PCR profiling of tumor-associated microorganisms in tissue of primary epithelial malignant tumors of colorectal origin: associations with key clinicopathological characteristics.

INTRODUCTION: Colorectal cancer is one of the most common malignancies worldwide, and microbiome research has strong potential to advance understanding of tumor biology and to support biomarker development and microbiome-targeted interventions. Most colorectal cancer microbiome studies rely on stool or mucosal sampling, but routine pathology archives contain abundant formalin-fixed, paraffin-embedded primary tumor tissue that could enable scalable assessment of tumor-associated microorganisms.

METHODS: We profiled tumor-associated microorganisms in primary colorectal adenocarcinoma specimens from 192 patients using a targeted quantitative PCR panel and evaluated associations with clinicopathological variables (tumor differentiation grade, primary tumor T category, and disease stage) using non-parametric tests, ordinal regression, regularized logistic regression, and within-panel profile ordination with permutation-based inference.

RESULTS: The most consistent signals were linked to tumor differentiation rather than stage. Tumors in the G2-G3 versus G1 comparison showed higher total bacterial load and a higher detection frequency of Ruminococcus spp., with both associations remaining significant after false discovery rate control; regression analyses corroborated these findings. A regularized logistic model achieved moderate discrimination for high-grade (G2-G3) disease (mean area under the receiver operating characteristic curve ≈0.71) and yielded a compact signature dominated by total bacterial load and detection of Ruminococcus spp., with additional contributions from low-frequency taxa that warrant cautious interpretation. In contrast, models targeting primary tumor T category, invasiveness (T1-T2 vs T3-T4), or overall stage showed low discriminative performance (area under the curve ≤0.59), and within-panel profile distances did not reveal robust global separation of groups.

CONCLUSIONS: Targeted quantitative PCR profiling of archived primary tumor tissue identifies reproducible microbiome signals that track tumor differentiation grade more strongly than stage, suggesting that tissue bacterial burden and selected taxa may reflect microenvironmental features associated with the G2-G3 versus G1 contrast. Broader tumor microbiome profiling should be required to capture diversity and refine clinically informative signatures.