Detection Of Rare Tumor Cells Research Articles

Tetrahedral framework nucleic acids linked CRISPR/Cas13a signal amplification system for rare tumor cell detection

The sensitive and accurate detection of rare tumor cells provides precise diagnosis and dynamic assessment information in various tumor spectrums. However, rare tumor cells assay is still a challenge due to the exceedingly rare presence in the blood. In this research, we develop a fluorescent approach for the identification of rare tumor cells based on a combination of immunosorbent capture and a three-step signal amplification strategy. First, rare tumor cells are captured by immunoadsorption on 96-well plates. Second, self-synthesized tetrahedral framework nucleic acids (tFNAs) spontaneously anchor into the lipid bilayer of rare tumor cells, resulting in a “one to more” amplification effect. Then, the double-stranded DNA (dsDNA) binds to the vertices of the tFNAs and generates a large amount of target RNA by T7 polymerase, which is the secondary signal amplification. Finally, the target RNA activates the collateral cleavage ability of CRISPR/Cas13a, and the reporter RNA is cleaved for third signal amplification. The detection limit of the proposed method is down to 1 cell mL−1. Furthermore, the tFNAs-Cas13a system is also shown to be capable of detecting rare tumor cells in spiked-in samples and clinical blood samples. This platform enables speedy detection of rare tumor cells with high sensitivity and good specificity, and shows great potential for tumor diagnosis.

Magnetic-Based Enrichment of Rare Cells from High Concentrated Blood Samples.

Here, we tested two magnetic-bead based systems for the enrichment and detection of rare tumor cells in concentrated blood products. For that, the defined numbers of cells from three pancreatic cancer cell lines were spiked in 108 peripheral blood mononuclear cells (PBMNCs) concentrated in 1 mL, mimicking diagnostic leukapheresis (DLA) samples, and samples were processed for circulating tumor cells (CTC) enrichment with the IsoFlux or the KingFisher systems, using different types of magnetic beads from the respective technology providers. Beads were conjugated with different anti-EpCAM and MUC-1 antibodies. Recovered cells were enumerated and documented by fluorescent microscopy. For the IsoFlux system, best performance was obtained with IsoFlux CTC enrichment kit, but these beads compromised the subsequent immunofluorescence staining. For the KingFisher system, best recoveries were obtained using Dynabeads Biotin Binder beads. These beads also allowed one to capture CTCs with different antibodies and the subsequent immunofluorescence staining. KingFisher instrument allowed a single and streamlined protocol for the enrichment and staining of CTCs that further prevented cell loss at the enrichment/staining interface. Both IsoFlux and KingFisher systems allowed the enrichment of cell line cells from the mimicked-DLA samples. However, in this particular experimental setting, the recovery rates obtained with the KingFisher system were globally higher, the system was more cost-effective, and it allowed higher throughput.

Detection of Circulating Tumor Cells (CTCs) in Malignant Pleural Mesothelioma (MPM) with the "Universal" CTC-Chip and An Anti-Podoplanin Antibody NZ-1.2.

Circulating tumor cell (CTC) is a potentially useful surrogate of micro-metastasis, but detection of rare tumor cells contaminated in a vast majority of normal hematologic cells remains technical challenges. To achieve effective detection of a variety of CTCs, we have developed a novel microfluidic system (CTC-chip) in which any antibody to capture CTCs is easily conjugated. In previous studies, we employed an antibody (clone E-1) against podoplanin that was strongly expressed on mesothelioma cells. The CTC-chip coated by the E-1 antibody (E1-chip) provided a modest sensitivity in detection of CTCs in malignant pleural mesothelioma (MPM). Here, to achieve a higher sensitivity, we employed a novel anti-podoplanin antibody (clone NZ-1.2). In an experimental model, MPM cells with high podoplanin expression were effectively captured with the CTC-chip coated by the NZ-1.2 antibody (NZ1.2-chip). Next, we evaluated CTCs in the peripheral blood sampled from 22 MPM patients using the NZ1.2-chip and the E1-chip. One or more CTCs were detected in 15 patients (68.2%) with the NZ1.2-chip, whereas only in 10 patients (45.5%) with the E1-chip. Of noted, in most (92.3%, 12/13) patients with epithelioid MPM subtype, CTCs were positive with the NZ1.2-chip. The CTC-count detected with the NZ1.2-chip was significantly higher than that with the E1-chip (p = 0.034). The clinical implications of CTCs detected with the NZ1.2-chip will be examined in a future study.

Detection of circulating tumor cells with a novel microfluidic system in malignant pleural mesothelioma.

