Effect of Inhaled Budesonide in Smokers with Bronchial DysplasiaEffect of Inhaled Budesonide in Smokers with Bronchial DysplasiaBC Cancer Research Centre

675 West 10th Avenue

Vancouver BC, V5Z 1L3

ph:604-675-8000 ext.7703

email: rchari@bccrc.ca

AbstractAbstract MethodsMethods

BackgroundBackground

ObjectivesObjectives

HypothesesHypotheses

Raj Chari1,2, Kim Lonergan1, Luc Girard4, John Minna4, Adi Gazdar4, Ruisheng Yao3, Ming You3, Raymond Ng2, Wan Lam1,2, Calum MacAulay1,2, Stephen Lam1,2

[1]British Columbia Cancer Agency, Vancouver, BC, Canada, [2]University of British Columbia, Vancouver, BC, Canada, [3]Washington University in St. Louis, St. Louis, MO, USA, [4]UT Southwestern Medical Centre4, Dallas, TX, USA

ConclusionsConclusions

AcknowledgementsAcknowledgements

Background: Budesonide, an inhaled corticosteroid used in the treatment of asthma, has been shown to be an effective chemopreventive agent in an animal model of adenocarcinoma [Carcinogenesis 1997 Oct 18(10):2015-7]. In humans, although inhaled budesonide for 6 months was not effective in regression of bronchial dysplasia, a significantly higher rate of resolution of CT detected small lung nodules was observed, some of which may represent pre-neoplastic lesions in the peripheral lung [Clinical Cancer Research 2004 Oct 1 10(19): 6502-11]. Despite being used as a drug for treatment of asthma and COPD for over two decades, the in-vivo effects of inhaled steroids on gene expression profiles of bronchial epithelial cells in smokers are still poorly understood.Objective: The objective of this study is to characterize the effects of inhaled budesonide on the gene expression profiles of bronchial cells from current and former smokers with bronchial dysplasia.Methods: Bronchial cells were obtained before and after six months of treatment with budesonide 800 mcg BID by inhalation. After two rounds of linear amplification of the extracted RNA, the gene expression profiles were analyzed using the Affymetrix U133A microarray chip.Results: Using Principal Component Analysis, the effect of active smoking was found to be stronger than the effect of budesonide. In current smokers, more Phase 1 enzyme genes were up-regulated compared with former smokers. However, Phase 2 enzyme genes were up-regulated in former smokers but down-regulated in current smokers. Specifically, CYP1B1 was shown to have a two-fold increase in current smokers after Budesonide treatment and approximately a 1.5-fold decrease in former smokers after treatment. In a separate analysis, it was found that genes up-regulated in current smokers had the tendency to be down-regulated in former smokers after treatment. As well, potential genes have been identified which correlate with the response to Budesonide treatment.Conclusions: A differential effect of Budesonide on gene expression profile was found between current and former smokers. Furthermore, we have characterized potential genes which correlate with the level of response. Targeting genes and pathways that correlate with patient response may be beneficial in screening of new chemopreventive agents.Supported by NIH contract N01-CN85188, & NCI SPORE CA 70907 & NIH 1P01 CA096964

ResultsResults

Supported by NIH contract N01-CN85188, & NCI SPORE CA 70907 & NIH 1P01 CA096964 & Genome Canada/Genome BC

Chemopreventive agents for lung cancer may exhibit a differential effect in current versus former or never smokersPrevious microarray studies showed different gene expression profiles between current and former smokersBudesonide is one of the most effective chemopreventive agents in animal models of lung cancer but was found to be ineffective in regression of bronchial dysplasia in smokers [Clinical Cancer Research 2004 Oct 1 10(19): 6502-11]

SAGE identifies differentially expressed genes not captured by previous microarray analysisCurrent and former smokers respond differently to inhaled budesonideRegression of bronchial dysplasia after budesonide is associated with different gene expression profiles versus non-responders

To determine the effect of active tobacco smoking on gene expression profiles in human bronchial epithelial cells retrieved by bronchial brushing during bronchoscopy To compare the gene expression profiles obtained by Affymetrix U133A array with Serial Analysis of Gene Expression (SAGE)To characterize the effects of inhaled budesonide on the gene expression profiles of bronchial cells from current versus former smokers with bronchial dysplasiaTo identify genes associated with regression of bronchial dysplasia after treatment with inhaled budesonide

Serial Analysis of Gene Expression (SAGE)Serial Analysis of Gene Expression (SAGE)

SAGE deduces expression profile based on the relative abundance of sequence tags.

