↓ Figure 1. Digital tile-based quantification of SOX2 spatial heterogeneity in early esophageal adenocarcinoma. (a) Representative SOX2-immunostained carcinoma field demonstrating the tile grid overlay used for digital segmentation. Each tile corresponds to a fixed-size analytical unit from which pixel-level optical density measurements were extracted. (b) Heatmap of tile mean SOX2 intensity values, illustrating marked regional variability in nuclear expression across the tumor field. High-intensity tiles (red–orange) correspond to clusters of SOX2-overexpressing glands, whereas low-intensity tiles (light yellow) represent areas with reduced transcriptional activity. (c) Histogram of tile intensities with calculated standard deviation (SD) and Gini coefficient. SD reflects overall variability, while the Gini index quantifies inequality of SOX2 distribution, capturing clonal and spatial heterogeneity within the neoplastic epithelium. These metrics formed the basis of the SOX2_Tile_SD and SOX2_Tile_Gini variables used in downstream analyses. SOX2: SRY-box transcription factor 2.
↓ Figure 2. Aberrant SOX2 expression across Barrett’s dysplasia and early adenocarcinoma. (a) High-grade Barrett’s dysplasia exhibiting strong, aberrant nuclear SOX2 expression within dysplastic glandular structures, representing a high-risk molecular phenotype (IHC, × 290). (b) Another case of high-grade Barrett’s dysplasia showing intense nuclear SOX2 positivity diffusely involving crowded and architecturally complex glands, consistent with marked lineage deregulation (IHC, × 300). (c) Low-grade Barrett’s dysplasia with unexpectedly high SOX2 expression in glandular epithelium, indicating early activation of a high-risk transcriptional pathway despite only low-grade morphologic atypia (IHC, × 390). (d) Intramucosal adenocarcinoma displaying diffuse nuclear SOX2 overexpression in infiltrative malignant glands, consistent with advanced lineage reprogramming and a phenotype associated with progression (IHC, × 460). IHC: immunohistochemistry; SOX2: SRY-box transcription factor 2.
↓ Figure 3. Variable CD44 immunohistochemical expression in early intramucosal esophageal adenocarcinoma. (a) Carcinoma demonstrating partial membranous CD44 expression, with patchy staining in a subset of infiltrative malignant glands, indicating heterogeneous retention of a stem-cell–associated phenotype (IHC, × 270). (b) Carcinoma showing complete absence of CD44 immunoreactivity, with entirely negative malignant glands despite preserved internal controls, reflecting loss of CD44-mediated adhesion pathways (IHC, × 300). (c) Carcinoma with moderate membranous CD44 staining, involving a wider population of atypical glands and micropapillary clusters, suggesting expansion of CD44-positive tumor subclones (IHC, × 440). CD44: cluster of differentiation 44; IHC: immunohistochemistry.
