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.     

Circulating tumor cell isolation,culture, and downstream molecular analysis

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.     

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.     

循环肿瘤细胞识别技术研究现状

循环肿瘤细胞(CTCs)对恶性肿瘤传播转移有重要影响,因此CTCs识别技术的出现与不断进步有着重要的临床意义,准确可靠的CTCs识别技术将为尽早确诊肿瘤、指导个性化治疗方案、诊断微小残留病变以及评估抗癌药物的敏感性提供强有力的工具。本文针对核酸检测法、免疫细胞化学术、流式细胞术和基于表征特性图像识别技术等CTC识别技术的发展情况进行了综述总结,比较各种技术的优缺点,对现阶段该领域存在的问题进行了讨论,并对CTCs识别的技术发展方向作了进一步的展望,为学者们提供更广的研究思路。     

Tumour biomarkers—Tracing the molecular function and clinical implication

In recent years, with the increase in cancer mortality caused by metastasis, and with the development of individualized and precise medical treatment, early diagnosis with precision becomes the key to decrease the death rate. Since detecting tumour biomarkers in body fluids is the most non‐invasive way to identify the status of tumour development, it has been widely investigated for the usage in clinic. These biomarkers include different expression or mutation in microRNAs (miRNAs), circulating tumour DNAs (ctDNAs), proteins, exosomes and circulating tumour cells (CTCs). In the present article, we summarized and discussed some updated research on these biomarkers. We overviewed their biological functions and evaluated their multiple roles in human and small animal clinical treatment, including diagnosis of cancers, classification of cancers, prognostic and predictive values for therapy response, monitors for therapy efficacy, and anti‐cancer therapeutics. Biomarkers including different expression or mutation in miRNAs, ctDNAs, proteins, exosomes and CTCs provide more choice for early diagnosis of tumour detection at early stage before metastasis. Combination detection of these tumour biomarkers may provide higher accuracy at the lowest molecule combination number for tumour early detection. Moreover, tumour biomarkers can provide valuable suggestions for clinical anti‐cancer treatment and execute monitoring of treatment efficiency.     

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.     

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.

Methodology/Principal Findings

Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.

Conclusions/Significance

We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.     

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.     

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 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.     

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.     

Liquid biopsy in pancreatic cancer – Current perspective and future outlook

Pancreatic cancer is a lethal condition with a rising incidence and often presents at an advanced stage, contributing to abysmal five-year survival rates. Unspecific symptoms and the current lack of biomarkers and screening tools hamper early diagnosis. New technologies for liquid biopsies and their respective evaluation in pancreatic cancer patients have emerged over recent years. The term liquid biopsy summarizes the sampling and analysis of circulating tumor cells (CTCs), small extracellular vesicles (sEVs), and tumor DNA (ctDNA) from body fluids. The major advantages of liquid biopsies rely on their minimal invasiveness and repeatability, allowing serial sampling for dynamic insights to aid diagnosis, particularly early detection, risk stratification, and precision medicine in pancreatic cancer. However, liquid biopsies have not yet developed into a new pillar for clinicians' routine armamentarium. Here, we summarize recent findings on the use of liquid biopsy in pancreatic cancer patients. We discuss current challenges and future perspectives of this potentially powerful alternative to conventional tissue biopsies.     

A label-free microfluidic chip for the highly selective isolation of single and cluster CTCs from breast cancer patients

BackgroundCirculating tumor cells (CTCs) existing in peripheral blood can be used to predict the prognosis and survival of cancer patients. The study was designed to detect circulating tumor cells and circulating tumor single cell genes by applying microfluidic chip technology. It was used to explore the clinical application value in breast cancer.MethodsWe have developed a size-based CTCs sorting microfluidic chip, which contains a hexagonal array and a micro-pipe channel array to isolate and confirm both single CTCs and CTCs clusters. The sorting performance of the as-fabricated chip was tested by analyzing the clinical samples collected from 129 breast cancer patients and 50 healthy persons.ResultsIn this study, the chip can detect different immunophenotypes of CTCs in breast cancer patients. It was found that the new microfluidic device had high sensitivity (73.6%) and specificity (82.0%) in detecting CTCs. By detecting the blood samples of 129 breast cancer patients and 50 healthy blood donors, it was found that the number of CTCs was not associated with clinical factors such as age, gender, pathological type, and tumor size of breast cancer patients (P > 0.05), but was associated with TNM staging of breast cancer, with or without metastasis (P < 0.005). There was a statistically significant difference in the number of CTCs between luminal A (ER+/PR+/HER2-) and HER-2+ (ER-/PR-/HER2+) (P < 0.05). The best cut-off level distinguished by CTC between the breast cancer patients and the healthy persons was 3.5 cells/mL, with 0.845 for AUC-ROC, 0.790–0.901 for 95% CI, 73.6% for sensitivity, and 82% for specificity (P = 0.000). The combination of CTC, CEA, CA125 and CA153 can provide more effective breast cancer screening.ConclusionsThe CTCs analysis method presented here doesn''t rely on the specific antibody, such as anti-EpCAM, which would avoid the missed inspection caused by antibody-relied methods and offer more comprehensive biological information for clinical breast cancer diagnosis and treatment.     

