Frank Schembri

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.

MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium

We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3′-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.

Characterizing the Impact of Smoking and Lung Cancer on the Airway Transcriptome Using RNA-Seq

Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-Seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high-molecular-weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without (NC) lung cancer undergoing lung nodule resection surgery. RNA-Seq libraries were prepared using 2 distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-Seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-Seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine–cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-Seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-Seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking-related changes in the inflammatory genes S100A8 and S100A9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3A1). Quantitative real-time PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-Seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention. Cancer Prev Res; 4(6); 803–17. ©2011 AACR.

Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium.

Previous studies have shown that physiological responses to cigarette smoke can be detected via bronchial airway epithelium gene expression profiling and that heterogeneity in this gene expression response to smoking is associated with lung cancer. In this study, we sought to determine the similarity of the effects of tobacco smoke throughout the respiratory tract by determining patterns of smoking-related gene expression in paired nasal and bronchial epithelial brushings collected from 14 healthy nonsmokers and 13 healthy current smokers. Using whole genome expression arrays, we identified 119 genes whose expression was affected by smoking similarly in both bronchial and nasal epithelium, including genes related to detoxification, oxidative stress, and wound healing. While the vast majority of smoking-related gene expression changes occur in both bronchial and nasal epithelium, we also identified 27 genes whose expression was affected by smoking more dramatically in bronchial epithelium than nasal epithelium. Both common and site-specific smoking-related gene expression profiles were validated using independent microarray datasets. Differential expression of select genes was also confirmed by RT-PCR. That smoking induces largely similar gene expression changes in both nasal and bronchial epithelium suggests that the consequences of cigarette smoke exposure can be measured in tissues throughout the respiratory tract. Our findings suggest that nasal epithelial gene expression may serve as a relatively noninvasive surrogate to measure physiological responses to cigarette smoke and/or other inhaled exposures in large-scale epidemiological studies.

Noninvasive method for obtaining RNA from buccal mucosa epithelial cells for gene expression profiling

Swabs and scrapings from the buc-cal mucosa in the mouth have been used to obtain DNA from epithelial cells for genetic studies (1,2). RNA has been obtained from resected tissues and from biopsy samples of mouth epithelium in various disease states to measure gene expression (3,4). How-ever, RNA has not been extracted from scrapings of buccal mucosa because ribonucleases that are present in saliva rapidly degrade epithelial cell RNA (5) during collection. To collect intact RNA from buc-cal mucosal epithelium for studies of the biologic effect of smoking on the airway epithelium, we developed a relatively noninvasive method for ob-taining small amounts of RNA from the mouth. We measured the expres-sion of selected genes in individual subjects using real-time PCR and used a recently described mass spectrometry method that requires only nanogram amounts of total RNA for analysis and lends itself to high-throughput analysis of hundreds of genes (6).Initially, we used a micropipet tip cut lengthwise to collect epithelial cells from the buccal mucosa in a relatively noninvasive fashion. We subsequently designed a standardized plastic tool that is concave with serrated edges. It is 5/16 inches wide and 1 6/16 inches long with a 3 inch handle that can be broken off when the scraping tool with the collected cells is inserted into a 2-mL mi-crofuge tube containing 1 mL RNAlater™ solution (Qiagen, Valencia, CA, USA). The tool has two features that allow the collection of a significant amount of good-quality RNA from the buccal mucosa: a finely serrated edge that can scrape off several layers of epithelial cells and a concave surface that collects the cells. Using gentle pressure, the serrated edge was scraped (10 times) against the buccal mucosa on the inside of the cheek, and the cells collected were immediately immersed in 1 cm

MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis

Significance MicroRNAs are small noncoding RNAs that negatively regulate gene expression and have been implicated in a variety of cellular processes. Using small RNA sequencing, we identified microRNA 4423 (miR-4423) as a primate-specific microRNA whose expression is largely restricted to airway epithelium and which functions as a regulator of airway epithelium differentiation and a repressor of lung carcinogenesis. Understanding miR-4423’s role in airway development may provide insights into primate-specific aspects of airway biology and the evolution of primate-specific tumor suppressors. Moreover, this study opens the possibility that microRNAs might be useful for the early detection of lung cancer in the proximal airway and that miR-4423 mimetics might also be used as therapeutic agents to specifically target lung cancer. Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis.

