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Although lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here, we applied single-cell whole-genome sequencing of proximal bronchial basal cells from 33 participants aged between 11 and 86 years with smoking histories varying from never-smoking to 116 pack-years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers. When plotted against smoking pack-years, mutations followed the linear increase in cancer risk until about 23 pack-years, after which no further increase in mutation frequency was observed, pointing toward individual selection for mutation avoidance. Known lung cancer-defined mutation signatures tracked with both age and smoking. No significant enrichment for somatic mutations in lung cancer driver genes was observed.

Introduction The burden of asthma morbidity with co-existing atopy among the racial/ethnic minorities in the socio-economically disadvantaged NYC borough of the Bronx is unusually high. The multidisciplinary Montefiore Asthma Center (MAC) provides guideline-based treatment to this high-risk population through the joint efforts of Allergists/Immunologists, Pulmonologists, and on-site health educators. Methods The objective of this prospective, observational study was to define the demographic and clinical characteristics of severe asthma, evaluate improvement in asthma severity and lung function through the course of treatment at the MAC, and describe the asthma phenotypes of the patients managed at the MAC. Adults with severe asthma receiving treatment at the MAC were followed from their first to their last visit at the MAC. Patient demographics, along with asthma severity and co-existing allergies, were assessed. Possible phenotypes were defined (based on presence or absence of atopy, age at asthma onset, and blood eosinophil counts). Results 227 patients were included in the final analysis, of which 55.5% were Hispanic and 33.9% identified as non-Hispanic Black. Ninety-one percent (91%) of our cohort was found to be atopic and allergic rhinoconjunctivitis (ARC) was the most commonly identified co-existing allergic condition (86.3%). Mean Asthma Control Test (ACT) scores improved from 11.1 (± 4.9) at the initial visit to 14.8 (± 6.1) at the last visit. The spirometric values did not improve despite treatment at MAC. Conclusion A multidisciplinary severe asthma center is an ideal setting to phenotype patients and offer personalized guideline-based management and education to adults with severe asthma.

Background Aerosolized Azacitidine has been shown to inhibit orthotopic lung cancer growth and induce re-expression of methylated tumor suppressor genes in murine models. We hypothesized that inhaled Azacitidine is safe and effective in reversing epigenetic changes in the bronchial epithelium secondary to chronic smoking. Patients and Methods We report the first in human study of inhaled Azacitidine. Azacitidine in aqueous solution was used to generate an aerosol suspension of 0.25–5 μm particle size. Main inclusion criteria: Stage IV or recurrent NSCLC with predominantly lung involvement, ≥1 prior systemic therapy, ECOG PS 0–1, and adequate pulmonary function. Patients received inhaled Azacitidine daily on days 1–5 and 15–19 of 28-day cycles, at 3 escalating doses (15, 30 and 45 mg/m2 daily). The primary objective was to determine the feasibility and tolerability of this new therapeutic modality. The key secondary objectives included pharmacokinetics, methylation profiles and efficacy. Results From 3/2015 to 2/2018, eight patients received a median number of 2 (IQR = 1) cycles of inhaled Azacitidine. No clinically significant adverse events were observed, except one patient treated at the highest dose developed an asymptomatic grade 2 decreased DLCO which resolved spontaneously. One patient receiving 12 cycles of therapy had an objective and durable partial response, and two patients had stable disease. Plasma Azacitidine was only briefly detectable in patients treated at the higher doses. Moreover, in 2 of 3 participants who agreed and underwent pre- and post-treatment bronchoscopy, the global DNA methylation in the bronchial epithelium decreased by 24 % and 79 % post-therapy, respectively. The interval between last inhaled treatment and bronchoscopy was 3 days. Conclusions Inhaled Azacitidine resulted in negligible plasma levels compared to the previously reported subcutaneous administration and was well-tolerated. The results justify the continued development of inhaled Azacitidine at non-cytotoxic doses for patients with lung-confined malignant and/or premalignant lesions.

