scholarly journals The Discordance of Gene Mutations between Circulating Tumor Cells and Primary/Metastatic Tumor

2019 ◽  
Vol 15 ◽  
pp. 21-29 ◽  
Author(s):  
Qi Wang ◽  
Lanbo Zhao ◽  
Lu Han ◽  
Xiaoqian Tuo ◽  
Sijia Ma ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Gene Mutations ◽  
Metastatic Tumor

scholarly journals A Modified Method to Isolate Circulating Tumor Cells and Identify by a Panel of Gene Mutations in Lung Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382199527
Author(s):  
Helin Wang ◽  
Jieqing Wu ◽  
Qi Zhang ◽  
Jianqing Hao ◽  
Ying Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Detection Rate ◽  
Target Genes ◽  
White Blood Cells ◽  
Gene Mutations ◽  
Copy Number Variations ◽  
Immunomagnetic Beads ◽  
Membrane Rupture

The CellSearch system is the only FDA approved and successful used detection technology for circulating tumor cells(CTCs). However, the process for identification of CTCs by CellSearch appear to damage the cells, which may adversely affects subsequent molecular biology assays. We aimed to explore and establish a membrane-preserving method for immunofluorescence identification of CTCs that keeping the isolated cells intact. 98 patients with lung cancer were enrolled, and the efficacy of clinical detection of CTCs was examined. Based on the CellSearch principle, we optimized an anti-EpCAM antibody and improved cell membrane rupture. A 5 ml peripheral blood sample was used to enrich CTCs with EpCAM immunomagnetic beads. Fluorescence signals were amplified with secondary antibodies against anti-EpCAM antibody attached on immunomagnetic beads. After identifying CTCs, single CTCs were isolated by micromanipulation. To confirm CTCs, genomic DNA was extracted and amplified at the single cell level to sequence 72 target genes of lung cancer and analyze the mutation copy number variations (CNVs) and gene mutations. A goat anti-mouse polyclonal antibody conjugated with Dylight 488 was selected to stain tumor cells. We identified CTCs based on EpCAM+ and CD45+ cells to exclude white blood cells. In the 98 lung cancer patients, the detection rate of CTCs (≥1 CTC) per 5 ml blood was 87.76%, the number of detections was 1–36, and the median was 2. By sequencing 72 lung cancer-associated genes, we found a high level of CNVs and gene mutations characteristic of tumor cells. We established a new CTCs staining scheme that significantly improves the detection rate and allows further analysis of CTCs characteristics at the genetic level.

Consistency evaluation of lung adenocarcinoma tissue and circulating tumor cells related gene mutation detection based on multi-site immunomagnetic beads

2022 ◽  
pp. 088532822110658
Author(s):  
Keying Xue ◽  
Bingqing Luo ◽  
Xiaoqing Li ◽  
Weixian Deng ◽  
Chunyan Zeng ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Lung Adenocarcinoma ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Targeted Therapies ◽  
Sanger Sequencing ◽  
Gene Mutations ◽  
Genetic Mutations ◽  
Separation System ◽  
Tumor Tissues

This study was designed to investigate the feasibility of genetic testing using circulating tumor cells (CTCs) instead of tumor tissues in lung adenocarcinoma to break through its limitation. Separation system for epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and Vimentin expressing CTCs was constructed and used to capture CTCs in the blood samples of 57 patients with lung adenocarcinoma. Genetic mutations of genes involved in targeted therapies were detected by next-generation sequencing (NGS) in tissues from these patients. Blood CTC samples with the gene mutations identified by tissue-NGS were selected and corresponding gene mutations were detected by Sanger sequencing. The specificity of the CTC separation system was 95.48% and the sensitivity was 90.85%. The average number of CTCs in the blood of patients with lung adenocarcinoma was 12.47/7.5 mL. Comparison of the tissue-NGS with the CTC-Sanger sequencing showed that the consistencies of genetic mutations of EGFR ( n = 24), KRAS ( n = 9), TP53 ( n = 19), BRAF ( n = 1), ERBB2 ( n = 2), and PIK3CA ( n = 3) were 92.31%, 75.00%, 86.36%, 50.00%, 66.67%, and 75.00%, with an overall consistency of 84.06%. The CTC separation system established in this study shows high specificity and sensitivity. CTCs can be used as a suitable alternative to tumor tissues that are difficult to obtain in clinical practice and overcome the difficulties in tumor tissue collection, which is of significance in guiding clinical medication and individualized treatment with significant clinical application value in terms of genetic testing for targeted therapies in tumor treatment.

