Kahkeshan Hijazi

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.

Bridging the clinical gaps: genetic, epigenetic and transcriptomic biomarkers for the early detection of lung cancer in the post-National Lung Screening Trial era

Lung cancer is the leading cause of cancer death worldwide in part due to our inability to identify which smokers are at highest risk and the lack of effective tools to detect the disease at its earliest and potentially curable stage. Recent results from the National Lung Screening Trial have shown that annual screening of high-risk smokers with low-dose helical computed tomography of the chest can reduce lung cancer mortality. However, molecular biomarkers are needed to identify which current and former smokers would benefit most from annual computed tomography scan screening in order to reduce the costs and morbidity associated with this procedure. Additionally, there is an urgent clinical need to develop biomarkers that can distinguish benign from malignant lesions found on computed tomography of the chest given its very high false positive rate. This review highlights recent genetic, transcriptomic and epigenomic biomarkers that are emerging as tools for the early detection of lung cancer both in the diagnostic and screening setting.

Abstract 1419: Characterization of the evolution of immune response in lung squamous carcinogenesis

Molecular profiling of from pre-invasive bronchial lesions, and particularly the evolution of immune response, may identify promising new biomarkers for early detection and targets for chemoprevention/treatment of lung cancer. mRNA expression was analyzed (Agilent microarrays) from fresh frozen human bronchial biopsies (N = 122, 77 patients) at successive morphological stages of lung squamous carcinogenesis. Modules of co-expressed genes were identified among the stage associated genes, selected using a linear mixed-effects model adjusting for smoking status, gender, and history of cancer as fixed effects and patient as a random effect. Cell type specific signatures were applied to the genes co-expressed in these modules in order to identify the immune response signal through the stages of lung carcinogenesis. Genes expression alterations were grouped into 4 successive molecular steps. Based on their behavior across the 4 steps, 8 modules of genes with different patterns were observed: gene modules primarily up-regulated among the early or late steps, up-regulated in a linearly across the 4 steps, as well as down- then up-regulated (biphasic). Genes signing the presence of activating CD8 and CD4 T cells, as well as some memory B cells were progressively and linearly up-regulated from metaplasia through all stages of carcinogenesis. At the later stages, the transition to high-grade dysplasia, the most significant gene expression changes included immune/inflammatory response genes. At this stage a very strong adaptive immune response is noted involving T cells - mainly Th1 and T regulators - and dendritic cells. In addition, immunosuppressive signals, corresponding to negative co-inhibitory molecules, also significantly appears at the high-grade dysplasia stage. The adaptive CD4/CD8 related immune response starts from the earlier stages (metaplasia) of lung carcinogenesis. The significant modification of inflammatory/immune response genes in association with high-grade lesions suggests, at this critical stage of carcinogenesis, a major role of the surrounding microenvironment with an adaptive immune response, driven by Th1 and Treg cells, and but also, before tumor invasion across the basal membrane, a tumor versus host immunosuppressive response. Citation Format: Celine Mascaux, Mihaela Angelova, Jennifer Beane, Kahkeshan Hijazi, Geraldine Antoine, Karen Willard Gallo, Vincent Ninane, Arsene Burny, Jean-Paul Sculier, Avrum Spira, Jerome Galon. Characterization of the evolution of immune response in lung squamous carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1419.

Abstract A27: Airway molecular alterations associated with premalignant lesion progression and lung cancer development

Recently, the National Lung Screening Trial reported a 20% reduction in lung cancer mortality and lung cancer chemoprevention trials targeting the arachidonic acid pathway have demonstrated decreases in lung cancer associated markers. These studies highlight possibilities for future reductions in lung cancer mortality through early detection and chemoprevention. Our group has previously identified smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells that can serve as a clinically-relevant biomarker for the early detection of lung cancer. Here, in an effort that could lead to markers of lung cancer risk, we identify changes in these cells associated with regression of premalignant airway lesions. Airway epithelial cells were collected via bronchoscopy from patients with bronchial dysplasia at baseline, on-treatment, and post-treatment with green tea extract (GTE) or placebo ranging from 2 to 6 months (n=27 patients, n=63 samples). RNA from the samples was processed and hybridized to Affymetrix Human Gene 1.0 ST microarrays. A linear mixed effect model was used to identify a gene expression signature predictive of subsequent dysplasia regression. Further a paired t-test was used to identify genes associated with dysplasia regression over time. Using gene set enrichment analysis (GSEA) we identified that the baseline dysplasia regression signature was enriched (FDR<0.05) among genes whose altered expression was associated with: dysplasia regression over time; the presence or future development of lung cancer; and human bronchial biopsies at successive morphological stages of lung squamous carcinogenesis. The genes associated with dysplasia regression were validated small cohorts of independent samples from chemoprevention trials testing Sulindac, Myo-inositol, and GTE. Analysis of the Connectivity Map identified compounds that reverse the dysplasia regression signature in vitro and are therefore candidate chemoprevention agents. Our studies suggest that the airway “field of injury” is modulated by bronchial premalignant lesions. The molecular signatures identified may be important tools for stratifying high-risk smokers for chemoprevention trials, as surrogate endpoints of efficacy in these trials, and for identification of novel molecular targets for chemoprevention. In addition, the molecular signature of regression of airway dysplasia may have additional utility as a biomarker predictive of the presence of or future lung cancer development. Citation Format: Jennifer Ebel Beane, Kahkeshan Hijazi, Katrina Steiling, Gang Liu, Sherry Zhang, Stephen Lam, Marc Lenburg. Airway molecular alterations associated with premalignant lesion progression and lung cancer development. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A27.

