Distinguishing Smoking-Related Lung Disease Phenotypes Via Imaging and Molecular Features.

BACKGROUND: Chronic tobacco smoke exposure results in a broad range of lung pathologies including emphysema, airway disease and parenchymal fibrosis as well as a multitude of extra-pulmonary comorbidities. Prior work using CT imaging has identified several clinically relevant subgroups of smoking related lung disease, but these investigations have generally lacked organ specific molecular correlates. RESEARCH QUESTION: Can CT imaging be used to identify clinical phenotypes of smoking related lung disease that have specific bronchial epithelial gene expression patterns to better understand disease pathogenesis? STUDY DESIGN AND METHODS: Using K-means clustering, we clustered participants from the COPDGene study (n = 5,273) based on CT imaging characteristics and then evaluated their clinical phenotypes. These clusters were replicated in the Detection of Early Lung Cancer Among Military Personnel (DECAMP) cohort (n = 360), and were further characterized using bronchial epithelial gene expression. RESULTS: Three clusters (preserved, interstitial predominant and emphysema predominant) were identified. Compared to the preserved cluster, the interstitial and emphysema clusters had worse lung function, exercise capacity and quality of life. In longitudinal follow-up, individuals from the emphysema group had greater declines in exercise capacity and lung function, more emphysema, more exacerbations, and higher mortality. Similarly, genes involved in inflammatory pathways (tumor necrosis factor-α, interferon-ß) are more highly expressed in bronchial epithelial cells from individuals in the emphysema cluster, while genes associated with T-cell related biology are decreased in these samples. Samples from individuals in the interstitial cluster generally had intermediate levels of expression of these genes. INTERPRETATION: Using quantitative CT imaging, we identified three groups of individuals in older ever-smokers that replicate in two cohorts. Airway gene expression differences between the three groups suggests increased levels of inflammation in the most severe clinical phenotype, possibly mediated by the tumor necrosis factor-α and interferon-ß pathways. CLINICAL TRIAL REGISTRATION: COPDGene (NCT00608764), DECAMP-1 (NCT01785342), DECAMP-2 (NCT02504697).

Gene Expression Alterations in the Bronchial Epithelium of e-Cigarette Users.

BACKGROUND: Although e-cigarette (ECIG) use has increased in the United States, their potential health effects remain uncertain. Understanding the effects of tobacco cigarette (TCIG) smoke on bronchial airway epithelial gene expression have previously provided insights into tobacco-related disease pathogenesis. Identifying the impact of ECIGs on airway gene expression could provide insights into their potential long-term health effects. We sought to compare the bronchial airway gene-expression profiles of former TCIG smokers now using ECIGs with the profiles of former and current TCIG smokers. METHODS: We performed gene-expression profiling of bronchial epithelial cells collected from current TCIG smokers (n = 9), current ECIG users who are former TCIG smokers (n = 15), and former TCIG smokers (n = 21). We then compared our findings with previous studies of the effects of TCIG use on bronchial epithelium, as well an in vitro model of ECIG exposure. RESULTS: Among 3,165 genes whose expression varied between the three study groups (q < 0.05), we identified 468 genes altered in ECIG users relative to former smokers (P < .05). Seventy-nine of these genes were up- or down-regulated concordantly among ECIG and TCIG users. We did not detect ECIG-associated gene-expression changes in known pathways associated with TCIG usage. Genes downregulated in ECIG users are enriched among the genes most downregulated by exposure of airway epithelium to ECIG vapor in vitro. CONCLUSIONS: ECIGs induce both distinct and shared patterns of gene expression relative to TCIGs in the bronchial airway epithelium. The concordance of the genes altered in ECIG users and in the in vitro study suggests that genes altered in ECIG users are likely to be changed as the direct effect of ECIG exposure.

Detection of early lung cancer among military personnel (DECAMP) consortium: study protocols.

