Comprehensive study of the intratumoral microbiome in early- vs. late-onset colorectal cancer: Final analysis of COSMO CRC.
Authors: Weinberg Benjamin Adam , Wang Hongkun , Geng Xue , Suter Robert K. , Sortur Shrayus , Green Myra E. , Shokralla Shadi , Bakhshi Emily , Chaldekas Krysta , Harris Brent T. , Clagett Dionyssia , Marshall John 
Published: June 2023

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Background: Although colorectal cancer (CRC) incidence has declined overall, CRC in individuals under age 45 (early-onset CRC, EOCRC) has risen dramatically. Obesity and diabetes may partially explain this epidemiologic shift; however, many patients (pts) with EOCRC are neither obese nor diabetic. Certain bacteria disrupt colonic luminal integrity and promote inflammation, leading to oncogenic mutations in colonic epithelial cells. Fusobacterium nucleatum ( F. nuc) promotes CRC by suppressing immune response within the tumor microenvironment, activating the β-catenin pathway, and causing chemoresistance due to autophagy. The intratumoral microbiome (MB) in pts with EOCRC may differ from pts with late-onset CRC (LOCRC). Methods: We compared the intratumoral MB in pts with CRC diagnosed before age 45 (EOCRC) and after age 65 (LOCRC) in a prospective/retrospective study between 2017 and 2022. Primary and metastatic tumors were included. DNA was extracted from tumors and analyzed using 16S ribosomal gene sequencing. We compared the frequency of F. nuc and other bacterial and fungal DNA in tumors EOCRC vs. LOCRC pts. Next-generation tumor sequencing and diet questionnaire data were available for some pts. Results: Tumors from 36 EOCRC pts (median age 38 years) and 27 LOCRC pts (median age 72 years) were analyzed. In total, 917 unique bacterial and fungal species were detected. F. nuc was found in 30.6% of EOCRC and 29.6% of LOCRC (p = 0.94). Cladosporium sp. was seen more commonly in EOCRC (30.6% vs. 11%, p = 0.04), whereas Pseudomonas luteola (2.8% vs. 22.2%), Ralstonia sp. (22.2% vs. 48.1%), and Moraxella osloensis (19.4% vs. 44.4%) were seen more commonly in LOCRC (p < 0.05). Clostridium perfringens (11.1%), Escherichia coli (11.1%), Leptotrichia hofstadii (11.1%) , Mycosphaerella sp. (11.1%), Neodevriesia modesta (11.1%), Penicillium sp. (11.1%), and Leptosphaeria sp. (11.1%) were seen exclusively in LOCRC (p < 0.05). There was no significant difference in median MB diversity in EOCRC vs. LOCRC (43 vs. 45 organisms per pt). Median follow up from time of diagnosis was 39.6 months (mo). Twenty-three EOCRC pts (64%) and 19 LOCRC pts (70%) are alive. Median overall survival (mOS) was 75.5 mo (95 CI: 40 - NA) in EOCRC and 60 mo (95% CI: 50.5 - NA) in LOCRC. There was no significant difference in OS in LOCRC vs. EOCRC (Log-rank test p = 0.85). Pts with F. nuc positive tumors (N = 17) had a mOS of 75.5 mo (95 CI: 23 – NA), whereas pts with F. nuc negative tumors (N = 46) had a mOS of 60 mo (95% CI: 50.5 – NA). There was no significant difference in OS in F. nuc positive vs. F. nuc negative groups (Log-rank test p = 0.87). Conclusions: There are significant differences in the intratumoral microbiome in EOCRC and LOCRC. These findings warrant larger, prospective studies to elucidate the role the intratumoral microbiome plays in the carcinogenesis, the tumor immune microenvironment, and responsiveness to specific therapies.