Custom biomarker investigation of circulating tumor cells using RarePlex® Developer Kits
Investigating a new biomarker on circulating tumor cells has meant building a multiplexed assay from scratch; this ACTC 2019 poster reports 8 markers added to a base epithelial CTC panel using RarePlex® Developer Kits, with default antigen retrieval and the expected subcellular localization.
Presented by RareCyte and the University of Washington.
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- 8 biomarkers were added to a three-channel CTC detection base without re-optimizing antigen retrieval. HER2, ER, PR, EGFR, Ki67, AR, ARv7 and PSMA were each tested on top of a base epithelial panel of a nuclear dye, an anti-CD45 antibody to exclude white blood cells, and cocktailed cytokeratin and EpCAM antibodies, with one or two user-selected markers combining with the Panel Kit under default antigen retrieval conditions.
- Positivity cut-offs were set on marker-negative cells, then read across a known expression range. Four cell lines spiked into three separate healthy donors scored 100.0, 99.6, 77.0 and 3.0 percent HER2-positive for BT474 (high), MDA-MB-453 (medium), OVCAR3 (low) and MDA-MB-468 (negative), the negative line setting the mean fluorescence intensity cut-off that was then applied to the other three.
- The custom assays carried through to patient samples, including two marker states within one patient. Individual CTCs from 2 prostate and 2 breast cancer patients were scored for AR with ARv7 and for ER with HER2; one breast cancer patient returned 13 ER+/HER2+ and 3 ER+/HER2- CTCs, mirroring the two tumors recorded for that patient, while ARv7 staining stayed nuclear as expected.
