Katrina Steiling

Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer

Lung cancer is the leading cause of death from cancer in the US and the world. The high mortality rate (80–85% within 5 years) results, in part, from a lack of effective tools to diagnose the disease at an early stage. Given that cigarette smoke creates a field of injury throughout the airway, we sought to determine if gene expression in histologically normal large-airway epithelial cells obtained at bronchoscopy from smokers with suspicion of lung cancer could be used as a lung cancer biomarker. Using a training set (n = 77) and gene-expression profiles from Affymetrix HG-U133A microarrays, we identified an 80-gene biomarker that distinguishes smokers with and without lung cancer. We tested the biomarker on an independent test set (n = 52), with an accuracy of 83% (80% sensitive, 84% specific), and on an additional validation set independently obtained from five medical centers (n = 35). Our biomarker had ∼90% sensitivity for stage 1 cancer across all subjects. Combining cytopathology of lower airway cells obtained at bronchoscopy with the biomarker yielded 95% sensitivity and a 95% negative predictive value. These findings indicate that gene expression in cytologically normal large-airway epithelial cells can serve as a lung cancer biomarker, potentially owing to a cancer-specific airway-wide response to cigarette smoke.

The Field of Tissue Injury in the Lung and Airway

The concept of field cancerization was first introduced over 6 decades ago in the setting of oral cancer. Later, field cancerization involving histologic and molecular changes of neoplasms and adjacent tissue began to be characterized in smokers with or without lung cancer. Investigators also described a diffuse, nonneoplastic field of molecular injury throughout the respiratory tract that is attributable to cigarette smoking and susceptibility to smoking-induced lung disease. The potential molecular origins of field cancerization and the field of injury following cigarette smoke exposure in lung and airway epithelia are critical to understanding their potential impact on clinical diagnostics and therapeutics for smoking-induced lung disease.

A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment

RATIONALE Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function. OBJECTIVES We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy. METHODS Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays. MEASUREMENTS AND MAIN RESULTS We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts. CONCLUSIONS Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.

Comparison of proteomic and transcriptomic profiles in the bronchial airway epithelium of current and never smokers.

Background Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear. Methodology/Principal Findings Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in ≤5% of airway samples from a previously published dataset. Conclusions/Significance 1D-PAGE coupled with LC-MS/MS effectively profiled the airway epithelium proteome and identified proteins expressed at different levels as a result of cigarette smoke exposure. While there was a strong correlation between protein and transcript detection within the same sample, we also identified proteins whose corresponding transcripts were not detected by microarray. This noninvasive approach to proteomic profiling of airway epithelium may provide additional insights into the field of injury induced by tobacco exposure.

DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways

DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illuminas Infinium HM27 and Affymetrixs Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2-related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.

Airway gene expression in COPD is dynamic with inhaled corticosteroid treatment and reflects biological pathways associated with disease activity

Background A core feature of chronic obstructive pulmonary disease (COPD) is the accelerated decline in forced expiratory volume in one second (FEV1). The recent Groningen and Leiden Universities study of Corticosteroids in Obstructive Lung Disease (GLUCOLD) study suggested that particular phenotypes of COPD benefit from fluticasone±salmeterol by reducing the rate of FEV1 decline, yet the underlying mechanisms are unknown. Methods Whole-genome gene expression profiling using the Affymetrix Gene ST array (V.1.0) was performed on 221 bronchial biopsies available from 89 COPD patients at baseline and after 6 and 30 months of fluticasone±salmeterol and placebo treatment in GLUCOLD. Results Linear mixed effects modelling revealed that the expression of 138 genes decreased, whereas the expression of 140 genes significantly upregulated after both 6 and 30 months of treatment with fluticasone±salmeterol versus placebo. A more pronounced treatment-induced change in the expression of 50 and 55 of these 278 genes was associated with a lower rate of decline in FEV1 and Saint George Respiratory Questionnaire, respectively. Genes decreasing with treatment were involved in pathways related to cell cycle, oxidative phosphorylation, epithelial cell signalling, p53 signalling and T cell signalling. Genes increasing with treatment were involved in pathways related to focal adhesion, gap junction and extracellular matrix deposition. Finally, the fluticasone-induced gene expression changes were enriched among genes that change in the airway epithelium in smokers with versus without COPD in an independent data set. Conclusions The present study suggests that gene expression in biological pathways of COPD is dynamic with treatment and reflects disease activity. This study opens the gate to targeted and molecular phenotype-driven therapy of COPD.

