Cell-in-Cell Structures in Colorectal Cancer: A Proposed Assessment Method and Correlation with Established Poor Prognostic Factors

<b>Background</b>: Cell-in-cell (CIC) structure is a histological picture of a whole cell inside another cell. Homotypic CIC structures formed by cancer cells are consistently demonstrated to be a factor of poor prognosis and resistance to chemo- and immunotherapy in colorectal cancer (CRC). However, the absence of a standardized counting method limits the use of this factor in the applied research. <b>Objective</b>: To propose an adapted method for quantifying CIC structures in CRC surgical specimens and to evaluate their correlation with established adverse prognostic factors. <b>Methods</b>: A total of 250 histological slides of surgical specimens from 58 patients with pT1-pT4 colorectal adenocarcinoma were studied. Identification of tumor cells and visualization of CIC structures were performed by immunohistochemistry (CK20). Quantitative assessment was performed on digital scans of H&E stained slides. Quantitative assessment was performed on digital slide scans stained with H&E. CIC structures were counted in 5 fields of view corresponding to a ×40 objective (0.975 mm²). A correlation analysis of CIC structures with CRC poor prognosis factors was performed. <b>Results</b>: Immunohistochemical study (CK20) confirmed the formation and prevalence of homotypic structures (95%) over heterotypic ones (5%) (<i>p</i> < 0.001). This finding informed the evaluation of H&E-stained slides and the formulation of criteria for CIC structure identification. A significant predominance of CIC structures in the invasive front was established compared to the tumor central zone (16.7 ± 5.2 and 1.2 ± 1.3 per 5 fields of view, respectively, <i>p</i> < 0.0001). Correlation analysis revealed weak but statistically significant relationships with the tumor-stromal ratio, the tumor buds number and the density of tumor-infiltrating lymphocytes. No correlations were found with the right- or left-sided location, pTNM, grading, lymphovascular and perineural invasion. <b>Conclusions</b>: The paper presents the adapted CIC structures counting method for surgical specimens of CRC, defines the criteria of the CIC, and demonstrates a higher number of CIC structures in the tumor invasive front. Weak correlations between the CIC structures and established factors of CRC poor prognosis are obtained.