Detection of rare tumor cells circulating in the blood (CTCs) presents technical challenges. CellSearch, the only approved system for clinical use, fails to capture epithelial cell adhesion molecule‐negative CTCs such as malignant pleural mesothelioma (MPM). We have developed a novel microfluidic device (CTC‐chip) in which any Ab to capture CTCs is conjugated. The CTC‐chip was coated with an Ab against podoplanin that is abundantly expressed on MPM. Circulating tumor cell‐detection performance was evaluated in experimental models in which MPM cells were spiked in blood sampled from a healthy volunteer and in clinical samples drawn from MPM patients. The CTC‐chip showed superior CTC‐detection performance over CellSearch in experimental models (sensitivity, 63.3%‐64.5% vs 0%‐1.1%; P < .001) and in clinical samples (CTC‐positivity, 68.8% vs 6.3%; P < .001). A receiver operating characteristic (ROC) analysis showed that the CTC test provided a significant diagnostic performance in discrimination of unresectable disease from resectable disease (area under the ROC curve, 0.851; P = .003). The higher CTC count (≥2 cells/mL) was significantly associated with a poor prognosis (P = .030). The novel CTC‐chip enabled sensitive detection of CTCs, which provided significant diagnostic and prognostic information in MPM.

Abstract 3574: Analysis of whole genome and transcriptome sequencing in single cell

Abstract The rapid advancement in Next Generation Sequencing (NGS) technology has allowed extensive genetic profiling and analysis of tumor tissues. While accurate tumor characterization is crucial for cancer therapy, current sequencing methods are unable to resolve the genetic differences between the heterogeneous cell populations within tumor. By profiling specific subpopulations of cells from a larger heterogeneous population, single cell sequencing can improve the efficiency of cancer treatments through early detection of rare tumor cells, monitoring of circulating tumor cells, and accurate profiling of intra-tumor heterogeneity. In this study, we compared commercially available single cell amplification methods and sequencing platforms for whole genome and whole transcriptome sequencing. We isolated single cells from a human breast cancer cell line, MCF7, and human embryonic kidney cell line, HEK293, by micromanipulation, and their whole genome and transcriptome were sequenced. For whole genome sequencing, the performance of Repli-g (Qiagen), Ampli1 (Slicon Biosystems) and WGA4 (Sigma) were assessed for whole genome amplification. Next, we used TruSeq DNA Library Kit and Ion Plus Fragment Library Kit for library preparation, and whole genome sequencing was performed on HiSeq2000 and Proton. The sequencing data were analyzed by SAMtools/GATK and tmap-f3. For whole transcriptome sequencing, cDNA was synthesized from single cells using SMARTer ultra low input RNA kit (Clontech), and the cDNA library was prepared with Nextera XT library prep kit (Illumina) and Ion Total RNA-Seq Kit v2 (Life Technologies). Sequencing was performed on HiSeq2000 and Proton, and analyzed with Tophat and Cufflink. We show that the percentage of mappable reads and regions with more than 1X coverage in whole genome sequencing were similar to that of the genomic DNA data, but the coverage rate of regions with more than 10X was lower in single cell data on both NGS platforms. In the RNA-Seq, the gene detection rate in single cell and more than 100 cells were about 65% and 85% compare to total RNA, respectively. Citation Format: Nak-Jung Kwon, Woo Chung Lee, Jiwoong Kim, Hyeri Kim, Ahreum Seong, Bong Cho Kim, Doo Hyun Park, Kap-Seok Yang. Analysis of whole genome and transcriptome sequencing in single cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2014-3574

Multiplex detection of lung cancer cells at the single-molecule level

We develop a simple and sensitive method for multiplex detection of lung cancer cells at the single-molecule level, with a detection limit of 15 cells per mL for A549 cells and 4 cells per mL for H23 cells, without the involvement of any sequence-based amplification. This method holds great potential for further application in early clinical diagnosis, especially for the detection of rare tumor cells.

Multicolor detection of rare tumor cells in blood using a novel flow cytometry‐based system

The presence and number of circulating tumor cells (CTCs) in the blood of patients with solid tumors are predictive of their clinical outcomes. To date, the CellSearch system is the only US Food and Drug Administration-approved CTC enumeration system for advanced breast, prostate, and colon cancers. However, sensitivity issues due to epithelial cellular adhesion molecule (EpCAM)-based enrichment and limited capability for subsequent molecular analysis must be addressed before CTCs can be used as predictive markers in the clinical setting. We have developed a multicolor CTC detection system using cross-contamination-free flow cytometry, which permits the enumeration and characterization of CTCs for multiple molecular analyses. Tumor cell lines with different expression levels of EpCAM were spiked into peripheral blood obtained from healthy donors. Spike-in samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, and this was followed by fixation and labeling with CD45-Alexa Fluor 700, EpCAM-phycoerythrin, cytokeratin (CK)-fluorescein isothiocyanate antibodies, and/or 7-aminoactinomycin D for nuclei staining. Excellent detection (slope = 0.760-0.888) and a linear performance (R(2) = 0.994-0.998) were noted between the observed and expected numbers of tumor cells, independent of EpCAM expression. The detection rate was markedly higher than that obtained using the CellSearch system, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Additionally, the incorporation of an epithelial-mesenchymal transition (EMT) marker allowed us to detect EpCAM-/CK- cells and EMT-induced tumor cells. Taken together, our multicolor CTC detection system may be highly efficient in detecting previously unrecognized populations of CTCs.

