Novel multi-parameter assays for investigational phenotyping of circulating tumor cells
Identifying a circulating tumor cell proves it is there but not what it expresses; this Next Gen Dx 2018 poster presents two assay frameworks that free two or three of six fluorescence channels for investigational biomarkers, and confirms malignancy by retrieving the cells one at a time for sequencing.
Presented by RareCyte / University of Washington.
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- Two panel frameworks split the same six fluorescence channels differently. The CTC-4 panel spends four channels identifying epithelial CTCs — nuclear stain, CD45, EpCAM and cytokeratin — leaving 2 channels for investigational biomarkers; the CTC-3 panel combines EpCAM and cytokeratin into a single cocktail channel, freeing up to 3.
- Four investigational assays were built on those frameworks and run on cell lines and patient samples. CTC-4 assays addressed prostate cancer (AR / AR-V7) and immune checkpoint inhibition (PD-L1 / IRF1); CTC-3 assays addressed breast cancer (Ki-67 / ER / HER2) and mesenchymal differentiation (vimentin / EGFR), with vimentin present in 2 of the prostate CTCs and 3 of the breast CTCs identified from patient samples.
- Single cells retrieved from a patient carried the known TP53 mutation, confirming they were malignant. CTCs were picked one at a time with CytePicker from a prostate cancer patient with TP53 G244S, then whole-genome amplified and Sanger sequenced; cells diverging from the canonical four-marker epithelial pattern, and epithelial-mesenchymal CTCs, returned allelic frequencies up to 100%.
