Intratumoral Escherichia Is Associated With Improved Survival to Single-Agent Immune Checkpoint Inhibition in Patients With Advanced Non-Small-Cell Lung Cancer

Abstract

PURPOSEThe impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients with non-small-cell lung cancer (NSCLC) is unknown. Preclinically, intratumoral Escherichia is associated with a proinflammatory tumor microenvironment and decreased metastases. We sought to determine whether intratumoral Escherichia is associated with outcome to ICI in patients with NSCLC.PATIENTS AND METHODSWe examined the intratumoral microbiome in 958 patients with advanced NSCLC treated with ICI by querying unmapped next-generation sequencing reads against a bacterial genome database. Putative environmental contaminants were filtered using no-template controls (n = 2,378). The impact of intratumoral Escherichia detection on overall survival (OS) was assessed using univariable and multivariable analyses. The findings were further validated in an external independent cohort of 772 patients. Escherichia fluorescence in situ hybridization (FISH) and transcriptomic profiling were performed.RESULTSIn the discovery cohort, read mapping to intratumoral Escherichia was associated with significantly longer OS (16 v 11 months; hazard ratio, 0.73 [95% CI, 0.59 to 0.92]; P = .0065) in patients treated with single-agent ICI, but not combination chemoimmunotherapy. The association with OS in the single-agent ICI cohort remained statistically significant in multivariable analysis adjusting for prognostic features including PD-L1 expression (P = .023). Analysis of an external validation cohort confirmed the association with improved OS in univariable and multivariable analyses of patients treated with single-agent ICI, and not in patients treated with chemoimmunotherapy. Escherichia localization within tumor cells was supported by coregistration of FISH staining and serial hematoxylin and eosin sections. Transcriptomic analysis correlated Escherichia-positive samples with expression signatures of immune cell infiltration.CONCLUSIONRead mapping to potential intratumoral Escherichia was associated with survival to single-agent ICI in two independent cohorts of patients with NSCLC.

Figure 1.. Intratumoral Escherichia is associated with improved overall survival in univariable and multivariable analyses in patients treated with single-agent immune checkpoint inhibition (MSK discovery cohort).

A. Overall survival in Escherichia-positive vs negative groups in patients treated with single-agent immune checkpoint blockade. B. Multivariable analysis for overall survival in patients treated with single-agent immune checkpoint inhibition. Escherichia-pos; Escherichia-positive group. Escherichia-neg; Escherichia-negative group. ECOG PS; Eastern Cooperative Oncology Group performance status. F; female. M; male. OS; overall survival. mOS; median OS. MSK; Memorial Sloan Kettering Cancer Center.

Figure 2.. Intratumoral Escherichia is associated with improved overall survival in multivariable analyses in patients treated with single-agent immune checkpoint inhibition (FMI validation cohort).

A. Overall survival in Escherichia-positive vs negative groups in patients treated with single-agent immune checkpoint blockade. B. Multivariable analysis for overall survival in patients treated with single-agent immune checkpoint inhibition Escherichia-pos; Escherichia-positive group. Escherichia-neg; Escherichia-negative group. ECOG PS; Eastern Cooperative Oncology Group performance status. F; female. M; male. OS; overall survival. mOS; median OS. FMI; Foundation Medicine. The FMI analyses have been adjusted for delayed entry to mitigate left truncation bias. As such, not all patients are displayed as at risk at Time 0 in the bottom at-risk tables.

Figure 3.. Intratumoral visualization of Escherichia coli

A. Colon adenocarcinoma, resection specimen from sigmoid colon, 13237 Escherichia reads. B. LUAD, section from left lower lobe resection; 0 Escherichia reads. C. LUAD, resection specimen from right middle lobe; 24 Escherichia reads. D. LUAD, biopsy of bladder metastasis, 166 Escherichia reads. Fluorescence (top sections): Blue - DAPI; Red - E.Coli. Hematoxylin and eosing staining shown below. All images captured at same magnification. Representative images shown.

Figure 4.. Bulk RNAsequencing reveals potential associations between intratumoral Escherichia and tumor microenvironment.

A. Volcano plot showing differentially expressed genes (DEGs) between Escherichia-positive (pos) and Escherichia-negative (neg) samples. Grey dots represent non-significant value; green dots represent DEG significant by p-value but not by fold-change threshold; blue dots represent DEG significant by fold-change threshold but not by p-value; red dots represent significant DEG by both fold-change and p-value threshold. B. Boxplots of relevant DEGs between Escherichia-positive and Escherichia-negative samples. P-value by Wilcox test. C. Volcano plot showing cell type enrichment between Escherichia-pos and Escherichia-neg samples. D. Pathway enrichment analysis between Escherichia-pos and Escherichia-neg samples. All p-values adjusted (p-adj) for false discovery rate.