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

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 Arnnis (R)) brand ImageStream (R) 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 gamma H2AX 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 gamma H2AX 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.

Automated summary

Detection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. This study evaluated the suitability of the Arnnis brand ImageStream X MkII (ISX) imaging flow cytometer for protein expression analysis and treatment monitoring in CTCs. The results showed that the ISX-based protocol was sensitive and specific in detecting phospho-EGFR and gamma H2AX foci in CTCs, and allowed assessment of protein expression heterogeneity within the CTC population.