Genetic regulation of gene expression in the lung identifies CST3 and CD22 as potential causal genes for airflow obstruction

Background COPD is a complex chronic disease with poorly understood pathogenesis. Integrative genomic approaches have the potential to elucidate the biological networks underlying COPD and lung function. We recently combined genome-wide genotyping and gene expression in 1111 human lung specimens to map expression quantitative trait loci (eQTL). Objective To determine causal associations between COPD and lung function-associated single nucleotide polymorphisms (SNPs) and lung tissue gene expression changes in our lung eQTL dataset. Methods We evaluated causality between SNPs and gene expression for three COPD phenotypes: FEV1% predicted, FEV1/FVC and COPD as a categorical variable. Different models were assessed in the three cohorts independently and in a meta-analysis. SNPs associated with a COPD phenotype and gene expression were subjected to causal pathway modelling and manual curation. In silico analyses evaluated functional enrichment of biological pathways among newly identified causal genes. Biologically relevant causal genes were validated in two separate gene expression datasets of lung tissues and bronchial airway brushings. Results High reliability causal relations were found in SNP–mRNA–phenotype triplets for FEV1% predicted (n=169) and FEV1/FVC (n=80). Several genes of potential biological relevance for COPD were revealed. eQTL-SNPs upregulating cystatin C (CST3) and CD22 were associated with worse lung function. Signalling pathways enriched with causal genes included xenobiotic metabolism, apoptosis, protease–antiprotease and oxidant–antioxidant balance. Conclusions By using integrative genomics and analysing the relationships of COPD phenotypes with SNPs and gene expression in lung tissue, we identified CST3 and CD22 as potential causal genes for airflow obstruction. This study also augmented the understanding of previously described COPD pathways.

Interaction of Cigarette Exposure and Airway Epithelial Cell Gene Expression

Cigarette smoking is responsible for lung cancer and chronic obstructive pulmonary disease (COPD), the leading cause of death from cancer and the second-leading cause of death in the United States. In the United States, 46 million people smoke, with an equal number of former smokers. Moreover, 20-25% of current or former smokers will develop either disease, and smokers with one disease are at increased risk for developing the other. There are no tools for predicting risk of developing either disease; no accepted tools for early diagnosis of potentially curable lung cancer; and no tools for defining molecular pathways or molecular subtypes of these diseases, for predicting rate of progression, or for assessing response to therapy at a biochemical or molecular level. This review discusses current studies and the future potential of measuring global gene expression in epithelial cells that are in the airway field of injury and of using the genomic information derived to begin to answer some of the above questions.

Updates and Controversies in the Rapidly Evolving Field of Lung Cancer Screening, Early Detection, and Chemoprevention

Lung cancer remains the leading cause of cancer-related death in the United States. Cigarette smoking is a well-recognized risk factor for lung cancer, and a sustained elevation of lung cancer risk persists even after smoking cessation. Despite identifiable risk factors, there has been minimal improvement in mortality for patients with lung cancer primarily stemming from diagnosis at a late stage when there are few effective therapeutic options. Early detection of lung cancer and effective screening of high-risk individuals may help improve lung cancer mortality. While low dose computerized tomography (LDCT) screening of high risk smokers has been shown to reduce lung cancer mortality, the high rates of false positives and potential for over-diagnosis have raised questions on how to best implement lung cancer screening. The rapidly evolving field of lung cancer screening and early-detection biomarkers may ultimately improve the ability to diagnose lung cancer in its early stages, identify smokers at highest-risk for this disease, and target chemoprevention strategies. This review aims to provide an overview of the opportunities and challenges related to lung cancer screening, the field of biomarker development for early lung cancer detection, and the future of lung cancer chemoprevention.

Airway Gene Expression in Chronic Obstructive Pulmonary Disease

Although cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD), only a subset of smokers develops this disease. There is significant clinical, radiographic, and pathologic heterogeneity within smokers who develop COPD that likely reflects multiple molecular mechanisms of disease. It is possible that variations in the individual response to cigarette smoking form the basis for the distinct clinical and molecular phenotypes and variable natural history associated with COPD. Using the biologic premise of a molecular field of airway injury created by cigarette smoking, this response to tobacco exposure can be measured by molecular profiling of the airway epithelium. Noninvasive study of this field effect by profiling airway gene expression in patients with COPD holds important implications for our understanding of disease heterogeneity, early disease detection, and identification of novel disease-modifying therapies.