Abstract 5066: Single-cell characterization of airway field alterations in patients with lung squamous premalignant lesions
Authors: Regan D. Conrad, Conor Shea, Lukas Kalinke, Kitty De Jong, Sherry Zhang, Zhihan Li, Gang Liu, Erin Kane, Emily Siedlecki, Kelsey Simon, Kate E. Otter, Kate Gowers, Mark Hennon, Sai Yendamuri, Steven Dubinett, Avrum Spira, Mary Reid, Sam Janes, Marc Lenburg, Sarah Mazzilli, Joshua Campbell, Jennifer Beane
Journal: Cancer Research (2025)

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide, and there is a critical need for early detection strategies to reduce its burden. In high-risk individuals, low-dose computed tomography (LDCT) reduces lung cancer mortality by detecting cancer at earlier stages. Gene expression obtained from bronchial and nasal brushings effectively discriminates benign from malignant lung nodules detected via LDCT. Lung premalignant lesions (PMLs), precursors to cancer, represent an opportunity for further mortality reduction, but detection methods remain underexplored. This study uses single-cell RNA sequencing (scRNA-seq) to investigate molecular and cellular alterations in the airway field of patients with PMLs. Endobronchial biopsies from lung sites with suspect PMLs and bronchial and nasal brushes from normal-appearing epithelium were collected from high-risk patients (n=46). Epithelial and immune cells were profiled using scRNA-seq via the CEL-Seq2 protocol (n=9405 biopsy, n=3416 bronchial brush, and n=2002 nasal brush cells). Cell and gene clustering were performed via Celda. Co-expressed gene modules and cell-type abundance differences by sample type, smoking status, and lesion histology were identified with differential composition analysis and linear mixed models. Gene module associations with phenotypes were validated in independent bronchial brushes profiled by bulk mRNA-seq (n=89 samples, n=30 patients). Clustering of epithelial cells (40 cell clusters, 70 gene modules) showed nasal brushes are enriched in cells from 9 cell clusters (p&amp;lt;0.001) with high expression of detoxification and mucous production gene modules. Nine gene modules associated with smoking status (p&amp;lt;0.001) across sample types were enriched for xenobiotic metabolism genes. The effect of smoking on these gene modules was strongest in bronchial biopsy samples, decreased in bronchial brushes and weakest in nasal brushes. This stepwise decrease was significant for each pairwise comparison (p &amp;lt;= 0.001). Five basal cell clusters enriched with carcinoma in situ lesion or tumor cells (termed high-grade basal cells) expressed 7 gene modules linked to keratinization and inflammation (p&amp;lt;0.01). Cells from a bronchial brush sample clustered with high-grade basal cells. The expression of a gene module upregulated in high-grade basal cells was associated with the worst lesion histology sampled during the procedure (p=0.006) in bronchial brushes profiled by bulk RNA-seq. This study identifies smoking and lesion severity associated molecular and cellular alterations in the airway fields of patients with PMLs. These findings suggest that airway field brushings may be useful in detecting the presence of high-grade PMLs. Citation Format: Regan D. Conrad, Conor Shea, Lukas Kalinke, Kitty De Jong, Sherry Zhang, Zhihan Li, Gang Liu, Erin Kane, Emily Siedlecki, Kelsey Simon, Kate E. Otter, Kate Gowers, Mark Hennon, Sai Yendamuri, Steven Dubinett, Avrum Spira, Mary Reid, Sam Janes, Marc Lenburg, Sarah Mazzilli, Joshua Campbell, Jennifer Beane. Single-cell characterization of airway field alterations in patients with lung squamous premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5066.