CTCs which were not available before. This review introduces some commonly used protein markers in CTC investigations and summarizes a few advanced technologies which have been successfully applied for studying CTC biology at the protein level.     

Classification of Circulating Tumor Cells by Epithelial-Mesenchymal Transition Markers

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.     

Circulating tumor cells: highlight on practical implications

Circulating tumor cells (CTCs) are cells of presumed epithelial origin, whose prognostic and predictive value in metastatic cancer patients has recently been demonstrated. To date, the count of CTCs through the CellSearch? system represents a valid approach for monitoring disease status in patients with metastatic colorectal, breast, and prostate cancer; in these cancer types, a rise in the CTC count at any time during treatment predicts a poor outcome. Nevertheless, the clinical utility of monitoring CTC counts remains controversial, and what to do when CTC counts rise during therapy still remains an unanswered question. In this report, we suggest how to integrate CTC counts with their molecular characterization to better translate biologic information obtained on CTCs into daily clinical practice.     

Molecular Biomarker Analyses Using Circulating Tumor Cells

Background

Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.

Methodology/Principal Findings

Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.

Conclusions/Significance

Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.     

Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.     

以微小RNA作为新型标志物检测循环肿瘤细胞及其临床意义

循环肿瘤细胞（circulating tumor cell,CTC）是随血液循环一起转运的实体肿瘤细胞,与实体肿瘤的发展、转移、复发和预后等关系密切。然而,CTC数量的稀少使有效检测CTC具有较大的挑战性。微小RNA（microRNA,miRNA）作为一类新发现的基因表达调控分子,在肿瘤的发生、发展、转归等过程中起着重要的作用。CTC关联性miRNA的研究为CTC的检测和肿瘤的诊治开创了新思路。该文介绍了CTC的临床意义和主要分析方法,在CTC关联性miRNA与肿瘤诊断、治疗和预后等方面总结了这类新型肿瘤细胞标志物的研究进展。     

Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell

Background

Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.

Methodology/Principal Findings

Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL.

Conclusions/Significance

The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.     

Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells

The occurrence of either regional or distant metastases is an indicator of poor prognosis for cancer patients. The mechanism of their formation has not yet been fully uncovered, which limits the possibility of developing new therapeutic strategies. Nevertheless, the discovery of circulating tumor cells (CTCs), which are responsible for tumor dissemination, and cancer stem cells (CSCs), required for tumor growth maintenance, shed light on the metastatic cascade. It seems that CTCs and CSCs are not necessarily separate populations of cancer cells, as CTCs generated in the process of epithelial-mesenchymal transition (EMT) can bear features characteristic of CSCs. This article describes the mechanisms of CTC and CSC formation and characterizes their molecular hallmarks. Moreover, we present different types of EMT occurring in physiological and pathological conditions, and we demonstrate its crucial role in providing CTCs with a CSC phenotype. The article delineates molecular changes acquired by cancer cells undergoing EMT that facilitate metastasis formation. Deeper understanding of those processes is of fundamental importance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice.     

Utilizing circulating tumor cells: challenges and pitfalls

Circulating tumor cells (CTC) detected in the blood of cancer patients could be used for risk-stratification, molecular subclassification and as an intermediate end-point in therapeutic efficacy studies. Most studies to date have focused on enumeration of CTC in advanced cancer patients but further development of CTC evaluation technologies could allow expansion into early disease, monitoring of treatment response, and selection of patients for targeted therapies based on a CTC derived signature. This review discusses the challenges faced in achieving these goals, including the potential absence of CTC in patients with no blood-borne metastases, CTC intra-patient molecular heterogeneity, ex vivo loss of CTC viability, and the biological differences between CTC and metastatic tissue.     

Detection and isolation of circulating tumor cells: Principles and methods

Efforts to improve the clinical management of several cancers include finding better methods for the quantitative and qualitative analysis of circulating tumor cells (CTCs). However, detection and isolation of CTCs from the blood circulation is not a trivial task given their scarcity and the lack of reliable markers to identify these cells. With a variety of emerging technologies, a thorough review of the exploited principles and techniques as well as the trends observed in the development of these technologies can assist researchers to recognize the potential improvements and alternative approaches. To help better understand the related biological concepts, a simplified framework explaining cancer formation and its spread to other organs as well as how CTCs contribute to this process has been presented first. Then, based on their basic working-principles, the existing methods for detection and isolation of CTCs have been classified and reviewed as nucleic acid-based, physical properties-based and antibody-based methods. The review of literature suggests that antibody-based methods, particularly in conjunction with a microfluidic lab-on-a-chip setting, offer the highest overall performance for detection and isolation of CTCs. Further biological and engineering-related research is required to improve the existing methods. These include finding more specific markers for CTCs as well as enhancing the throughput, sensitivity, and analytic functionality of current devices.     

