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

PURPOSE This study aims to establish a highly adaptable workflow downstream of microfluidic enrichment for facilitating systematic CTC enumeration and genetic discovery. METHODS To 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. RESULTS The 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 < 0.01). Serial CTC monitoring of the patients revealed reduction in CTC numbers after treatments, suggesting its clinical utility in pharmacodynamic studies. Deep sequencing of CTC samples revealed somatic mutations in TP53 and ESR1. CONCLUSIONS The 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.