Reply to: Caution Regarding the Specificities of Pan-Cancer Microbial Structure

The cancer microbiome field tremendously accelerated following the release of our manuscript nearly three years ago, including direct validation of our cancer type-specific conclusions in independent, international cohorts and the tumor microbiome's adoption into the hallmarks of cancer. Disentangling contamination signals from biological signals is an important consideration for this research field. Therefore, despite numerous, high-impact, peer-reviewed research papers that either validated our conclusions or extended them using data we released, we carefully considered criticism raised by Gihawi et al . about potential mishandling of contaminants, batch effects, and machine learning approaches—all of which were central topics in our manuscript. Nonetheless, a close examination of each concern alongside the original manuscript and re-analyses of our published data strongly demonstrates the robustness of the original findings. To remove all doubt, however, we have reproduced all key conclusions from the original manuscript using only overlapping bacterial genera identified in a highly decontaminated, multi-cancer, international cohort (Weizmann Institute of Science, WIS), with or without batch correction, and with multiclass machine learning analyses to mitigate class imbalances. Our published pan-cancer mycobiome manuscript also affirms these findings using updated, state-of-the-art methods. We also note that every analysis shown here was possible using public data and code that we had already provided. ### Competing Interest Statement Clarity Genomics, the employer of E.K., did not provide funding for this study. G.D.S.-P. and R.K. are inventors on a US patent application (PCT/US2019/059647) submitted by The Regents of the University of California and licensed by Micronoma; that application covers methods of diagnosing and treating cancer using multi-domain microbial biomarkers in blood and cancer tissues. G.D.S.-P. and R.K. are founders of and report stock interest in Micronoma. G.D.S.-P. has filed several additional US patent applications on cancer bacteriome and mycobiome diagnostics that are owned by The Regents of the University of California or Micronoma. R.K. additionally is a member of the scientific advisory board for GenCirq, holds an equity interest in GenCirq, and can receive reimbursements for expenses up to US $5,000 per year.