Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients
Current Issues in Molecular Biology. 2022;44(2):750-763. DOI 10.3390/cimb44020052. PMID 35723337. PMCID PMC8928994.
How to cite
AMA
Barbirou M, Miller A, Manjunath Y, Ramirez AB, Ericson NG, Staveley-O'Carroll KF, et al. Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients. Curr Issues Mol Biol. 2022;44(2):750-763. doi:10.3390/cimb44020052APA
Barbirou, M., Miller, A., Manjunath, Y., Ramirez, A. B., Ericson, N. G., Staveley-O'Carroll, K. F., et al. (2022). Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients. Current Issues in Molecular Biology, 44(2), 750-763. https://doi.org/10.3390/cimb44020052BibTeX
@article{barbirou2022single,
title = {Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients},
author = {Barbirou, Mouadh and Miller, Amanda and Manjunath, Yariswamy and Ramirez, Arturo B and Ericson, Nolan G and Staveley-O'Carroll, Kevin F and others},
journal = {Current Issues in Molecular Biology},
volume = {44},
number = {2},
pages = {750--763},
year = {2022},
doi = {10.3390/cimb44020052}
}Lung cancer is the leading cause of cancer death, and tissue biopsies are invasive, usually taken only once, and hard to repeat as the disease changes. A simple blood draw that captures whole tumor cells could profile the cancer again and again without another operation.
Researchers drew blood from 20 non-small-cell lung cancer patients and 11 cancer-free long-term smokers, found the rare tumor cells circulating in it, and lifted them out one cell at a time. Each single cell was then sequenced across 65 cancer genes.
Patients carried far more of these cells than the smokers did, and the sequenced cells shared mutations in genes that drive lung cancer, showing that single circulating tumor cells can be read one by one for precision oncology.
Key findings
- The workflow detected more circulating tumor cells in cancer patients than in cancer-free smokers. CTCs were found in 12 of 20 NSCLC patients (60%) versus 2 of 11 control smokers (18%), and the counts were significantly higher in the cancer group (p = 0.0132, Mann-Whitney test).
- Single tumor cells were retrieved one at a time and sequenced across 65 cancer genes. From seven patients, 36 single cells (23 CTCs and 13 white blood cells) were individually retrieved and sequenced, revealing 644 somatic variants shared across all CTCs within a patient, with predicted oncogenic changes in seven genes including TP53, KRAS, and ERBB2.
- Blinded spike-in tests showed the workflow recovered tumor cells accurately before any patient sample was run. Known numbers of lung cancer cells added to healthy blood were recovered in linear proportion (linear regression r² = 0.999), establishing analytic accuracy for the enrichment-free approach.
The AccuCyte–CyteFinder workflow in the methods
“Phlebotomies were performed and blood (7.5 mL) was collected in AccuCyte BCT tubes and shipped overnight to RareCyte Inc. (Seattle, WA, USA) for CTC enumeration and single-cell retrieval of CTCs and WBCs in NSCLC patients. Processing was performed using the AccuCyte sample preparation system to isolate nucleated cells and spread them evenly onto SuperFrost ™ Plus Microscope Slides (Fisherbrand ™ , Fisher Scientific, Hampton, NH, USA). The slides were air-dried at room temperature and banked for later staining (stored at −20 °C). Enumeration and retrieval of CTCs and WBCs were performed using CyteFinder ® instrument based on CF405, Sytox Orange, CF647, and QD800 tags to target the Pre-label, Nucleus, CK/EpCAM, and CD45, respectively. Slide images were analyzed by CyteMapper ® software. Then, cells were individually retrieved and dispensed in PCR tubes for downstream NGS. CTCs were defined by nuclear size ≥8 μm in diameter, presence of a well-defined and visible cytoplasm, and immunofluorescence staining in the corresponding channels of predicted biomarkers (CK+ and/or EpCAM+, CD45-, DAPI+ nucleus).”
— Barbirou et al., Current Issues in Molecular Biology (2022), Methods, “CTC Enumeration with NSCLC Cell Line Cells Spiked into Healthy Human Blood”
Disclosure: RareCyte is listed as an author affiliation on the publication cited above.
Disclosure: RareCyte is named in the competing-interests statement of the publication cited above.
Why it matters for The AccuCyte–CyteFinder workflow users
If you are weighing an enrichment-free route to circulating tumor cells, this study shows the AccuCyte–CyteFinder workflow doing the full job from blood tube to sequenced cell. Blood was drawn into AccuCyte tubes, and rather than selecting cells by an epithelial marker first, the sample preparation system isolated every nucleated cell by density and spread it across a slide. The CyteFinder instrument then imaged the whole slide to find candidate tumor cells and, just as important, retrieved each one individually for sequencing. That last step is what separates enumeration from genomics: a counted cell tells you how many, a retrieved cell tells you what is inside it. Here the retrieved single cells carried mutations in known lung cancer genes, so the workflow delivered analysis-ready material rather than only a number. For your own work the point is that one run can both quantify rare cells and hand you intact single cells for downstream sequencing, without an antigen-capture step deciding in advance which cells you get to study.