Background Sarcoidosis is believed to represent a genetically primed, abnormal immune response to an antigen exposure or inflammatory trigger, with both genetic and environmental factors playing a role in disease onset and phenotypic expression. In a population of firefighters with post-World Trade Center (WTC) 9/11/2001 (9/11) sarcoidosis, we have a unique opportunity to describe the clinical course of incident sarcoidosis during the 15 years postexposure and, on average, 8 years following diagnosis. Methods Among the WTC-exposed cohort, 74 firefighters with post-9/11 sarcoidosis were identified through medical records review. A total of 59 were enrolled in follow-up studies. For each participant, the World Association of Sarcoidosis and Other Granulomatous Diseases organ assessment tool was used to categorize the sarcoidosis involvement of each organ system at time of diagnosis and at follow-up. Results The incidence of sarcoidosis post-9/11 was 25 per 100,000. Radiographic resolution of intrathoracic involvement occurred in 24 (45%) subjects. Lung function for nearly all subjects was within normal limits. Extrathoracic involvement increased, most prominently joints (15%) and cardiac (16%) involvement. There was no evidence of calcium dysmetabolism. Few subjects had ocular (5%) or skin (2%) involvement, and none had beryllium sensitization. Most (76%) subjects did not receive any treatment. Conclusions Extrathoracic disease was more prevalent in WTC-related sarcoidosis than reported for patients with sarcoidosis without WTC exposure or for other exposure-related granulomatous diseases (beryllium disease and hypersensitivity pneumonitis). Cardiac involvement would have been missed if evaluation stopped after ECG, 48-h recordings, and echocardiogram. Our results also support the need for advanced cardiac screening in asymptomatic patients with strenuous, stressful, public safety occupations, given the potential fatality of a missed diagnosis.

BACKGROUND: Nasal polyps influence the burden of aspirin exacerbated respiratory disease (AERD) by contributing to eicosanoid production. AERD is diagnosed through graded aspirin challenges. It is not known how sinus surgery affects aspirin challenge outcomes. OBJECTIVE: To investigate the effects of endoscopic sinus surgery (ESS) on aspirin-induced reaction severity and on the levels of eicosanoids associated with these reactions. METHODS: Twenty-eight AERD patients were challenged with aspirin before and 3-4 weeks after ESS. Respiratory parameters and plasma and urine levels of eicosanoids were compared before and after challenges. RESULTS: Before ESS, AERD diagnosis was confirmed in all study patients by aspirin challenges that resulted in hypersensitivity reactions. After ESS, reactions to aspirin were less severe in all patients and twelve out of twenty-eight patients (43%, p

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

Asthma prevalence is reportedly higher among U.S.-born relative to foreign-born Hispanics/Latinos. Little is known about rates of asthma onset before and after relocation to the U.S. in Latinos. Asthma rates were examined by U.S. residence and country/territory of origin.In 2015-2016, age at first onset of asthma symptoms was analyzed, defined retrospectively from a cross-sectional survey in 2008-2011, in relation to birthplace and U.S. residence among 15,573 U.S.-dwelling participants (aged 18-76 years) in the Hispanic Community Health Study/Study of Latinos.Cumulative incidence of asthma through age 30 years ranged from 7.9% among Mexican background individuals to 29.4% among those of Puerto Rican background. Among those born outside the U.S. mainland, the adjusted hazard for asthma was 1.52-fold higher (95% CI=1.25, 1.85) after relocation versus before relocation to the U.S. mainland, with heterogeneity in this association by Hispanic/Latino background (p-interaction0.0001). Among foreign-born Dominicans and Mexicans, rates of asthma were greater after relocation versus before relocation (adjusted hazard ratio [AHR] for after versus before relocation, 2.42, 95% CI=1.44, 4.05 among Dominicans; AHR=2.90, 95% CI=2.02, 4.16 among Mexicans). Puerto Ricans had modestly increased asthma onset associated with U.S. mainland residence (AHR=1.52, 95% CI=1.06, 2.17). No similar increase associated with U.S. residence was observed among Central/South American immigrants (AHR=0.94, 95% CI=0.53, 1.67). Asthma rates among Cuban immigrants were lower after relocation (AHR=0.45, 95% CI=0.24, 0.82).The effect of relocation to the U.S. on asthma risk among Hispanics is not uniform across Hispanic/Latino groups.

The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3'UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitative polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA.

Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

Aspirin-exacerbated respiratory disease (AERD) is diagnosed through graded aspirin challenges that induce hypersensitivity reactions and eicosanoid level changes. It is not known whether diagnostically useful changes also occur after low-dose aspirin challenges that do not induce hypersensitivity reactions.To investigate the utility of low-dose oral aspirin challenges for diagnosing AERD by measuring different clinical parameters and eicosanoid changes.Sixteen patients with AERD and 13 patients with aspirin-tolerant asthma underwent oral challenges with low-dose (20 or 40 mg) aspirin and diagnostic oral graded aspirin challenges (up to 325 mg of aspirin). Forced expiratory volume in 1 second, nasal peak flow, the fraction of exhaled nitric oxide (FeNO), and eicosanoid levels in plasma and urine were analyzed.In patients with AERD but not in those with aspirin-tolerant asthma, 40-mg aspirin challenges induced a significant mean (SEM) decrease from baseline in FeNO (19% [5.1%]; P = .001) without causing any hypersensitivity reaction. The FeNO decrease also occurred after higher-dose aspirin challenges (27.8% [4.9%]; P.001). The sensitivity and specificity of 40-mg aspirin-induced FeNO changes for identifying AERD were 90% and 100% with an area under the curve of 0.98 (95% CI, 0.92-1.00). The low-dose challenge also induced a significant leukotriene E4 urine increase in patients with AERD (from 6.32 [0.08] to 6.91 [0.15] log-pg/mg creatinine; P.001), but the sensitivity and specificity of these changes were less than for the FeNO changes.The low-dose aspirin-induced decrease in FeNO in patients with AERD may be useful for the diagnosis of aspirin allergy without inducing a hypersensitivity reaction.clinicaltrials.gov Identifier: NCT01320072.

Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element-mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)-specific gene expression-based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4'-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H(2)O(2) induced nuclear translocation of nuclear factor erythroid 2-related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.

The number of predicted human microRNAs in Sanger miRBase currently stands at over a thousand, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both). In addition, contrasting results obtained when either stably expressing or transiently transfecting members of the miR-200 family illustrate limitations in the verification methods currently in use. In this article we suggest that instead of allowing computational predictions to drive investigation, it would be desirable, when possible, to systematically evaluate microRNA targets using inducible, stable, ectopic expression. The advantage of stable lines ectopically expressing microRNA(s) is that they allow an analysis of changes to both the proteome and the transcriptome. This would allow verification of targets, improve the design of prediction algorithms and greatly increase our understanding of the outcome of microRNA/mRNA interaction.

DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified by liquid chromatography-electrospray ionization/multistage tandem mass spectrometry (LC-ESI/MS/MS(n)) in saliva samples from 37 human volunteers on unrestricted diets. The N-(deoxyguanosin-8-yl) (dG-C8) adducts of the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and the aromatic amine, 4-aminobiphenyl (4-ABP), were characterized and quantified by LC-ESI/MS/MS(n), employing consecutive reaction monitoring at the MS(3) scan stage mode with a linear quadrupole ion trap (LIT) mass spectrometer (MS). DNA adducts of PhIP were found most frequently: dG-C8-PhIP was detected in saliva samples from 13 of 29 ever-smokers and in saliva samples from 2 of 8 never-smokers. dG-C8-AalphaC and dG-C8-MeIQx were identified solely in saliva samples of three current smokers, and dG-C8-4-ABP was detected in saliva from two current smokers. The levels of these different adducts ranged from 1 to 9 adducts per 10(8) DNA bases. These findings demonstrate that PhIP is a significant DNA-damaging agent in humans. Saliva appears to be a promising biological fluid in which to assay DNA adducts of tobacco and dietary carcinogens by selective LIT MS techniques.

Purpose: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated. Experimental Design: The “RNA” extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, β-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase (“no-RT”) to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA. Results: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated “RNA” extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, β-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non–RNA-specific primers on saliva samples yielded PCR products for 36B4, β-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the “no-RT” and “+RT” conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva. Conclusions: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.

