Entosis in Colorectal, Lung, and Breast Cancer: Associations with Clinicopathological Features, Patient Outcomes, and Copy Number Alteration Landscape

Objective: This study examined the frequency of entosis in solid tumors of various origins (colorectal cancer, breast cancer, and lung cancer) and its association with clinical and pathological characteristics. It also examined survival and copy number alterations (CNAs) in genes associated with stem cells. The aim was to assess the potential prognostic value of entotic events in tumors. Methods: A total of 238 patients were included: 96 with colorectal cancer (CRC), 45 with lung cancer (LC), and 97 with breast cancer (BC). Entotic cell-in-cell (CIC) structures were evaluated on hematoxylin–eosin–stained slides using Mackay’s criteria. A CIC frequency >0.1 per 20 high-power fields was considered positive. Clinicopathological parameters, overall survival (CRC), metastasis-free survival (LC and BC), and CNA profiles of stemness-related genes were analyzed. Amplifications of MAP1LC3A and other chromosomal loci were assessed. Results: CRC demonstrated the highest entosis rate, more than two-fold higher compared with BC and LC (p < 0.05). Entosis correlated with high tumor grade (G3) in CRC (p = 0.03). In LC, CIC-positive tumors were more frequent in patients with lymph-node metastases (p = 0.02), whereas in BC, the opposite trend was observed (p = 0.02). It was noted that in patients with stage III–IV LC, the frequency of entosis was significantly higher than in patients with stage I–II cancer (p = 0.03). CIC-positive status was associated with poorer overall survival in CRC (p = 0.03) and reduced metastasis-free survival in LC (p = 0.011). In breast cancer, no statistically significant survival differences were observed. Tumors harboring two or more stemness-gene amplifications showed significantly higher entosis frequency regardless of tumor site. A strong association was identified between entosis and MAP1LC3A amplification. Conclusions: Enosis is not a random morphological phenomenon but a process associated with unfavorable tumor characteristics, high malignancy, reduced survival, and amplification of stem cell-related genes. The results of this study confirm the working hypothesis that entosis may contribute to the emergence of aneuploid clones of tumor cells, including those containing amplifications of stem cell-associated genes. This positions entosis as a potential factor in tumor genetic heterogeneity, which is particularly important in the context of therapeutic selection pressure. The observed association between high entosis frequency and the presence of ≥2 stem cell gene amplifications, as well as its association with poor prognosis in colorectal and lung cancer, highlights its potential value as a prognostic indicator. Furthermore, MAP1LC3A amplification data may serve as a molecular marker of entotic activity and a potential therapeutic target.