Gut microbiome profiles in locally advanced rectal cancer after total neoadjuvant therapy: comparisons with healthy controls and associations with survival

The gut microbiome plays a key role in colorectal carcinogenesis, but its composition and prognostic relevance in locally advanced rectal cancer (LARC) remain underexplored. This study aimed to assess microbial diversity, identify distinct bacterial signatures in LARC patients compared with healthy controls, and evaluate associations between microbiome composition and clinical outcomes. In this prospective cohort study, baseline stool samples from 62 LARC patients prior to TNT initiation were compared with samples from 30 healthy controls using 16 S rRNA gene sequencing. Alpha diversity (Shannon and Gini–Simpson indices) and beta diversity (Bray–Curtis dissimilarity with PERMANOVA) were assessed. Differential taxa were identified using linear discriminant analysis effect size (LEfSe). Survival analyses were performed within the LARC cohort, including exploratory Kaplan–Meier stratified by optimal cutpoint–defined Proteobacteria abundance. At baseline, LARC patients demonstrated significantly reduced microbial alpha diversity compared with healthy controls (Shannon index, p < 0.0001) and distinct microbial community composition (PERMANOVA, p = 0.001). Phylum-level analysis revealed reduced Bacillota and increased Bacteroidota and Fusobacteriota in LARC patients. In survival analyses, alpha and beta diversity did not differ significantly between Survivor and Non-survivor groups. However, Proteobacteria were significantly enriched in non-survivors ( p = 0.002), along with higher relative abundance of Euryarchaeota and Elusimicrobia. LEfSe analysis identified distinct microbial signatures associated with survival, with enrichment of Faecalibacterium duncaniae and Saccharofermentans acetigenes in survivors and Clostridium oceanicum and Veillonella denticariosi in non-survivors. An optimal cutpoint of 4.5% identified a high-risk group with markedly reduced overall survival on Kaplan–Meier analysis. LARC patients exhibit marked baseline gut microbiome dysbiosis compared with healthy individuals. Although global diversity measures were not predictive of survival, specific taxonomic shifts—particularly enrichment of Proteobacteria—were associated with poorer outcomes. Given the small number of events ( n = 10) and the exploratory, data-derived cutpoint used to define abundance groups, these findings should be regarded as candidate associations requiring validation in larger, independent cohorts rather than as an established prognostic biomarker; they nonetheless point to a possible link between baseline dysbiosis and inflammatory pathways in patients undergoing TNT.