An exfoliated buccal cell biomarker assay for antioxidant gene transcript levels was used to measure inter-tissue concordance with lung, and inter-subject variability in a lung cancer case-control study.First, qualitative RNA-specific RT-PCR was used to compare expression in exfoliated buccal cells with that in laser microdissected lung tissue remote from the tumor from 14 individuals providing both specimens.There was complete [100% for quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GPX), and superoxide dismutase 1 (SOD1)], or predominant [85.7% for catalase (CAT)] inter-tissue concordance for qualitative expression. Second, quantitative real-time RT-PCR for antioxidant enzyme transcript levels was performed in exfoliated buccal samples from these same 14 individuals, as well as 28 additional individuals providing buccal cells only, for a total of 42 buccal specimens (19 current smokers and 23 ex- or never-smokers), of whom 26 (61.39%) had a new diagnosis of lung cancer.Wide inter-individual expression differences for each gene transcript (10(1)-10(4)-fold) were observed in the exfoliated buccal cells, unrelated to smoking and case-control status. In multivariate analyses, family history of tobacco-related malignancy correlated inversely with buccal NQO1 and CAT mRNA levels (p=0.003, p0.001, respectively). This antioxidant expression trait may relate to family risk of cancer, but is notably unrelated to oxidant challenges inherent in cigarette smoke.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (χ2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.

Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original "RNA" extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence-which lacks introns but contains a poly(A) tail-for commonly studied internal reference genes beta-actin and GAPDH, and target genes GSTM1, GSTP1, and others. These pseudogene sequences therefore confound standard-design "RNA-specific" PCR primer pairs which rely, for cDNA versus gDNA specificity, on the pair-spanning introns, or one of the individual primer oligos spanning an exon/exon splice site, because these features are lacking in processed pseudogene sequences. The result is false RT-PCR positives for these "housekeeper" genes in total RNA extracts; the gDNA processed pseudogene is mistaken for mRNA gene transcript. A universal RT primer has been designed that targets the poly(A) tail of mRNA and adds a unique tag sequence not otherwise existing in the human genome. Genomic DNA does not incorporate this RT-inserted unique tag. PCR is then performed using a transcript-specific forward primer and a reverse primer that is identical to the unique tag incorporated at RT. Only cDNA made with this RT primer is compatible with this reverse PCR primer, thus eliminating confounding signal from contaminating gDNA. This method performs RNA-specific qualitative and quantitative evaluation of gene expression, while preserving the sensitivity of standard RT-PCR techniques. Applications to low-copy transcripts in human samples are demonstrated.

Estrogen receptor (ER) expression in human lung has been understudied, particularly in light of its potential biological importance in the female lung cancer epidemic. Reverse transcription-polymerase chain reaction was used to probe mRNA expression of wild-type ERalpha and ERbeta and their splice variants in human bronchogenic tumor and adjacent nontumor specimens. In tumor tissue from 13 women and 13 men, ERalpha was expressed in 85% of women versus 15% in men [P=0.001]. ERbeta was expressed equally in tumors from women versus men [92% vs. 69%, P=ns]. Both ERalpha and beta forms were expressed simultaneously in the lung tumors of 77% of women versus 15% of men [P=0.005]. Among adjacent nontumor lung specimens, 31% of the women expressed ERalpha mRNA versus 0% of men [P=0.101], and 39% of women expressed ERbeta mRNA versus 31% of men [P=ns]; only one woman and no men expressed both ERalpha and beta in nontumor tissue. Females expressed ERalpha [P=0.017], ERbeta [P=0.013], and ERalpha+beta [P=0.002] more frequently in tumor versus nontumor tissue, whereas in males expression of ERalpha, beta and both alpha+beta was not clearly different for tumor versus nontumor tissue. In specimens expressing ERalpha mRNA, the transcript lacking exon 7 (delta7) was the major splice variant with varying contributions from the transcripts delta4, delta3+4, delta5 and others unidentified. Alternative splicing of ERbeta mRNA was observed, but not to as great an extent as for ERalpha mRNA. ERalpha promoter usage in tumors varied among individuals. When the ER receptors were co-expressed in tumors, ERalpha was quantitatively more abundant in the majority of cases than ERbeta. Within this small group of 26 patients, no correlation was found between age, smoking history, plasma nicotine, cotinine, estradiol concentrations or histopathologic type with tumor or nontumor estrogen receptor status of any type. However, several positive correlations imply that: (1) ERalpha expression occurs more often in the lungs of women than men; (2) ERbeta is expressed with approximately equal frequency in the lungs of both genders; and (3) tumors display a higher frequency of both receptor types than nontumors in women. We hypothesize that these putative gender-dependent differences in ERalpha and ERbeta expression could contribute unique phenotypic characteristics to lung cancer development or progression in women.