Combination of Circulating Tumor Cells with Serum Carcinoembryonic Antigen Enhances Clinical Prediction of Non-Small Cell Lung Cancer

Circulating tumor cells (CTCs) have emerged as a potential biomarker in the diagnosis, prognosis, treatment, and surveillance of lung cancer. However, CTC detection is not only costly, but its sensitivity is also low, thus limiting its usage and the collection of robust data regarding the significance of CTCs in lung cancer. We aimed to seek clinical variables that enhance the prediction of CTCs in patients with non-small cell lung cancer (NSCLC). Clinical samples and pathological data were collected from 169 NSCLC patients. CTCs were detected by CellSearch and tumor markers were detected using the Luminex xMAP assay. Univariate analyses revealed that histology, tumor stage, tumor size, invasiveness, tumor grade and carcinoembryonic antigen (CEA) were associated with the presence of CTCs. However, the level of CTCs was not associated with the degree of nodal involvement (N) or tumor prognostic markers Ki-67, CA125, CA199, Cyfra21-1, and SCCA. Using logistic regression analysis, we found that the combination of CTCs with tumor marker CEA has a better disease prediction. Advanced stage NSCLC patients with elevated CEA had higher numbers of CTCs. These data suggest a useful prediction model by combining CTCs with serum CEA in NSCLC patients.     

Current and emerging applications of liquid biopsy in pan-cancer

Cancer morbidity and mortality are growing rapidly worldwide and it is urgent to develop a convenient and effective method that can identify cancer patients at an early stage and predict treatment outcomes. As a minimally invasive and reproducible tool, liquid biopsy (LB) offers the opportunity to detect, analyze and monitor cancer in any body fluids including blood, complementing the limitations of tissue biopsy. In liquid biopsy, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are the two most common biomarkers, displaying great potential in the clinical application of pan-cancer. In this review, we expound the samples, targets, and newest techniques in liquid biopsy and summarize current clinical applications in several specific cancers. Besides, we put forward a bright prospect for further exploring the emerging application of liquid biopsy in the field of pan-cancer precision medicine.     

Blood-based DNA methylation signatures in cancer: A systematic review

DNA methylation profiles are in dynamic equilibrium via the initiation of methylation, maintenance of methylation and demethylation, which control gene expression and chromosome stability. Changes in DNA methylation patterns play important roles in carcinogenesis and primarily manifests as hypomethylation of the entire genome and the hypermethylation of individual loci. These changes may be reflected in blood-based DNA, which provides a non-invasive means for cancer monitoring. Previous blood-based DNA detection objects primarily included circulating tumor DNA/cell-free DNA (ctDNA/cfDNA), circulating tumor cells (CTCs) and exosomes. Researchers gradually found that methylation changes in peripheral blood mononuclear cells (PBMCs) also reflected the presence of tumors. Blood-based DNA methylation is widely used in early diagnosis, prognosis prediction, dynamic monitoring after treatment and other fields of clinical research on cancer. The reversible methylation of genes also makes them important therapeutic targets. The present paper summarizes the changes in DNA methylation in cancer based on existing research and focuses on the characteristics of the detection objects of blood-based DNA, including ctDNA/cfDNA, CTCs, exosomes and PBMCs, and their application in clinical research.     

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.     

X-ray enabled detection and eradication of circulating tumor cells with nanoparticles

The early detection and eradication of circulating tumor cells (CTCs) play an important role in cancer metastasis management. This paper describes a new nanoparticle-enabled technique for integrated enrichment, detection and killing of CTCs by using magnetic nanoparticles and bismuth nanoparticles, X-ray fluorescence spectrometry, and X-ray radiation. The nanoparticles are modified with tumor targeting agents and conjugated with tumor cells through folate receptors over-expressed on cancer cells. A permanent micro-magnet is used to collect CTCs suspended inside a flowing medium that contains phosphate buffered saline (PBS) or whole blood. The characteristic X-ray emissions from collected bismuth nanoparticles, upon excitation with collimated X-rays, are used to detect CTCs. Results show that the method is capable of selectively detecting CTCs at concentrations ranging from 100-100,000cells/mL in the buffer solution, with a detection limit of ～100CTCs/mL. Moreover, the dose of primary X-rays can be enhanced to kill the localized CTCs by radiation induced DNA damage, with minimal invasiveness, thus making in vivo personalized CTC management possible.     

Clinical proteomics-driven precision medicine for targeted cancer therapy: current overview and future perspectives

Cancer is a common disease that is a leading cause of death worldwide. Currently, early detection and novel therapeutic strategies are urgently needed for more effective management of cancer. Importantly, protein profiling using clinical proteomic strategies, with spectacular sensitivity and precision, offer excellent promise for the identification of potential biomarkers that would direct the development of targeted therapeutic anticancer drugs for precision medicine. In particular, clinical sample sources, including tumor tissues and body fluids (blood, feces, urine and saliva), have been widely investigated using modern high-throughput mass spectrometry-based proteomic approaches combined with bioinformatic analysis, to pursue the possibilities of precision medicine for targeted cancer therapy. Discussed in this review are the current advantages and limitations of clinical proteomics, the available strategies of clinical proteomics for the management of precision medicine, as well as the challenges and future perspectives of clinical proteomics-driven precision medicine for targeted cancer therapy.