BACKGROUND: Lung cancer is the leading cause of cancer-related death due in large part to our inability to diagnose it at an early and potentially curable stage. Screening for lung cancer via low dose computed tomographic (LDCT) imaging has been demonstrated to improve mortality but also results in a high rate of false positive tests. The identification and application of non-invasive molecular biomarkers that improve the performance of CT imaging for the detection of lung cancer in high risk individuals would aid in clinical decision-making, eliminate the need for unnecessary LDCT follow-up, and further refine the screening criteria for an already large high-risk population. METHODS: The Detection of Early Lung Cancer Among Military Personnel (DECAMP) consortium is conducting two multicenter prospective studies with the goals of developing an integrated panel of both airway and blood-based molecular biomarkers that discriminate benign and malignant indeterminate nodules detected on CT scan as well as predict the future development of lung cancer in high-risk individuals. To achieve these goals, DECAMP is compiling an extensive array of biospecimens including nasal brushings, serum, plasma and intrathoracic airway samples (bronchial brushings and bronchial biopsies) from normal-appearing airway epithelium. DISCUSSION: This bank of samples is the foundation for multiple DECAMP efforts focused on the identification of those at greatest risk of developing lung cancer as well as the discrimination of benign and malignant pulmonary nodules. The clinical, imaging and biospecimen repositories will serve as a resource for the biomedical community and their investigation of the molecular basis of chronic respiratory disease. TRIAL REGISTRATION: Retrospectively registered as NCT01785342 - DECAMP-1: Diagnosis and Surveillance of Indeterminate Pulmonary Nodules (DECAMP-1). Date of Registration: February 7, 2013. Retrospectively registered as NCT02504697 - DECAMP-2: Screening of Patients With Early Stage Lung Cancer or at High Risk for Developing Lung Cancer (DECAMP-2). Date of Registration: July 22, 2015.

Assessment of a Highly Multiplexed RNA Sequencing Platform and Comparison to Existing High-Throughput Gene Expression Profiling Techniques.

The need to reduce per sample cost of RNA-seq profiling for scalable data generation has led to the emergence of highly multiplexed RNA-seq. These technologies utilize barcoding of cDNA sequences in order to combine multiple samples into a single sequencing lane to be separated during data processing. In this study, we report the performance of one such technique denoted as sparse full length sequencing (SFL), a ribosomal RNA depletion-based RNA sequencing approach that allows for the simultaneous sequencing of 96 samples and higher. We offer comparisons to well established single-sample techniques, including: full coverage Poly-A capture RNA-seq, microarrays, as well as another low-cost highly multiplexed technique known as 3' digital gene expression (3'DGE). Data was generated for a set of exposure experiments on immortalized human lung epithelial (AALE) cells in a two-by-two study design, in which samples received both genetic and chemical perturbations of known oncogenes/tumor suppressors and lung carcinogens. SFL demonstrated improved performance over 3'DGE in terms of coverage, power to detect differential gene expression, and biological recapitulation of patterns of differential gene expression from in vivo lung cancer mutation signatures.

Molecular Impact of Electronic Cigarette Aerosol Exposure in Human Bronchial Epithelium.

Little evidence is available regarding the physiological effects of exposure to electronic cigarette (ECIG) aerosol. We sought to determine the molecular impact of ECIG aerosol exposure in human bronchial epithelial cells (HBECs). Gene-expression profiling was conducted in primary grown at air liquid interface and exposed to 1 of 4 different ECIG aerosols, traditional tobacco cigarette (TCIG) smoke, or clean air. Findings were validated experimentally with quantitative polymerase chain reaction and a reactive oxygen species immunoassay. Using gene set enrichment analysis, signatures of in vitro ECIG exposure were compared with those generated from bronchial epithelial brushings of current TCIG smokers and former TCIG smokers currently using ECIGs. We found 546 genes differentially expressed across the ECIG, TCIG, and air-exposed groups of HBECs (ANOVA; FDR q < .05; fold change > 1.5). A subset of these changes were shared between TCIG- and ECIG-exposed HBECs. ECIG exposure induced genes involved in oxidative and xenobiotic stress pathways and increased a marker of reactive oxygen species production in a dose-dependent manner. ECIG exposure decreased expression of genes involved in cilia assembly and movement. Furthermore, gene-expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users (q < .01). In summary, our data suggest that ECIG aerosol can induce gene-expression changes in bronchial airway epithelium in vitro, some of which are shared with TCIG smoke. These changes were generally less pronounced than the effects of TCIG exposure and were more pronounced in ECIG products containing nicotine than those without nicotine. Our data further suggest that the gene-expression alterations seen with the in vitro exposure system reflects the physiological effects experienced in vivo by ECIG users.