51 Seeing Beyond H&E: Polychromatic PolScope Microscopy Visualizes Distinct Collagen Fiber Signatures in Colorectal Adenocarcinoma

Introduction/Objective While tumor-associated collagen signatures (TACS) have demonstrated diagnostic and prognostic value in breast and pancreatic cancers, their investigation in colorectal disease has been limited. Addressing this gap, we present the first application of a novel microscopic technology, the Polychromatic PolScope Module (PPM), which allows direct assessment of collagen fiber orientation on routine H&E-stained slides using a unique approach to capture birefringence across all fiber angles. This study explores distinct collagen patterns in colorectal adenocarcinoma identified by PPM, focusing on its potential to resolve the persistent diagnostic challenge of differentiating early-stage (pT1) adenocarcinoma from pseudoinvasion in adenomas. Methods/Case Report Colonic tissue samples selected from benign cases (including normal tissue and abnormal benign entities such as diverticula, colonic granulation tissue, and fibrotic colonic mucosa post-injury), adenoma cases with pseudoinvasion, and colorectal adenocarcinoma cases (including pT1, pT2, and pT3 stages) were examined using a PPM attached to an inverted Olympus microscope (BX60F5). Collagen fiber organization, morphology, distribution relative to glandular structures, and interaction with other tissue components were systematically assessed and compared across the groups based on the obtained findings. Regions of interest and recurring collagen signatures were photographed using CellSens software. Results In normal colonic mucosa, the lamina propria exhibited minimal collagen signal and collagen was only faintly visible surrounding individual glands, likely highlighting the basement membrane. Within the submucosa, collagen density varied, but the collagen fibers were predominantly characterized as wavy, short, polychromatic, and directional. Regarding collagen organization and arrangement in benign entities, it was typically organized, often circumferentially wrapping around groups of glands without directly contacting individual glands. Conversely, pT2/T3 colonic adenocarcinomas displayed a distinct collagen signature characterized by collagen fibers traversing malignant cells, perpendicular collagen fibers to malignant glands, collagen directly adjacent glands, collagen encapsulated by malignant glands, haphazard arrangements around the periphery of group of glands and between. Concerning collagen fiber morphology, benign collagen fibers were predominantly thin, wavy, polychromatic, and short. In contrast, a wide range of collagen morphologies was observed in malignant conditions, including straight, monochromatic, thick bundles or needle-shaped fibers with irregular corrugation. In all pseudoinvasion cases, although the collagen fiber architecture was distorted compared to completely normal tissue, the malignant collagen features were not present. Overall, collagen morphology in these cases still resembled that of benign conditions. In contrast, pT1 colonic adenocarcinomas showed variable collagen characteristics, exhibiting some similarity to pT2/T3 adenocarcinomas. Conclusion This project demonstrates that PPM reveals distinct collagen features capable of differentiating between benign colonic tissue and advanced colorectal adenocarcinoma. The absence of these features in pseudoinvasion suggests that collagen remodeling may serve as a critical indicator of true invasion. The variability observed in pT1 adenocarcinoma underscores the potential utility of detailed collagen analysis in this diagnostically challenging category, warranting further investigation to determine if specific collagen patterns can reliably distinguish it from pseudoinvasion.