Background Aspirin desensitization followed by daily aspirin provides therapeutic benefits to patients with aspirin-exacerbated respiratory disease (AERD). It is not well understood how eicosanoid levels change during aspirin treatment. Objective To investigate associations between clinical outcomes of aspirin treatment and plasma eicosanoid levels in patients with AERD. Methods Thirty-nine patients with AERD were offered aspirin treatment (650 mg twice daily) for 4 weeks. Respiratory parameters and plasma levels of multiple eicosanoids were recorded at baseline and after 4 weeks of aspirin therapy using the Asthma Control Test and Rhinoconjunctivitis Quality of Life Questionnaire. Respiratory function was evaluated using the FEV 1 and nasal inspiratory peak flow. Results After aspirin treatment, respiratory symptoms improved in 16 patients, worsened in 12 patients, and did not change in 4 patients. Seven patients were unable to complete the desensitization protocol. Patients with symptom improvement had higher baseline plasma 15-hydroxyeicosatetraenoic acid (15-HETE) levels than did patients with symptom worsening: 7006 pg/mL (interquartile range, 6056-8688 pg/mL) versus 4800 pg/mL (interquartile range, 4238-5575 pg/mL), P = .0005. Baseline 15-HETE plasma levels positively correlated with the change in Asthma Control Test score ( r = 0.61; P = .001) and in FEV 1 after 4 weeks of aspirin treatment ( r = 0.49; P = .01). It inversely correlated with Rhinoconjunctivitis Quality of Life Questionnaire score ( r = −0.58; P = .002). Black and Latino patients were more likely to have symptom worsening on aspirin or fail to complete the initial desensitization than white, non-Latino patients ( P = .02). Conclusions In patients with AERD, low baseline 15-HETE plasma levels and black or Latino ethnicity are associated with worsening of respiratory symptoms during aspirin treatment.

We evaluated the importance of tumor cell selection for generating gene signatures in non–small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor–nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.

One in ten tobacco smokers develops bronchogenic carcinoma over a lifetime. The study of susceptibility of an individual and a population to lung cancer traditionally has been limited to the study of tobacco smoke dose and family history of cancer. New insights into lung carcinogenesis have made the study of molecular markers of risk possible in human populations in the emerging field of molecular epidemiology. This review summarizes data addressing the relationships of human lung cancer to polymorphisms of phase I procarcinogen-activating and phase II-deactivating enzymes and intermediate biomarkers of DNA mutation, such as DNA adducts, oncogene and tumor suppressor gene mutation, and polymorphisms. These parameters are reviewed as they relate to tobacco smoke exposure, procarcinogen metabolizing polymorphisms, and the presence of lung cancer. Problem areas in biomarker validation, such as cross-sectional data interpretation, tissue source, race, statistical power, and ethical implications are ad...

Ever since a lung cancer epidemic emerged in the mid-1900 s, the epidemiology of lung cancer has been intensively investigated to characterize its causes and patterns of occurrence. This report summarizes the key findings of this research.A detailed literature search provided the basis for a narrative review, identifying and summarizing key reports on population patterns and factors that affect lung cancer risk.Established environmental risk factors for lung cancer include smoking cigarettes and other tobacco products and exposure to secondhand tobacco smoke, occupational lung carcinogens, radiation, and indoor and outdoor air pollution. Cigarette smoking is the predominant cause of lung cancer and the leading worldwide cause of cancer death. Smoking prevalence in developing nations has increased, starting new lung cancer epidemics in these nations. A positive family history and acquired lung disease are examples of host factors that are clinically useful risk indicators. Risk prediction models based on lung cancer risk factors have been developed, but further refinement is needed to provide clinically useful risk stratification. Promising biomarkers of lung cancer risk and early detection have been identified, but none are ready for broad clinical application.Almost all lung cancer deaths are caused by cigarette smoking, underscoring the need for ongoing efforts at tobacco control throughout the world. Further research is needed into the reasons underlying lung cancer disparities, the causes of lung cancer in never smokers, the potential role of HIV in lung carcinogenesis, and the development of biomarkers.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)–non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p

