Salivary Microbial Signature Highlighting Actinomyces as a Predictor of Immune-Checkpoint Inhibitor Response in Advanced Non–Small Cell Lung Cancer.

Background Immune checkpoint inhibitors (ICIs) have improved survival in advanced non-small cell lung cancer (NSCLC), yet reliable biomarkers beyond programmed death-ligand 1 (PD-L1) expression remain limited. Increasing evidence links the gut microbiome to ICI activity, but the predictive value of the oral microbiome is poorly defined. Methods We prospectively analyzed baseline saliva from 71 stage IV NSCLC patients treated with anti-PD-1/PD-L1 monotherapy. After quality control, 70 samples underwent 16S rRNA gene sequencing of the V1–V3 region. Microbial diversity, differential abundance (LEfSe, Mann-Whitney/Kruskal-Wallis with false-discovery-rate correction) and survival associations (Kaplan-Meier; Cox proportional-hazards with LASSO-based variable selection and 1000-fold bootstrap validation) were examined. An optimal genus-level cut-off was defined by receiver-operating-characteristic analysis. Results α-diversity and β-diversity did not differ between responders (progression-free survival (PFS) ≥ 12 months; n = 18) and non-responders (n = 52). Differential‑abundance profiling revealed a graded enrichment of the phylum Actinobacteria across all lower ranks—class Actinobacteria, order Actinomycetales, family Actinomycetaceae and genus Actinomyces —in non‑responders (LEfSe LDA > 3.5; p = 0.001 for each level; FDR ≤ 0.049). ROC analysis identified an Actinomyces abundance of 11% (AUC = 0.768; sensitivity 0.94; specificity 0.44) as the optimal stratification point, classifying patients into low (≤ 11%, n = 46) and high (> 11%, n = 24) groups. High abundance predicted shorter PFS (median 3 vs 4 months; HR = 2.16, 95% CI 1.21–3.88, p = 0.009) and overall survival (OS) (median 5 vs 9 months; HR = 2.61, 95% CI 1.48–4.61, p < 0.001) after multivariable adjustment for ECOG status, treatment line, corticosteroid and opioid use, smoking, histology and metastatic sites. Bootstrap validation confirmed model stability: median bootstrap HRs were 2.56 (PFS) and 2.63 (OS), with narrow percentile CIs (PFS 1.57–4.49; OS 1.40–6.34) that overlapped the original estimates. Conclusions Oral microbiome signature characterized by high Actinomyces abundance is independently associated with poorer ICI outcomes in NSCLC. Saliva profiling is non-invasive and, when combined with tumour PD-L1 and clinical factors, may refine patient stratification.