scholarly journals Sensitive cytometry based system for enumeration, capture and analysis of gene mutations of circulating tumor cells

2016 ◽  
Vol 107 (3) ◽  
pp. 307-314 ◽  
Author(s):  
Takeshi Sawada ◽  
Masaru Watanabe ◽  
Yuu Fujimura ◽  
Shigehiro Yagishita ◽  
Tatsu Shimoyama ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Gene Mutations

scholarly journals Multifunctional Gold Nano-Cytosensor With Quick Capture, Electrochemical Detection, and Non-Invasive Release of Circulating Tumor Cells for Early Cancer Treatment

2021 ◽  
Vol 9 ◽  
Author(s):  
Rui Zhang ◽  
Qiannan You ◽  
Mingming Cheng ◽  
Mingfeng Ge ◽  
Qian Mei ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Treatment Options ◽  
Metastatic Tumor ◽  
Quantitative Detection ◽  
Cell Capture ◽  
Primary Tumors ◽  
Relative Standard

Circulating tumor cells (CTCs) are metastatic tumor cells that shed into the blood from solid primary tumors, and their existence significantly increases the risk of metastasis and recurrence. The timely discovery and detection of CTCs are of considerable importance for the early diagnosis and treatment of metastasis. However, the low number of CTCs hinders their detection. In the present study, an ultrasensitive electrochemical cytosensor for specific capture, quantitative detection, and noninvasive release of EpCAM-positive tumor cells was developed. The biosensor was manufactured using gold nanoparticles (AuNPs) to modify the electrode. Three types of AuNPs with controllable sizes and conjugated with a targeting molecule of monoclonal anti-EpCAM antibody were used in this study. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) of the cytosensors were performed to evaluate the cell capture efficiency and performance. The captured 4T1 cells by the AuNPs hindered electron transport efficiency, resulting in increased EIS responses. The cell capture response recorded using EIS or DPV indicated that the optimal AuNPs size should be 17 nm. The cell capture response changed linearly with the concentration range from 8.0 × 10 to 1 × 107 cells/mL, and the limit of detection was 50 cells/mL. After these measurements, glycine-HCl (Gly-HCl) was used as an antibody eluent to destroy the binding between antigen and antibody to release the captured tumor cells without compromising their viability for further clinical research. This protocol realizes rapid detection of CTCs with good stability, acceptable assay precision, significant fabrication reproducibility with a relative standard deviation of 2.09%, and good recovery of cells. Our results indicate that the proposed biosensor is promising for the early monitoring of CTCs and may help customize personalized treatment options.

Enumeration of circulating tumor cells using a non–EpCAM-dependent device.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23064-e23064
Author(s):  
Robert J. Amato ◽  
Reynolds Brobey ◽  
Yue Wang ◽  
Kent Murphy ◽  
Jeff Smith
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Cancer Cell ◽  
Cancer Biology ◽  
Metastatic Tumor ◽  
Tumor Burden ◽  
Cell Detection ◽  
Tumor Types ◽  
Cancer Cell Detection ◽  
Prospective Longitudinal

e23064 Background: One component of the metastatic process is the circulation of tumor cells. Identification of these circulating tumor cells (CTCs) provides important insights into cancer biology. There is enormous heterogeneity among CTCs, necessitating capture of both epithelial and non-epithelial subtypes. Detection and monitoring of CTCs will provide a non-invasive biomarker for the detection of early progression and assessing therapeutic response. Methods: The AxonDx nCyte™ System is designed to detect rare circulating cells from peripheral blood without enrichment or selection. An epi-fluorescence microscope scans pathology glass slides on which a sample has been applied, and high-resolution images of potential CTCs are captured at four emission wavelengths. The proprietary Cancer Cell Detection Cocktail is designed to detect CTCs from numerous solid tumors, including various stages (e.g., mesenchymal-epithelial transformation) and stem cells. The software distinguishes cancer-derived material (nucleus+/cytokeratin+/WBC marker-/ > 4 µm); from leukocytes (nucleus+/cytokeratin-/WBC marker+). Vimentin is used to distinguish epithelial from mesenchymal CTCs. We obtained informed consent from 32 genitourinary cancer patients to participate in this IRB-approved study. Red blood cells from 6 mL of whole blood were lysed and removed from 32 patient samples. Nucleated cells from the blood sample were fixed, permeabilized, and stained with AxonDx’s Cancer Cell Detection Cocktail. The prepared cells were dispensed onto two microscope slides, cover-slipped, and analyzed. Results: We detected CTCs in 32 of 32 samples (100%), in a range of 0.6–64.3 CTCs/mL. Number of CTCs correlated with metastatic tumor burden. Conclusions: The AxonDx nCYTE™ system can reliably detect CTCs from genitourinary patient samples. The AxonDx nCyte™ System has also successfully detected CTCs in other tumor types, e.g., breast, lung, CRC, and HCC. Because the system is not EpCAM-dependent, it captures the heterogeneous population of CTCs. Prospective longitudinal studies are ongoing to determine trends in CTC counts.

scholarly journals A Pilot Study for the Feasibility of Exome-Sequencing in Circulating Tumor Cells Versus Single Metastatic Biopsies in Breast Cancer

2020 ◽  
Vol 21 (14) ◽  
pp. 4826
Author(s):  
Pushpinder Kaur ◽  
Daniel Campo ◽  
Tania B. Porras ◽  
Alexander Ring ◽  
Janice Lu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Exome Sequencing ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Copy Number ◽  
Chromosomal Rearrangements ◽  
Gene Mutations ◽  
Clinical Samples ◽  
Breast Cancer Patients ◽  
Somatic Alterations

The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing (WES). We determined the level of genomic concordance for somatic nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs). The variant allele fractions (VAFs) of somatic variants were remarkably similar between amplified and non-amplified cell line samples as technical replicates. In clinical samples, a significant fraction of somatic variants had low VAFs in CTCs compared to metastases. The most frequently recurrent gene mutations in clinical samples were associated with an elevated C > T mutational signature. We found complex rearrangement patterns including intra- and inter-chromosomal rearrangements, singleton, and recurrent gene fusions, and tandem duplications. We observed high molecular discordance for somatic alterations between paired samples consistent with marked heterogeneity of the somatic landscape. The most prevalent copy number calls were focal deletion events in CTCs and metastases. Our results demonstrate the feasibility of an integrated workflow for the identification of a complete repertoire of somatic alterations and highlight the intrapatient genomic differences that occur between CTCs and metastases.

scholarly journals Molecular profiling of single circulating tumor cells from lung cancer patients

2016 ◽  
Vol 113 (52) ◽  
pp. E8379-E8386 ◽  
Author(s):  
Seung-min Park ◽  
Dawson J. Wong ◽  
Chin Chun Ooi ◽  
David M. Kurtz ◽  
Ophir Vermesh ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
High Efficiency ◽  
Tumor Biology ◽  
Metastatic Tumor ◽  
Lung Cancer Patients ◽  
Single Ctcs ◽  
Mutation Profiling

Circulating tumor cells (CTCs) are established cancer biomarkers for the “liquid biopsy” of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non–small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

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