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.     

High speed detection of circulating tumor cells

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.     

A general framework for analyzing tumor subclonality using SNP array and DNA sequencing data

Intra-tumor heterogeneity reflects cancer genome evolution and provides key information for diagnosis and treatment. When bulk tumor tissues are profiled for somatic copy number alterations (sCNA) and point mutations, it may be difficult to estimate their cellular fractions when a mutation falls within a sCNA. We present the Clonal Heterogeneity Analysis Tool, which estimates cellular fractions for both sCNAs and mutations, and uses their distributions to inform macroscopic clonal architecture. In a set of approximately 700 breast tumors, more than half appear to contain multiple recognizable aneuploid tumor clones, and many show subtype-specific differences in clonality for known cancer genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0473-4) contains supplementary material, which is available to authorized users.     

Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma

Background and aimsCirculating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients.MethodsTwenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR.ResultsCTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1–15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival.ConclusionsOncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.     

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。     

Detection and cultivation of circulating tumor cells in gastric cancer

Circulating tumor cells (CTCs) are important targets for treatment and critical surrogate markers when evaluating cancer prognosis and therapeutic response. A sensitive methodology for detecting CTCs in gastric cancer (GC) patients is needed. In this study we demonstrate a device for enrichment and cultivation of CTCs. In total, 22 patients with GC, all candidates for surgery, were enrolled in the study. Peripheral blood samples were collected before surgery, and patients were re-evaluated within operation and divided into two groups: resectable and non-resectable GC. A new size-based separation test for enrichment and cultivation of CTCs was used (MetaCell^®). In addition to cytomorphological analysis, gene expression of tumor associated genes (Cytokeratin-18, Cytokeratin-19, Cytokeratin-20, Cytokeratin-7, EPCAM, MUC1, HER2, EGFR) and of leukocyte markers (e.g. CD45, CD68) was tested in enriched CTC fractions. CTCs were detected in 59 % of the patients studied (n = 13/22). CTCs were detected in seven patients of the resection group (7/10, 70 %) and six of the non-resectable group (6/12, 50 %). Enrichment of the viable CTCs allowed subsequent successful cultivation in vitro. The cytomorphological characterization of the CTCs was a prerequisite of random gene expression testing in CTC-positive samples. In CTC-positive samples gene expression of cytokeratin 18 and 19 was elevated in comparison to the whole blood gene expression analysis. CTCs were found to be present in both resectable and non-resectable gastric cancer patients. The size-based separation platform for CTCs may be used for in vitro cultivation, as well as in subsequent molecular analysis if desired. The sensitivity of CTC-detection could be enhanced by the combination of cytomorphological and molecular analysis.     

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.     

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.     

Automated identification of circulating tumor cells by image cytometry

Presence of circulating tumor cells (CTC), as detected by the CellSearch System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 40×/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright-field images. Improved detection efficiency for CD45-APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated.     

单分子测序技术在肿瘤诊断中的应用研究进展

肿瘤的发生发展通常与多个基因的遗传突变、表达异常有关,全面深入地分析肿瘤基因组、转录组及表观遗传组学对于快速剖析疾病特定基因簇及修饰位点至关重要。之前,研究者们主要采用第二代测序技术进行有效信息的挖掘,然而随着研究的不断深入,第二代测序技术表现出诸如序列拼接困难、低丰度难以检出等缺点。因此,单分子测序技术以其独特的优势应运而生,文中主要对单分子测序技术在几种常见肿瘤中的研究现状进行了综述,并展望了其在临床诊断中的应用前景。     

Influence of adjuvant radiotherapy on circulating epithelial tumor cells and circulating cancer stem cells in primary non-metastatic breast cancer

