High-Grade Atypia in Circulating Tumour-Related Endothelial-Like Cells Serves as a Specific Marker for Malignant...

Conventional liquid biopsy primarily focuses on epithelial circulating tumour cells (CTCs). We previously demonstrated that a population of circulating cells in thoracic tumours express the vascular endothelial marker ERG. In this study, we defined these ERG-positive cells with endothelial morphology in liquid-based cytology (LBC) as "circulating tumor-related endothelial-like cells" (CELCs). Our aim was to establish a morphological grading system to distinguish malignancy-specific High-Grade CELC from benign or reactive CELC populations.

Preoperative arterial blood samples from 28 patients (25 malignant and 3 benign cases) were processed using a soft-microporous filtration system (S-MPF). Captured cells were evaluated by Papanicolaou staining. CELCs were categorized based on LBC morphology into "Low-Grade" (single cells or clusters < 15 cells) and "High-Grade" (three-dimensional morula-like clusters ≥ 15 cells with marked nuclear pleomorphism).

CELCs were identified in 13 cases (46.4%). While Low-Grade CELCs were observed in both cohorts (12 malignant, 1 benign), High-Grade clusters-representing definitive CTEC signatures-were exclusively identified in 8 malignant cases (5 primary lung cancers, 2 metastatic tumours, and 1 thymic cancer). No High-Grade features were detected in benign lesions, yielding a specificity of 100% (95% CI: 29.2%-100%) for malignancy.

Implementing an LBC-based morphological threshold of ≥ 15 cells allows malignancy-specific CELCs to be accurately distinguished from the broader population of CELCs. This grading system provides a practical and highly specific liquid biopsy framework, ensuring precise identification of tumour-associated endothelial clusters even amidst reactive non-malignant cells.

UMIN Clinical Trials Registry (UMIN-CTR, identifier: UMIN000058385).