Despite substantial progress in cancer microbiome research, recognized confounders and advances in absolute microbiome quantification remain underused; this raises concerns regarding potential spurious associations. Here we study the fecal microbiota of 589 patients at different colorectal cancer (CRC) stages and compare observations with up to 15 published studies (4,439 patients and controls total). Using quantitative microbiome profiling based on 16S ribosomal RNA amplicon sequencing, combined with rigorous confounder control, we identified transit time, fecal calprotectin (intestinal inflammation) and body mass index as primary microbial covariates, superseding variance explained by CRC diagnostic groups. Well-established microbiome CRC targets, such as Fusobacterium nucleatum, did not significantly associate with CRC diagnostic groups (healthy, adenoma and carcinoma) when controlling for these covariates. In contrast, the associations of Anaerococcus vaginalis, Dialister pneumosintes, Parvimonas micra, Peptostreptococcus anaerobius, Porphyromonas asaccharolytica and Prevotella intermedia remained robust, highlighting their future target potential. Finally, control individuals (age 22–80 years, mean 57.7 years, standard deviation 11.3) meeting criteria for colonoscopy (for example, through a positive fecal immunochemical test) but without colonic lesions are enriched for the dysbiotic Bacteroides2 enterotype, emphasizing uncertainties in defining healthy controls in cancer microbiome research. Together, these results indicate the importance of quantitative microbiome profiling and covariate control for biomarker identification in CRC microbiome studies.

尽管癌症微生物组研究取得了实质性进展，但公认的混杂因素和绝对微生物组定量方面的进展仍未得到充分利用；这引起了人们对潜在虚假关联的担忧。在这里，我们研究了 589 名处于不同结直肠癌 (CRC) 阶段的患者的粪便微生物群，并将观察结果与多达 15 项已发表的研究（总共 4,439 名患者和对照）进行了比较。使用基于 16S 核糖体 RNA 扩增子测序的定量微生物组分析，结合严格的混杂因素控制，我们确定了转运时间、粪便钙卫蛋白（肠道炎症）和体重指数作为主要微生物协变量，取代了 CRC 诊断组解释的方差。在控制这些协变量时，成熟的微生物组 CRC 目标（例如具核梭杆菌）与 CRC 诊断组（健康、腺瘤和癌）没有显着相关性。相比之下，阴道厌氧球菌、肺炎球菌、微单胞菌、厌氧消化链球菌、解糖卟啉单胞菌和中间普雷沃氏菌的关联性仍然强劲，凸显了它们未来的目标潜力。最后，符合结肠镜检查标准（例如，通过粪便免疫化学检测呈阳性）但没有结肠病变的对照个体（年龄 22-80 岁，平均 57.7 岁，标准差 11.3）富含菌群失调 Bacteroides2 肠型，强调定义的不确定性癌症微生物组研究中的健康控制。总之，这些结果表明定量微生物组分析和协变量控制对于 CRC 微生物组研究中生物标志物识别的重要性。