Serotonin Syndrome Associated With Clozapine Withdrawal

IMPORTANCE We describe a case of serotonin syndrome secondary to clozapine withdrawal and concomitant use of citalopram hydrobromide, a phenomenon that has been rarely reported. OBSERVATIONS This is a case report of a 47-year-old woman admitted to an academic medical center intensive care unit with coma, hypersalivation, hyperreflexia, and stimulus-induced clonus. The patient received a diagnosis of serotonin syndrome attributed to abrupt clozapine withdrawal with concomitant use of citalopram. She improved only minimally with supportive treatment (intravenous fluids, benzodiazapines, and withdrawal of selective serotonin-reuptake inhibitor) and received cyproheptadine hydrochloride on her third day of symptoms. Four hours after she received the loading dose of cyproheptadine, she was alert and oriented and at her baseline mental status, although some clonus remained. CONCLUSIONS AND RELEVANCE Serotonin syndrome can result from the abrupt withdrawal of a 5-hydroxytryptamine receptor 2A antagonist from a treatment regimen that also includes a medication that increases serotonin availability.

Detecting Noncoding RNA Expression: From Arrays to Next-Generation Sequencing

Detection and quantification of noncoding(nc) RNA species can present specific challenges as compared to mRNA. Among them, ncRNA sequences are generally less well annotated, can include extraordinarily small species such as miRNA or piRNA, can have repetitive sequences or have high GC content, or even have antisense expression. Despite this, many different traditional technologies have been adapted to measure ncRNAs and include those based on a priori knowledge of sequence such as probe detection (qRT-PCR) and hybridization (arrays), and those where such knowledge is not required (next-generation sequencing). A summary of these experimental techniques is reviewed in this chapter, including the available chemistries, throughput, starting material, and species interrogated. Subsequent focus is on the computational analysis for next-generation sequencing since this technology can not only detect and measure ncRNAs but more excitingly also lead to the discovery of new or isomeric forms.

Is There a TIME and Place for Thrombolytics in Malignant Pleural Effusions

Frank Schembri The Pulmonary Center Boston University School of Medicine Boston, MA 02118 J Scott Ferguson Division of Pulmonary and Critical Care University of Wisconsin School of Medicine and Public Health Madison, WI 53705 The use of intrapleural thrombolytics as an adjunct therapy to promote pleural drainage is probably best elucidated in patients with pleural infections 1 , but has also been studied for other indications including traumatic or nontraumatic retained hemothorax 2 and multiloculated malignant pleural effusions(MPE). Considering that malignant pleural effusions have increased pro-coagulant properties and depressed fibrinolytic activity similar to other causes of exudative pleuritis 3 , it is not surprising that over time many MPEs form fibrinous strands and multiple loculations. This presumably leads to difficulty with management since fibrinous material may preclude adequate drainage and lung expansion before pleurodeisis or impede tunneled pleural catheter function. A natural clinical question then is “can improved drainage with the use of fibrinolytics improve dyspnea and pleurodesis in the management of MPE?”

MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis - eScholarship

Significance MicroRNAs are small noncoding RNAs that negatively regulate gene expression and have been implicated in a variety of cellular processes. Using small RNA sequencing, we identified microRNA 4423 (miR-4423) as a primate-specific microRNA whose expression is largely restricted to airway epithelium and which functions as a regulator of airway epithelium differentiation and a repressor of lung carcinogenesis. Understanding miR-4423’s role in airway development may provide insights into primate-specific aspects of airway biology and the evolution of primate-specific tumor suppressors. Moreover, this study opens the possibility that microRNAs might be useful for the early detection of lung cancer in the proximal airway and that miR-4423 mimetics might also be used as therapeutic agents to specifically target lung cancer. Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis.