Tumor heterogeneity is a key determinant of progression, metastasis, and therapeutic resistance in lung cancer. Circulating tumor cells (CTCs) provide a minimally invasive means to explore this heterogeneity beyond primary tumor biopsies. In this study, we investigated the phenotypic diversity of circulating cells with tumor-associated features using a cytoblock-based enrichment approach followed by immunocytochemical analysis.

Peripheral blood samples from one hundred newly diagnosed lung cancer patients were enriched and processed to preserve rare circulating cells and stained with a panel of epithelial (pan-cytokeratins, EpCAM), organ-specific (TTF-1), and hematopoietic (CD45) markers. Typical CTCs were defined as nucleated epithelial marker–positive and CD45-negative cells. Circulating hybrid cells (CHCs) were identified based on co-expression of epithelial markers and CD45. In addition, rare nucleated cells lacking immunocytochemical staining but displaying atypical cytomorphology were documented as potential undifferentiated tumor cells, without definitive lineage attribution.

Overall, potential CTCs were morphologically detected in 98% of patients, with a subset exhibiting pronounced phenotypic heterogeneity. Non-epithelial phenotypes, including CHCs and marker-negative atypical cells, were frequently observed, particularly in patients with clinically advanced disease. These findings indicate that circulating tumor-associated cells in lung cancer extend beyond classical epithelial definitions and that epithelial-restricted detection strategies may underestimate this diversity. In conclusion, integrating morphological assessment with multiparametric immunocytochemical profiling allows a more comprehensive characterization of circulating tumor heterogeneity and may enhance the biological relevance of liquid biopsy approaches in lung cancer.