Abstract 3377: NSCLC subtype-associated molecular and cellular alterations in the normal-appearing bronchial epithelium
Authors: Shi Xingyi , Zhang Jiarui , Callori Steven , Campbell Joshua , Fine Denise , Gesthalter Yaron , Liu Gang , Xiao Xiaohui , Spira Avrum , Lenburg Marc , Beane Jennifer 
Published: September 2020

Abstract
Rationale: We have previously identified lung cancer-associated gene expression alterations in normal bronchial airway epithelial cells of ever smokers with and without lung cancer. It is unclear, however, whether or not gene expression in the bronchial airway also reflects non-small cell lung cancer (NSCLC) subtypes. In this study, we sought to identify airway gene expression differences between patients with squamous (LUSC) and adenocarcinoma (LUAD) NSCLC and determine if these reflect changes in cell type composition or cell type-specific expression.
Methods: Bronchial brushings from ever smokers with NSCLC from the AEGIS I (153 LUSC; 148 LUAD; 154 benign) & II (82 LUSC; 102 LUAD; 74benign) trials were profiled by whole-genome microarrays. The NSCLC-associated gene expression alterations were identified using a linear model adjusting for smoking status in AEGIS I and confirmed in AEGIS II via GSEA. Enriched biological pathways were identified using EnrichR. In parallel, bronchial airway brushings collected from ever smokers undergoing bronchoscopy for suspect lung cancer (17 subjects) were profiled via scRNA-seq using the CEL-Seq2 protocol (3264 cells). Seurat was used to correct for batch effects and identify distinct cell populations. Genes differentially expressed between cell populations using a negative binomial model were clustered into gene sets using WGCNA. Per patient metagene scores were computed across the datasets for each gene set, and differences in these scores between NSCLC subtypes were evaluated using a linear model adjusting for smoking status.
Results: 451 differentially expressed genes were found between LUSC v LUAD patients (FDR &lt; 0.05). Genes involved in ciliary function were enriched among genes up-regulated in LUAD whereas keratinization and cancer-associated pathway genes were enriched among LUSC up-regulated genes (FDR &lt; 0.05). In the scRNA-seq data, 15 populations of cells were identified that expressed known epithelial or immune cell type markers. Using WGCNA to cluster genes that distinguish the cell populations (5148 genes, FDR &lt; 0.05) identified 12 gene sets (GS). In AEGIS I, LUSC patients had higher expression of a mitochondrial GS expressed highly in basal cells, a secretory-cell GS, and lower expression of a ionocyte-specific GS than LUAD patients (p = 0.041, 0.0043 and 2.06e-06 respectively). Both LUAD and LUSC patients have lower expression levels of the immune GSs than the benign patients (p = 0.00034 and 0.0008). Concordant results were found in the AEGIS II samples with the same methods.
Conclusion: We leveraged bulk and single cell gene expression data and identified molecular differences in the airway field of injury among ever smokers with LUSC versus LUAD that are highly expressed specific cell populations. Our results suggest that there are different responses to injury in the normal-appearing airway that are associated with NSCLC subtype.
Citation Format: Xingyi Shi, Jiarui Zhang, Steven Callori, Joshua Campbell, Denise Fine, Yaron Gesthalter, Gang Liu, Xiaohui Xiao, Avrum Spira, Marc Lenburg, Jennifer Beane. NSCLC subtype-associated molecular and cellular alterations in the normal-appearing bronchial epithelium [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3377.