Association of an ectopic upper gastrointestinal microbiome with multiple primary lung cancers: A multi-site cross-sectional study

Background: The etiology of multiple primary lung cancers (MPLC), defined by their independent multifocal origins, remains largely unknown, particularly the contribution of the microbiome. Gastroesophageal reflux disease (GERD) is an established risk factor for lung cancer, but its specific association with MPLC and the potential for GERD-mediated translocation of upper gastrointestinal (UGI) microbiota into pulmonary nodules (PNs) has not been investigated. We hypothesized that GERD facilitates UGI-to-lung microbial translocation, contributing to a pro-carcinogenic microenvironment in MPLC. Materials and Methods: In this prospective case-control study (NCT06973499), GERD symptom burden was assessed in 149 MPLC, 41 solitary primary lung cancer (SPLC), and 119 healthy controls using the validated GERD-Q questionnaire. For a subset of 21 MPLC patients, we performed comprehensive multi-site microbiome profiling using PacBio full-length 16S rRNA gene sequencing on samples from seven anatomical compartments, including the UGI tract and multiple resected PNs. Rigorous contamination controls were employed. Microbial source tracking (SourceTracker2) was used to quantify UGI bacterial translocation, with strain-level identity confirmed via phylogenetic analysis. Results: MPLC patients exhibited a significantly higher prevalence of high-risk GERD symptoms (GERD-Q score >8) compared to healthy controls (26.2% vs. 16.0%; OR = 1.87, 95% CI 1.01-3.44, P=0.048); this association was not observed in SPLC patients (P=0.87). Crucially, microbial source tracking identified a significant, ectopic UGI-derived bacterial signature in 22.9% of PNs (8 of 35 nodules) from MPLC patients. Linking the symptom to the microbe, a post-hoc analysis revealed a strong trend where MPLC patients with a high GERD burden showed a greater proportional contribution of UGI microbiota to their PNs compared to those with a low GERD burden (P = 0.0692). Finally, high-resolution phylogenetic analysis confirmed shared, identical strains of Prevotella melaninogenica and Gemella haemolysans across UGI and pulmonary sites within the same individuals, providing definitive evidence of active translocation. Conclusion: This study provides the first direct, multi-site evidence linking GERD symptom burden to the active translocation of UGI microbiota into pulmonary nodules in MPLC patients. We introduce the 'GERD-microbial translocation-multifocal carcinogenesis' axis as a novel pathogenic pathway that may contribute to the etiology and heterogeneity of MPLC. Our findings identify the UGI-lung axis as a potential target for novel diagnostic and therapeutic strategies, suggesting that managing GERD or targeting the translocated pathobiome could represent a new paradigm for MPLC prevention or interception.