Assessment of 16S rRNA sequencing for analysis of circulating microbial DNA in colorectal cancer patients–proof of concept and early changes during experimental chemoimmunotherapy
Adriana M. Sanabria, Tonje Bjørnetrø, Anniken J. Fuglestad, Sebastian Meltzer, Anne Helene Køstner, Torben Lüders, Christian Kersten, Anne Hansen Ree
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Published: Jun 8, 2026
DOI: 10.3389/fmicb.2026.1802448
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Background Circulating microbial DNA (cmDNA) is an emerging biomarker in cancer, yet its analytical and clinical utility remains to be validated. Methods This study establishes a proof-of-concept for cmDNA sequencing and analysis using 16S rRNA amplicons in colorectal cancer (CRC) patient samples. Two cohorts were analyzed – the Test cohort ( n = 11) comparing plasma and serum from early-stage colon cancer patients to determine the optimal sample type, and the METIMMOX cohort ( n = 11) of patients with newly diagnosed metastatic CRC to explore the initial dynamics of cmDNA alterations during experimental therapy. The METIMMOX trial investigated alternating oxaliplatin-based chemotherapy and nivolumab in metastatic microsatellite-stable CRC, the major CRC entity essentially considered unresponsive to immune checkpoint inhibitors. For the metastatic CRC patients, plasma was used for cmDNA analysis at baseline and following the initial chemoimmunotherapy. Results Both plasma and serum were suitable for cmDNA profiling; however, plasma was preferred due to higher numbers of bacterial reads and lower proportion of unassigned reads. Compared to METIMMOX patients with long-lasting treatment response, exhibiting a stable initial cmDNA composition, the patients unresponsive to chemoimmunotherapy showed an increase in alpha diversity (observed amplicon sequence variants: p = 0.014, Chao1 species richness indices: p = 0.010, Shannon community evenness indices: p = 0.004) over the short period of time until treatment failure. Likewise, beta diversity estimation (by Bray-Curtis dissimilarity indices) indicated that the early treatment course influenced the cmDNA composition differently in patients with and without response. Conclusion This study demonstrates the feasibility of cmDNA sequencing in CRC patients and highlights its potential to uncover treatment-related microbial shifts that may serve as non-invasive biomarkers of therapeutic response or resistance.
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