Circulating tumour cells in clinical practice: Methods of detection and possible characterization

Circulating Tumour Cells (CTCs) can be released from the primary tumour into the bloodstream and may colonize distant organs giving rise to metastasis. The presence of CTCs in the blood has been documented more than a century ago, and in the meanwhile various methods have been described for their detection. Most of them require an initial enrichment step, since CTCs are a very rare event. The different technologies and also the differences among the screened populations make the clinical significance of CTCs detection difficult to interprete. Here we will review the different assays up to now available for CTC detection and analysis. Moreover, we will focus on the relevance of the clinical data, generated so far and based on the CTCs analysis. Since the vast majority of data have been produced in breast cancer patients, the review will focus especially on this malignancy.     

Characterization of a novel microfluidic platform for the isolation of rare single cells to enable CTC analysis from head and neck squamous cell carcinoma patients

Detailed examination of tumor components is leading‐edge to establish personalized cancer therapy. Accompanying research on cell‐free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter‐ and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully‐automated enrichment and single cell dispensing of CTCs from whole blood without pre‐processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR‐based confirmation of tumor‐related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real‐time monitoring.     

Statistical considerations for enumeration of circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) in patients with carcinomas are extremely rare. In metastatic breast cancer, the presence of >or=5 CTCs in 7.5 ml of blood has been associated with short survival. As this threshold has clinical implications, it is important to recognize the limitations associated with the detection and enumeration of CTCs. METHODS: Statistical analyses were performed on data generated from a multi-center clinical trial that utilized the CellSearchtrade mark System to isolate and enumerate CTCs in 7.5 ml blood samples. The statistical issues associated with each step of the process, from blood collection to final image analysis and CTC enumeration, were determined and implemented into a model. RESULTS: A model describing the statistics of the different process steps that are needed for the isolation and detection of CTCs was developed. The model uses the Poisson distribution for blood collection and empirically determined distributions for the isolation and identification of CTCs. The variability between readers was identified as one of the main sources of errors responsible for the current threshold level of five CTCs. CONCLUSIONS: Elimination of the errors made in the identification of tumor cells isolated from 7.5 ml of blood could potentially reduce the CTC threshold for the identification of patients with a poor prognosis from the current value of five CTCs to one CTC per 7.5 ml of blood.     

Circulating tumor cells as therapy-related biomarkers in cancer patients

Carcinomas (tumors of epithelial origin) are responsible for most of all new cancers in the industrialized countries. Due to the high mortality rate caused by the metastatic spread of aggressive cancer cells, there is an urgent demand in finding new biomarkers, which should detect early formation of metastases and monitor efficacy of systemic adjuvant therapy in a timely manner. It has been considered that the molecular analysis of cells which are shed from tumors into the blood system (circulating tumor cells (CTCs)) might provide new insights for the clinical management of cancer, probably far earlier than using traditional high-resolution imaging technologies. Clinical trials indicated that CTCs can be deployed for diagnostic, monitoring, and prognostic purposes. Furthermore, these cells are discussed to be suitable as predictive markers. In any case, identification of CTCs requires innovative and challenging technologies as detection methods should be specific, sensitive, standardized, and highly reproducible. Although many different approaches have been developed until now, only the CellSearch? method has been cleared by the American Food and Drug Administration. Although the detection of CTCs has already shown to have a prognostic impact in many tumor entities including breast, prostate, lung and colon cancer, ongoing and future studies are aimed to explore whether CTCs can be used for an individual therapy decision making including novel immunotherapeutic approaches. This review discusses (1) different detection strategies for CTCs, (2) their clinical impact, and (3) the potential use of CTCs guiding the treatment of individual cancer patients.     

Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal

Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow-up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program.     

Clinical Implications and Future Perspectives of Circulating Tumor Cells and Biomarkers in Clinical Outcomes of Colorectal Cancer

Colorectal cancer (CRC) is a major public health problem. Early CRC detection, pretherapeutic responsiveness prediction, and postoperative micrometastasis monitoring are the hallmarks for successful CRC treatment. Here, the methodologies used for detecting circulating tumor cells (CTCs) from CRC are reviewed. In addition to the traditional CRC biomarkers, the persistent presence of posttherapeutic CTCs indicates resistance to adjuvant chemotherapy and/or radiotherapy; hence, CTCs also play a decisive role in the subsequent relapse of CRC. Moreover, the genetic and phenotypic profiling of CTCs often differs from that of the primary tumor; this difference can be used to select the most effective targeted therapy. Consequently, studying CTCs can potentially individualize treatment strategies for patients with CRC. Therefore, CTC detection and characterization may be valuable tools for refining prognosis, and CTCs can be used in a real-time tumor biopsy for designing individually tailored therapy against CRC.