Succinct workflows for circulating tumor cells after enrichment: From systematic counting to mutational profiling

PurposeThis study aims to establish a highly adaptable workflow downstream of microfluidic enrichment for facilitating systematic CTC enumeration and genetic discovery.MethodsTo facilitate CTC enumeration, we established a CK/EPCAM-combined immunostaining strategy and an automated CTC analytical pipeline using an open-source image analyzer. By virtue of this workflow, we conducted a pilot study of 56 cancer patients and 21 healthy individuals using a high-throughput spiral microfluidic chip system. To facilitate genetic discovery of somatic mutations in CTCs, we integrated the CTC enumeration into next-generation sequencing and established a straightforward amplicon library comprising diversifier random sequences to sequence CTC samples.ResultsThe CTC staining and enumeration workflow achieved 80.4% sensitivity and 85.7% specificity (AUC = 0.87, p = 0.004, power = 0.985), as evaluated by ROC analysis. Univariate and multivariate analysis verified that the CTC (CK/EpCAM+CD45−), but not other cell populations, is a significant and independent biomarker for cancer patients (p TP53 and ESR1.ConclusionsThe significance of this report is to demonstrate a systematic and adaptable workflow to bridge the gap between the microfluidic enrichment and CTC analyses, which fosters broader applications of CTCs in both clinical settings and academic studies.