Tumour tissue-associated microbiome differences between colonic adenoma and carcinoma revealed by 5R 16S rRNA sequencing of formalin-fixed paraffin-embedded tissues: a case-control study.

Tumour-associated microbiota directly colonise lesion sites and interact with the local immune microenvironment, yet their compositional shifts during the adenoma-to-cancer transition remain poorly characterised, particularly in archival tissue. Using five-region 16S ribosomal RNA (5R 16S rRNA) sequencing with a four-category contamination control framework, we compared tumour-associated microbiomes in formalin-fixed paraffin-embedded (FFPE) tissues from 25 colonic tubular adenoma and 25 colon cancer patients in a case-control design. Alpha diversity was significantly reduced in the colon cancer group across three indices (Shannon, Simpson, and Pielou's evenness), reflecting shifts in community evenness rather than overall species loss. Beta diversity analysis, however, revealed no significant differences in global community composition between groups (ANOSIM: R = 0.026, p = 0.106; PERMANOVA: R 2 = 0.024, p = 0.137), indicating that observed compositional differences are taxon-specific rather than community-wide. Across four independent statistical methods (ANCOM-BC2, LEfSe, Mann-Whitney U test, and Fisher's exact test), Bacteroides caccae and Prevotella intermedia were consistently enriched in colon cancer tissue, suggesting compositional shifts in the tumour-associated microbiome that may be associated with colorectal carcinogenesis. Within the colon cancer group, Alistipes finegoldii abundance showed a negative correlation with tumour diameter across all colon cancer samples (Spearman r = -0.520, p = 0.008; n = 25, including 20 samples at the detection limit as tied ranks; only 5 samples had detectable abundance); given the very limited number of detectable samples, this finding should be regarded as strictly exploratory and requires prospective validation. These findings demonstrate that tumour-associated microbiome profiling in archival FFPE tissue is feasible under a rigorous contamination control framework, and provide preliminary evidence for taxon-specific compositional differences during colonic adenoma-to-carcinoma progression. The identified candidate taxa warrant prospective validation in larger cohorts and mechanistic investigation to clarify their roles in colon carcinogenesis.