Background : Glutathione S-transferase (GST) P1 is a major phase II xenobiotic-metabolizing enzyme in the human lung. Our laboratory had previously identified nine single nucleotide polymorphisms (SNPs) in the GSTP1 gene promoter, which were then grouped into three main haplotypes (Hap1, Hap2, and Hap3) based on statistical inference. Hap3 was found to display a high expression phenotype. The main objective of the current study was to test the association between GSTP1 promoter haplotypes with the risk of lung cancer after determining the promoter haplotypes experimentally through cloning and sequencing. Methods : We conducted a case–control analysis of 150 subjects with lung cancer and 329 controls with no personal history of the disease. The three statistically inferred GSTP1 promoter haplotypes were confirmed experimentally through cloning and sequencing. Haplotype-tagging SNPs were selected and GSTP1 haplotypes were tested for genetic association to lung cancer using unconditional logistic regression after adjusting for confounders. Statistical interaction between GSTP1 promoter haplotypes with either cigarette smoking or dietary fruit and vegetable intake were tested using the likelihood ratio test. Results : We did not find protective effects of Hap3 against lung cancer, despite an adequately powered design for this main effect. Homozygous variants of tagSNPs −1738 T>A and −354 G>T, which tag Hap2, showed an increased (but statistically non-significant) risk of lung cancer among all subjects as well as among individuals with low fruit and vegetable intake, compared to homozygous wildtypes for these SNPs. We did not find significant interactions between Hap2 and dietary intake of fruits and vegetables. Conclusions : Our results do not support significant main and modifying effects for GSTP1 promoter haplotypes on susceptibility to lung cancer in this population, but reinforce the protective effects of dietary intake of fruits and vegetables.

MicroRNAs are ∼22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, δEF1, Nil-2-α) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non–small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor–positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor–negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-cadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal. [Cancer Res 2007;67(17):7972–6]

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR-200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated approximately 3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against approximately 600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2'-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.

Phase II detoxification of carcinogens is reported to mediate some of the anticarcinogenesis effects of candidate chemopreventive agents. We explored the interaction between sequence variation in the GSTP1 gene promoter and candidate chemopreventive exposure in regulating human GSTP1 expression. Polymorphisms along 1.8 kb of the GSTP1 promoter were identified in leukocytes [peripheral blood mononuclear cells (PBMC)] from 40 Caucasian subjects. Ten promoter polymorphisms (9 previously unreported) displayed strong linkage disequilibrium, yielding identification of three frequently observed haplotypes [HAP1 (43%), HAP2 (36%), and HAP3 (8%)]. Each haplotype was cloned into luciferase reporter constructs and transfected into normal human bronchial epithelial cells. Basal HAP3 reporter activity was significantly elevated (1.8-fold) but decreased to the same levels as HAP2 and HAP1 with increasing concentrations of sulforaphane, benzyl isothiocyanate (BITC), and epigallocatechin gallate (EGCG). To confirm native HAP3 functionality, we quantitated mRNA expression in uncultured PBMCs and in laser microdissected normal lung epithelial cells (MNLEC) from the same patients. Basal mRNA expression was higher in HAP3 individuals [1.8-fold (PBMC) and 4-fold (MNLEC) for HAP3 heterozygotes and 2.3-fold (PBMC), and 15-fold (MNLEC) for the HAP3 homozygote] than in the other genotypes. PBMC GSTP1 mRNA expression correlated to MNLEC expression (R2 = 0.77). After culture and in vitro exposure to sulforaphane, BITC, or EGCG, the elevated GSTP1 mRNA expression of PBMCs from HAP3 individuals decreased to common expression levels. Elevated HAP3 function was confirmed at the protein level in PBMCs (5-fold higher for HAP3 heterozygotes and 7.6-fold for the HAP3 homozygote). These data suggest a potentially protective GSTP1 promoter haplotype and unpredicted inhibitory chemopreventive agent-haplotype interactions. (Cancer Res 2006; 66(12): 6439-48)

A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R=0.935, p=0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.

