Microbiome confounders and quantitative profiling challenge predicted microbial targets in colorectal cancer development

Despite substantial progress in cancer microbiome research, recognizedconfounders and advances in absolute microbiome quantification remain underused;this raises concerns regarding potential spurious associations. Here we study thefecal microbiota of 589 patients at different colorectal cancer (CRC) stages andcompare observations with up to 15 published studies (4,439 patients and controlstotal). Using quantitative microbiome profiling based on 16S ribosomal RNA ampliconsequencing, combined with rigorous confounder control, we identified transit time,fecal calprotectin (intestinal inflammation) and body mass index as primarymicrobial covariates, superseding variance explained by CRC diagnostic groups.Well-established microbiome CRC targets, such as Fusobacterium nucleatum, did not significantly associate with CRCdiagnostic groups (healthy, adenoma and carcinoma) when controlling for thesecovariates. In contrast, the associations of Anaerococcusvaginalis, Dialister pneumosintes,Parvimonas micra, Peptostreptococcus anaerobius, Porphyromonasasaccharolytica and Prevotellaintermedia remained robust, highlighting their future targetpotential. Finally, control individuals (age 22–80 years, mean57.7 years, standard deviation 11.3) meeting criteria for colonoscopy (forexample, through a positive fecal immunochemical test) but without colonic lesionsare enriched for the dysbiotic Bacteroides2 enterotype, emphasizing uncertainties indefining healthy controls in cancer microbiome research. Together, these resultsindicate the importance of quantitative microbiome profiling and covariate controlfor biomarker identification in CRC microbiome studies.