Abstract B07: Nasal epithelial gene expression profiles reflect dynamic biological response to smoking cessation

Rationale: There are few indicators of which cigarette smokers are at highest risk for developing lung cancer and COPD, and it is unclear why individuals remain at risk for decades after smoking-cessation. The kinetics of gene-expression alterations upon smoking-cessation might delineate distinct biological processes that are activated in response to and during recovery from tobacco smoke exposure. Furthermore, differences in this response or recovery might be associated with disease risk. We therefore sought to determine whether the kinetics of gene-expression alterations upon smoking-cessation could be detected in readily collected nasal epithelium. Methods: Nasal epithelial brushings were obtained from 8 active smokers enrolled in smoking-cessation programs. Brushings were obtained at baseline and at 4, 8, 16, and 24-weeks after smoking-cessation. Urine cotinine or exhaled carbon monoxide was used to assess tobacco abstinence. RNA extracted from the nasal brushings was processed and hybridized to Gene 1.0 ST Arrays. Gene expression changes associated with smoking-cessation were identified with a linear mixed effects model. Functional enrichment was determined using DAVID. Gene Set Enrichment Analysis (GSEA) was used to determine the relationship of nasal epithelial gene-expression changes associated with smoking-cessation to previous cross-sectional bronchial and nasal airway gene expression datasets of current, longer-term former and never smokers and to a previous published dataset of in-vitro exposure of cultured bronchial epithelial cells (NHBE) to cigarette smoke (CS). Results: The expression levels of 78 genes were associated with short-term smoking-cessation (FDR q<0.05, FC ≥ 1.75), with 6 genes up-regulated and 72 genes down-regulated with tobacco abstinence. A majority of the changes occur within 1-2 months of quitting. These genes were enriched in functional categories including oxidoreductase activity and metabolism of xenobiotic (p<0.05). Genes that decreased with smoking-cessation were enriched for genes whose expression levels in the airway datasets were higher in current smokers relative to never smokers or longer-term former smokers (FDR <10−3). Genes increased in active smokers that reversed with smoking-cessation were enriched among genes that are induced followed 15 minutes in-vitro exposure of NHBE to CS (FDR <5*10−3). Conclusion: Nasal epithelial gene expression profiles reflect the host response to smoking-cessation and are concordant with reversible changes identified in an independent bronchial airway dataset. Genes that reverse rapidly after smoking-cessation are inversely related to gene expression differences induced by acute cigarette smoke exposure in-vitro; suggesting that aspects of the gene-expression response to tobacco smoke both occur and reverse with rapid kinetics upon smoking. We have further found that genes with different biological functions revert to baseline with different dynamics following smoking-cessation. These data suggest that the smoking-induced field of injury can be measured non-invasively in nasal epithelium and leveraged to understand the kinetics of the reversible impact of smoking. These minimally invasive tools can be applied in population studies to assess the physiological impact of alternate tobacco products and other inhaled environmental exposures. Citation Format: Kahkeshan Hijazi, Bozena Malyszko, Xiaohui Zhang, Gang Liu, Yuriy Alekseyev, Yves-Martine Dumas, Louise Hertsgaard, Joni Jenson, Cindy Rohde, Dorothy K Hatsukami, Stephen S. Hecht, Daniel R Brooks, George O’Connor, Marc Lenburg, Katrina Steiling, Avrum Spira. Nasal epithelial gene expression profiles reflect dynamic biological response to smoking cessation. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B07.