Identification and Characterization of Effusion Tumor Cells (ETCs) From Remnant Pleural Effusion Specimens
Cancer Cytopathology. 2021;129(11):893-906. DOI 10.1002/cncy.22483.
How to cite
AMA
Zhu Y, Allard GM, Ericson NG, George TC, Kunder CA, Lowe AC. Identification and Characterization of Effusion Tumor Cells (ETCs) From Remnant Pleural Effusion Specimens. Cancer Cytopathol. 2021;129(11):893-906. doi:10.1002/cncy.22483APA
Zhu, Y., Allard, G. M., Ericson, N. G., George, T. C., Kunder, C. A., & Lowe, A. C. (2021). Identification and Characterization of Effusion Tumor Cells (ETCs) From Remnant Pleural Effusion Specimens. Cancer Cytopathology, 129(11), 893-906. https://doi.org/10.1002/cncy.22483BibTeX
@article{zhu2021identification,
title = {Identification and Characterization of Effusion Tumor Cells (ETCs) From Remnant Pleural Effusion Specimens},
author = {Zhu, Yili and Allard, Grace M. and Ericson, Nolan G. and George, Tad C. and Kunder, Christian A. and Lowe, Alarice C.},
journal = {Cancer Cytopathology},
volume = {129},
number = {11},
pages = {893--906},
year = {2021},
doi = {10.1002/cncy.22483}
}Pleural effusions, the fluid that builds up around the lungs in many patients with advanced cancer, are routinely drained and discarded, even though they often carry tumor cells. Because these samples usually hold few cancer cells, they are rarely used for the molecular testing that guides targeted treatment.
This study adapted a rare-cell detection method, first built for tumor cells in blood, to leftover pleural fluid. The team set quantitative staining thresholds to tell tumor cells apart from background cells, then applied them to remnant samples from 21 patients using multiplexed immunofluorescence imaging.
The assay identified cancer with high sensitivity and specificity, and pools of just five isolated tumor cells yielded driver mutations that matched each patient's clinical results.
Key findings
- The assay detected epithelial malignancy with 89.5% sensitivity and 100% specificity. Effusion tumor cells were found in 17 of 19 malignant pleural effusion samples and in none of the 6 samples that were benign on clinical cytology.
- Two fluorescence-intensity cutoffs separated tumor cells from background cells. Model tumor cells (n=80) and white blood cells (n=50) parted cleanly on EpCAM and CD45 staining, so a cell scoring above 100 arbitrary units for EpCAM and below 100 arbitrary units for CD45 was counted as a tumor cell.
- Molecular profiling of just 5 tumor cells recovered clinically concordant driver mutations. Pools of 5 confirmed effusion tumor cells were retrieved and sequenced, yielding pathogenic mutations such as KRAS G12A and ERBB2 S310F that were also reported by the patients' clinical molecular testing.
CyteFinder II HT in the methods
“We performed multiplexed fluorescent staining of the blood-sample control slides and TP slides using a custom RarePlex Staining Kit (RareCyte Inc). Briefly, the air-dried slides were fixed in 10% neutralized buffered formalin for 10 minutes and then blocked with 10% goat serum. The blocked slides were next subjected to the RarePlex Staining Kit and incubated sequentially with different RarePlex reagents in the dark. The slides were mounted with mounting media and coverslipped with imaging coverslips (Fisher Scientific). The slides were scanned at ×10 magnification using the CyteFinder II HT Instrument (RareCyte Inc).”
— Zhu et al., Cancer Cytopathology (2021), Methods, “Multiplexed Immunofluorescent Imaging Workflow”
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 CyteFinder II HT users
For anyone extending a rare-cell immunofluorescence assay to a difficult specimen, the hard part is finding a handful of tumor cells among thousands of background cells. This study leaned on whole-slide scanning to do exactly that. After staining, the CyteFinder II HT Instrument imaged each slide across four fluorescence channels, and every cell was scored against fixed intensity cutoffs for EpCAM and CD45 rather than by eye alone. Because the scanner reads the entire slide, the team could recover tumor cells from remnant effusions as small as 10 to 25 mL, well below the volume usually considered adequate, and still tie cell counts back to the clinical tumor cellularity. The same instrument then retrieved confirmed cells in five-cell pools for sequencing. For your own work, that is the useful part: the imaging step turns a sparse, low-yield sample into a quantitative, reviewable readout that also feeds molecular profiling.
