Multiparametric Phenotyping of Circulating Tumor Cells for Analysis of Therapeutic Targets, Oncogenic Signaling Pathways and DNA Repair Markers

SIMPLE SUMMARY: Detection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. In order to expand the clinical utility of this blood–based minimally invasive biomarker we established a protocol allowing multiparametric phenotyping of CTCs to analyze the expression levels of therapeutic target proteins. By applying this assay, we demonstrated intratumoral heterogeneity of PD–L1 expression in CTCs from head and neck cancer patients, an observation previously reported in tumor tissue specimens. We further verified the feasibility of applying the protocol to analyze the activation status of important oncogenic pathways and the extent of DNA repair following radiation. These promising preliminary results warrant further study and may lead to the implementation of this assay in clinical routine for improved treatment selection and monitoring. ABSTRACT: Detection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. Multiparametric phenotyping of CTCs could expand the area of application for this liquid biomarker. We evaluated the Amnis(®) brand ImageStream(®)X MkII (ISX) (Luminex, Austin, TX, USA) imaging flow cytometer for its suitability for protein expression analysis and monitoring of treatment effects in CTCs. This was carried out using blood samples from patients with head and neck squamous cell carcinoma (n = 16) and breast cancer (n = 8). A protocol for negative enrichment and staining of CTCs was established, allowing quantitative analysis of the therapeutic targets PD–L1 and phosphorylated EGFR (phospho–EGFR), and the treatment response marker γH2AX as an indicator of radiation–induced DNA damage. Spiking experiments revealed a sensitivity of 73% and a specificity of 100% at a cut–off value of ≥3 CTCs, and thus confirmed the suitability of the ISX-based protocol to detect phospho–EGFR and γH2AX foci in CTCs. Analysis of PD–L1/–L2 in both spiked and patient blood samples further showed that assessment of heterogeneity in protein expression within the CTC population was possible. Further validation of the diagnostic potential of this ISX protocol for multiparametric CTC analysis in larger clinical cohorts is warranted.