Background: There is an unmet need to identify biomarkers that directly reflect response to adjuvant radiotherapy (RT). Circulating epithelial tumor cells (CETCs) represent the liquid component of solid tumors and are responsible for metastatic relapse. CETC subsets with cancer stem cell characteristics, circulating cancer stem cells (cCSCs), play a pivotal role in the metastatic cascade. Monitoring the most aggressive subpopulation of CETCs could reflect the aggressiveness of the remaining tumor burden. There is limited data on the detection and monitoring changes in CETC and cCSC numbers during RT in early breast cancer.Methods: CETC numbers were analyzed prior to, at midterm and at the end of RT in 52 primary non-metastatic breast cancer patients. Hormone receptor status was determined in CETCs prior to and at the end of RT. For the identification of cCSCs cell suspensions from the peripheral blood of patients were cultured in vitro under conditions favoring growth of tumorspheres.Results: Hormone receptor status in CETCs before RT was comparable to that in primary tumor tissue. Prior to RT numbers of CETCs correlated with aggressiveness of primary tumors. cCSCs could be successfully identified and monitored during RT. Prior to RT patients treated with neoadjuvant chemotherapy had significantly higher numbers of CETCs and tumorspheres compared to patients after adjuvant chemotherapy. During RT, the number of CETCs decreased continuously in patients after neoadjuvant chemotherapy but not after adjuvant chemotherapy.Conclusion: Monitoring the number of CETCs and the CETC subset with cancer stem cell properties during RT may provide additional clinically useful prognostic information.     

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.     

The potential and challenges of nanopore sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.     

HER2-positive circulating tumor cells in breast cancer

Purpose

Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging.

Methods

Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®.

Results

Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03).

Conclusion

HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.     

Apoptosis of circulating tumor cells in prostate cancer patients.

BACKGROUND: The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. METHODS: In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole. RESULTS: In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. CONCLUSIONS: The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients.     

An electrical biosensor for the detection of circulating tumor cells

In this report, we demonstrate a semi-integrated electrical biosensor for the detection of rare circulating tumor cells (CTCs) in blood. The sample was first enriched through a combination of immunomagnetic isolation and size filtration. The integration of both methods provided a high enrichment performance with a recovery rate above 70%, even for very low numbers of cancer cells present in the original sample (10 spiked MCF7 cells in 0.5 mL of blood). In the same system, the sample was then transferred to a microchip for further magnetic concentration, followed by immunochemical trapping and electronic detection by impedance spectroscopy. Three levels of spiked CTC number (30±2, 124±29, 273±23) in 10 μL of filtered blood sample were distinguished by monitoring the impedance change of the microelectrode array (MEA). The integration of different functions in a single system provided a methodology to process milliliter-sized blood samples at the macroscale and interface with the microdimensions of a highly sensitive electronic detector. The results showed that the whole system was able to detect different levels of spiked cancer cells without the use of time- and cost-intensive fluorescence labeling and image analysis. This has the potential to provide clinicians with a standalone system to monitor changes in CTC numbers throughout therapy conveniently and frequently for efficient cancer treatments.     

Bioinformatics challenges of new sequencing technology

New DNA sequencing technologies can sequence up to one billion bases in a single day at low cost, putting large-scale sequencing within the reach of many scientists. Many researchers are forging ahead with projects to sequence a range of species using the new technologies. However, these new technologies produce read lengths as short as 35-40 nucleotides, posing challenges for genome assembly and annotation. Here we review the challenges and describe some of the bioinformatics systems that are being proposed to solve them. We specifically address issues arising from using these technologies in assembly projects, both de novo and for resequencing purposes, as well as efforts to improve genome annotation in the fragmented assemblies produced by short read lengths.     

Computational methods and challenges in analyzing intratumoral microbiome data

The human microbiome is intimately related to cancer biology and plays a vital role in the efficacy of cancer treatments, including immunotherapy. Extraordinary evidence has revealed that several microbes influence tumor development through interaction with the host immune system, that is, immuno–oncology–microbiome (IOM). This review focuses on the intratumoral microbiome in IOM and describes the available data and computational methods for discovering biological insights of microbial profiling from host bulk, single-cell, and spatial sequencing data. Critical challenges in data analysis and integration are discussed. Specifically, the microorganisms associated with cancer and cancer treatment in the context of IOM are collected and integrated from the literature. Lastly, we provide our perspectives for future directions in IOM research.