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Life sciences · Journal article
Frontiers in Cellular and Infection Microbiology · October 28, 2026
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Background Early primary resistance to anti-PD-1 therapy is common in advanced non-small cell lung cancer (NSCLC). Existing microbiome biomarkers largely rely on individual species and lack stability across populations. Objective The aim of this study was to construct the classical probiotic–butyrate axis score (PBAS), perform external validation, and evaluate its value for risk stratification of 90-day early primary resistance and PFS. Methods This study adopted a dual-cohort, patient-level secondary analysis design and reanalyzed pretreatment fecal shotgun metagenomic data from two public cohorts, including 70 and 40 patients in the discovery and external validation cohorts, respectively. An equally weighted PBAS was constructed based on predefined classical probiotic-associated taxa and the butyryl-CoA:acetate CoA-transferase ( but ) and the butyrate kinase ( buk ) genes. ANCOM-BC2 was used for differential analysis, Firth logistic regression for association analysis of early primary resistance, 2,000 patient-level bootstrap resampling for internal validation, and Cox proportional hazards models for PFS analysis. Results Early primary resistance occurred in 41/70 and 23/40 patients in the two cohorts, respectively. Classical probiotic-associated taxa, the butyrate functional subscore, and PBAS were lower in the resistant group than in the nonresistant group, whereas buk and Akkermansia muciniphila showed no significant differences. After adjustment for age, sex, and BMI, acetyl-CoA pathway potential was also reduced. For each 1-SD increase in PBAS, the OR for early primary resistance was 0.39 and 0.37 after adjustment for clinical variables. The bootstrap-corrected area under the receiver operating characteristic curve (AUC) was 0.733, and the external validation AUC was 0.726 (95% CI: 0.559–0.894). Increased PBAS was associated with a lower risk of progression or death, with HRs of 0.54 and 0.61 in the two cohorts, respectively (both p < 0.001). Conclusion Combined reductions in pretreatment classical probiotic-associated ecology and butyrate biosynthetic potential indicate a high risk of early primary resistance to anti-PD-1 therapy in advanced NSCLC and support PBAS as an exploratory outcome risk marker for cross-cohort risk stratification among patients receiving anti-PD-1 therapy, without establishing treatment-specific predictive value.