Intratumoral microbial networks as biomarkers for second primary oral cancer risk in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) survivors remain at elevated risk of developing second primary oral cancer (SPOC), yet the role of intratumoral microbiomes in SPOC emergence is not fully understood. We performed 16 S rRNA V3-V4 sequencing on tumor brushings from 28 ESCC patients (20 SPOC-negative, 8 SPOC-positive) to profile microbial diversity, taxonomic composition, functional potential, and interaction networks. Alpha diversity metrics (Chao1, Shannon) did not differ significantly between groups (p > 0.05), whereas sparse partial least squares-discriminant analysis of beta diversity robustly separated SPOC-positive from SPOC-negative tumors (p < 0.001), identifying 32 discriminant amplicon sequence variants (ASVs) linked to 41 differential KEGG pathways. Intratumoral Spearman correlation networks (|r| > 0.3, p < 0.05) between the ten most abundant genera and these pathways revealed two distinct modules: a SPOC-associated network centered on Prevotella pallens and P. scopos, enriched in carbohydrate metabolism, PI3K-Akt signaling, and glycosaminoglycan degradation; and a non-SPOC network anchored by Alcaligenaceae, Cyanobiaceae, Rhodobacteraceae, and Prevotella oris, associated with macrolide biosynthesis and aminobenzoate degradation. These findings demonstrate that specific intratumoral microbial interaction networks distinguish ESCC patients who develop SPOC, and highlight network-based microbial signatures as promising biomarkers for SPOC risk stratification.