The regulation of carcinogen metabolism machinery may involve proximate tobacco smoke exposure, hormonal and other endogenous coregulatory factors, and an individual's underlying genetic responsiveness. The mRNA and protein expression patterns of known carcinogen metabolism genes encoding the aromatic hydrocarbon receptor Ahr; the cytochromes P450 CYP1A1 and CYP1B1; glutathione S-transferases GSTM1, GSTM3, GSTP1, and GSTT1; and NADPH quinone oxidoreductase NQO1 were examined.Paired tumor and nontumor lung tissue from 45 subjects was subject to a recently devised RNA-specific qualitative reverse transcription-PCR strategy, as well as Western immunoblotting. Tobacco exposure measured by plasma biomarkers nicotine and cotinine, plasma estradiol levels, alpha and beta estrogen receptor (ER) expression in the lung, gender, age, and histological diagnosis were then analyzed using multivariate regression models.In nontumor lung tissue, multivariate models identified several correlates of mRNA expression: (a) CYP1B1 in females (positively: smoke status, P=0.024; ER-beta expression, P=0.024); (b) GSTT1 in females (positively: cotinine, P=0.007; negatively: age, P=0.001; ER-beta expression, P=0.005) and in males (positively: plasma estradiol, P=0.015; ER-beta expression, P=0.025); and (c) NQO1 in females (positively: smoke status, P=0.002) and in males (positively: ER-beta expression, P=0.001). CYP1A1 (mRNA, 9.1%) and GSTM1 (mRNA, 17.5%) are uncommonly expressed in human lung. Confirmation by Western immunoassayed protein is described. The results in nontumor tissue differed from that in tumor tissue.Regulation of carcinogen metabolism genes expressed in human lung seems impacted by hormonal and gender factors, as well as ongoing tobacco exposure. Expression differences between tumor and nontumor tissue in this pathway have both susceptibility and therapeutic implications.

Interindividual variation in the expression of the carcinogen- and estrogen-metabolizing enzymes cytochrome P4501B1 and 1A1 (CYP1B1 and CYP1A1) has been detected in human lung. To search for polymorphisms with functional consequences for CYP1B1 and CYP1A1 gene expression, we examined 1.5 kb of the promoter region of each gene. Genomic DNA from 21 Caucasian individuals was amplified by polymerase chain reaction (PCR) for direct cycle sequencing. Eight single nucleotide polymorphisms (SNPs) for CYP1B1 and 13 SNPs for CYP1A1 were found. The majority of polymorphisms occurred as multiSNP combinations for individual subjects. The wild-type sequences were cloned into a luciferase reporter construct. The most frequent polymorphisms were then recreated by iterative site-directed mutagenesis, replicating single polymorphisms and multiSNP combinations. These wild-type and variant constructs were functionally evaluated in transient transfection experiments employing exposures to either the index polycyclic aromatic hydrocarbon (PAH) inducer benzo[a]pyrene (B[a]P), a composite mixture of cigarette smoke extract (CSE), or the repressor chemopreventive agent trans-3,4,5-trihydroxystilbene (reseveratrol). Results indicated that all wild-type and variant constructs responded in qualitatively concordant fashion to the inducers and to the repressor. The CYP1B1 haplotypes and the majority of CYP1A1 haplotypes were shown to have no functional consequence, as compared to those of the wild-type promoter sequences. Two constructs of composite polymorphisms of CYP1A1 appeared to result in a statistically significant increase in basal promoter activity (1.38- and 1.50-fold, respectively), but the degree of functional impact was judged unlikely to be biologically important in vivo. We conclude that the observed promoter region polymorphisms in these genes are common, but are of unclear functional consequence.

Objective: To describe an unusual case of acute eosinophilic pneumonia (AEP) associated with hemodynamic instability. Design: Case report, clinical. Settings: Tertiary care intensive care unit (ICU). Patient: A single patient admitted to the ICU. Interventions: Intravenous corticosterolds. Measurements and Main Results: Resolution of distributive shock and respiratory failure. Conclusions: AEP with respiratory failure was first reported in 1989 as a distinct clinical entity. Patients with this variant of eosinophilic lung disease develop acute hypoxemic respiratory failure with a rapid response to treatment with corticosterolds. The characteristic feature of this syndrome is a predominance of eosinophils found in bronchoalveolar lavage fluid and lung biopsy. Despite the increasing number of reported cases, to our knowledge, distributive shock has not been reported as a feature of AEP. We report a unique case of AEP associated with shock and review the pertinent literature.

Background There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs. Methods To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5β promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)]. Results We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5β and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5β, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5β promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (padj = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1)~-215~-113 and (2) -84 ~+26); while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci. Conclusion Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.