A distinct immune–microbiome axis in early-onset colorectal cancer.

e15736 Background: The incidence of early-onset colorectal cancer (EOCRC; ≤50 years) is rising globally and is increasingly recognised as biologically distinct from late-onset colorectal cancer (LOCRC). Differences in tumour immune contexture and gene-expression programs have been described, and emerging data suggest a role for tumour-associated bacteria and bacterial genotoxins, including colibactin-producing (pks+) E. coli , in EOCRC. However, integrated analyses linking immune phenotype, transcriptomics, and tumour-resident microbiome, including bacterial toxin signatures, remain limited. Methods: Pre-treatment colorectal tumour samples from EOCRC patients (n = 19) were compared with LOCRC controls aged > 65 years (n = 196). RNA sequencing was used to assess differential host gene expression, gene-set enrichment, and consensus molecular subtypes (CMS). Tumour-resident microbiomes were profiled using meta-transcriptomic analysis of unmapped RNA-seq reads, including targeted assessment of bacterial genotoxin signatures. Microbial diversity, taxonomic abundance, and expression of E. coli –associated toxins, including pks⁺ and cytotoxic necrotising factor 1 ( CNF1 ), were compared between age groups. Results: EOCRC tumours showed a distinct transcriptomic profile characterised by enrichment of cell-cycle and proliferative gene sets and depletion of immune-related pathways. When CMS subtypes were grouped by immune phenotype, 94% of EOCRC tumours were immune-excluded (CMS2/3) compared to LOCRC tumours (p = 0.04), with markedly reduced representation of immune-rich CMS1/4 subtypes. Genes associated with chromosomal instability and WNT signalling, including HOXA11-AS , STMN2 , and DACH2 , were overexpressed in EOCRC, while immune-associated genes were enriched in LOCRC. Tumour-resident microbiome analysis revealed significantly reduced microbial species richness in EOCRC tumours (p < 0.01), with distinct enrichment of specific bacterial taxa despite similar overall beta diversity. Expression of E. coli –derived genotoxins was significantly associated with younger age, including colibactin (pks⁺), previously reported in EOCRC, and CNF1 , representing a novel association. Several bacterial taxa enriched in LOCRC tumours have been previously linked to immunogenic host responses. Conclusions: EOCRC is characterised by an immune-excluded tumour microenvironment, proliferative transcriptional programs, and a distinct tumour-resident microbiome, including enrichment of bacterial genotoxin signatures. Integration of known EOCRC-associated colibactin signals with the newly identified association of cnf1 suggests that microbial genotoxicity may contribute to age-related differences in tumour biology. These findings support EOCRC as a biologically distinct disease entity and highlight tumour–microbiome interactions as potential targets for risk stratification and prevention.