Consistent Microbial Signatures in the Pseudomyxoma Peritonei Tumor Microbiome Revealed by Cross-Laboratory Replication.

Recent studies suggest that tumor tissues may harbor distinct microbial communities; however,distinguishing true low-biomass microbial signals from environmental and laboratory contaminationremains technically challenging. Here, we establish a rigorous workflow to identify reproducibletumor-associated microbiomes using pseudomyxoma peritonei (PMP), a rare malignancycharacterized by dissemination of mucin-secreting cells throughout the peritoneal cavity. Previousreports identified a core microbiome in PMP tumors but did not adequately address contamination.We analyzed 70 PMP tumor samples, six normal peritoneal control tissues, and comprehensivelaboratory controls. In addition, DNA extraction and sequencing were independently replicated in aseparate laboratory for a subset of 25 samples. Using stringent quality-control criteria andcross-laboratory replication, we identified reproducible microbial signatures in six PMP tumors,including patient-specific taxa previously associated with colorectal cancer, as well as a limited set ofcore taxa likely attributable to surgical contamination. Notably, preoperative chemotherapy exposurewas associated with reduced tumor microbiome diversity.These findings demonstrate that independent laboratory replication is an effective strategy fordistinguishing biological signal from contamination in low-biomass tumor microbiome studies. Thisapproach provides a robust framework for improving confidence in tumor-associated microbialsignatures and advancing research in low-microbial-biomass systems