Future medical applications of single-cell sequencing in cancer

Advances in whole genome amplification and next-generation sequencing methods have enabled genomic analyses of single cells, and these techniques are now beginning to be used to detect genomic lesions in individual cancer cells. Previous approaches have been unable to resolve genomic differences in complex mixtures of cells, such as heterogeneous tumors, despite the importance of characterizing such tumors for cancer treatment. Sequencing of single cells is likely to improve several aspects of medicine, including the early detection of rare tumor cells, monitoring of circulating tumor cells (CTCs), measuring intratumor heterogeneity, and guiding chemotherapy. In this review we discuss the challenges and technical aspects of single-cell sequencing, with a strong focus on genomic copy number, and discuss how this information can be used to diagnose and treat cancer patients.

QS110. Fluorescence Expressing Viruses Enhance The Detection of Rare Tumor Cells in Human Whole Blood

Background: Cytologists are often asked to detect rare cancer cells in biologic specimens for diagnosis and staging of cancer, though the limits of detection even for the best cytologist is approximately 1 malignant cell in 20,000 normal cells. Many viruses are being engineered to selectively infect tumors as part of gene therapy strategies. We wondered if a herpes virus, NV1066, carrying the green fluorescence protein gene (GFP) can be used to selectively infect tumor and allow for enhanced detection of rare tumor cells in blood.

Novel Markers of Subclinical Disease for Ewing Family Tumors from Gene Expression Profiling

Targeting subclinical disease in the bone marrow is particularly relevant in metastatic Ewing family tumors (EFT) where cure is difficult. Genome-wide expression arrays can uncover novel genes differentially expressed in tumors over normal marrow/blood, which may have potentials as markers of subclinical disease. Gene expression array data were obtained on 28 EFT tumors using the Affymetrix U133 gene chip and compared with 10 normal blood samples. Ten genes with high tumor to blood ratios were identified. Quantitative reverse transcription-PCR was done to study (a) the dynamic range of detection of rare tumor cells, (b) the gene expression in normal blood/marrow samples, (c) the gene expression among EFT tumors, and (d) the detection and prognostic impact of marker positivity in histology-negative diagnostic marrows of EFT patients. Five of 10 genes (i.e., six-transmembrane epithelial antigen of the prostate 1 [STEAP1], cyclin D1 [CCND1], NKX2-2 transcription factor [NKX2-2], plakophilin 1 [PKP1], and transmembrane protein 47 [TMEM47]) were chosen for further analyses based on their steep linear dynamic range in detecting tumor cells seeded in normal mononuclear cells and on their homogeneous expression among EFT tumors. Prognostic effect was evaluated in 35 histology-negative diagnostic marrows. Marker negativity of STEAP1, CCND1, or NKX2-2, as well as three markers in combination, was strongly correlated with patient survival as well as survival without new metastases. This gene expression array-based approach identified novel markers that may be informative at diagnosis for risk group assessment. Their clinical utility needs to be tested in large patient cohorts.

STEAP1: A potential marker of metastatic disease for Ewing family tumors

10028 Background: Ewing Family Tumors (EFTs) metastasize by hematogeneous spread. Because of its dismal cure rate, targeting subclinical disease is particularly relevant. Genome-wide gene expression array analysis can uncover novel genes differentially expressed in tumors over normal marrow/blood, which may have clinical potentials as markers of minimal residual disease (MRD). Methods: Gene expression array data were obtained on 28 EFT tumors and 10 EFT cell lines using the Affymetrix U133 gene chip. Ten genes with high tumor to blood ratios were identified. Quantitative RT-PCR was performed to study (1) the dynamic range of detection of rare tumor cells (frequency of 10-7 to 10-4), (2) the gene expression in normal blood and marrow samples, (3) the gene expression among EFT tumors, and (4) the prognostic impact of marker positivity in EFT patients with histologically negative marrows. Results: Of the 10 genes, 5 genes, namely six transmembrane epithelial antigen of the prostate 1 [STEAP1], cyclin D1 [CCND1], plakophilin 1 [PKP1], transmembrane protein 47 [TMEM47], and NKX2.2 transcription factor [NKX2.2] were chosen for further analyses. This was based on their steep linear dynamic range in tumor cells seeded in normal mononuclear cells and consistently high expression among EFT tumor samples. Among 53 EFT patients with histologically negative marrow samples, a marker was classified as positive if the gene transcript level was above the upper limit of normal, defined as mean + 2SD of 26 normal blood and marrow samples. Of the 5 markers tested, STEAP1 positivity in marrow samples was highly correlated with survival with new metastasis in Kaplan Meier analysis (p=0.001). Conclusions: This gene expression array-based approach identified STEAP1 as a promising surrogate MRD marker of metastatic EFT with potential prognostic importance. Its clinical utility will need to be further tested in large patient cohorts. No significant financial relationships to disclose.

Detection of rare tumor cells in peripheral blood for early identification of recurrent disease

Detection of rare tumor cells in peripheral blood for early identification of recurrent disease