SAGE is not limited to genes spotted on the arraySAGE produces quantitative, discrete (digital) data values~150,000 tags enumerated per library

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Nla IIIdigestion

Linkerligation

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enzyme digestion

GTACCATG

CATGGTACLigation

Ditag

Amplification

GTACCATG CATG

GTAC

GTACCATG

NlaIII digestion

Ligation

Cloning, sequencing and bioinformatics analysis

Expression profile

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Nla IIIdigestion

Linkerligation

and “Tagging”

enzyme digestion

GTACCATG

CATGGTACLigation

Ditag

Amplification

GTACCATG CATG

GTAC

GTACCATG

NlaIII digestion

Ligation

Cloning, sequencing and bioinformatics analysis

Expression profile

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PlatformFormer

SmokersCurrent

Smokers

Affymetrix U133A 18 34

SAGE 11 9

Description of DatasetDescription of Dataset

Statistical MethodsStatistical Methods

A combination of the student’s t-test, fold-change and permutation test were used in the analysis Differentially expressed genes were selected at a significance level of p ≤ 0.001T-test and fold-change are commonly used in the literaturePermutation test

suitable test for a small sample sizenon-parametric testperforms well in identifying differentially expressed genes

Effect of BudesonideEffect of Budesonide

Filtering of dataFiltering of data

Affymetrix data filtered as explained by Spira et al. [Proc Natl Acad Sci U S A. 2004;101(27):10143-8]Data for SAGE tags was retained only if raw tag counts were greater than 1 in at least one library

Identify genes that show similar expression differences before and after treatment in 4 of 5 subjectsFold-change of ≥ 1.5 (log2 fold of 0.585) between baseline and six months Remove genes if similar expression pattern seen in the placebo armFindings validated in the dataset of the one-month study

Subject Background and ResponseSubject Background and Response

Smoking status

# on active arm

Clinical Response*

Former (n = 5) 2 1CR, 1PR

Current (n = 5) 3 1CR, 1PR, 1PD

Correlation of Gene Expression Correlation of Gene Expression with Clinical Responsewith Clinical Response

Response characterized as complete response (CR), partial response (PR) and progressive disease (PD)CR is defined as complete regression of all dysplastic lesions to hyperplasia or normal, PR is regression of some but not all dysplastic lesions; PD is appearance of new lesions or progression of dysplastic lesions by ≥ 2 grades after treatment Identify genes that have a ≥ 1.5 fold change in complete responders onlyMust also exhibit a gradient of expression between CR, PR and PD.

Identify genes which have ≥ 1.5 fold change exclusively in all former smokers but not in all current smokers and vice-versa Remove genes if similar expression pattern seen in placebo arm

Comparison of Affymetrix U133A and SAGEComparison of Affymetrix U133A and SAGE

Comparison of the number of probes or tags that are differentially expressed based on t-value, permutation test score, fold-change and all 3 criteria at significance level of p ≤ 0.001Using the 3 criteria

In current smokers, SAGE data shows 30 tags (mapping to 27 unique genes) and Affymetrix U133A gives 7 probes (mapping 5 unique genes) that are higher compared to former smokersIn former smokers, SAGE data suggests 22 tags (mapping to 18 unique genes) and Affymetrix U133A has none that are higher relative current smokers

Higher in Current SmokersHigher in Current Smokers Higher in Former SmokersHigher in Former Smokers

Budesonide Associated Expression ChangesBudesonide Associated Expression Changes

(Below) Sample of genes up-regulated and down-regulated after treatment in at least 4 of 5 subjectsAlso exhibit same pattern in at least 5 of 7 subjects in validation dataset (U133A 2.0)

Down-Regulated Up-Regulated

TRBC1 - T cell receptor beta constant 1 TOP2A - topoisomerase (DNA) II alpha 170kDa

CCL5 - chemokine (C-C motif) ligand 5HPGD - hydroxyprostaglandin dehydrogenase

15-(NAD)

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Human bronchial epithelial cells were obtained in 10 smokers with bronchial dysplasia by bronchial brushings before and after daily treatment with 1600 µg of inhaled budesonide/placebo for six months (Clin Cancer Res 2004;10:6502-6511). Specimens were also obtained from 7 subjects before and after one month treatment with budesonide

Effect of Active SmokingEffect of Active Smoking

Affymetrix U133A data was obtained from the paper by Spira et al. [Proc Natl Acad Sci U S A. 2004;101(27):10143-8]SAGE libraries were generated from human bronchial epithelial cells obtained by bronchial brushings during bronchoscopy of heavy smokers

SpecimensSpecimens

Evaluation CriteriaEvaluation Criteria

PlatformPlatform

Affymetrix U133A array was used to profile the samples obtained from the 10 subjects in the six month studyAffymetrix U133A 2.0 array was used for the 7 subjects in the one month study

Smoking Status and Response to Smoking Status and Response to Inhaled BudesonideInhaled Budesonide

Genes Correlating with Clinical ResponseGenes Correlating with Clinical Response

Effect of Budesonide Independent of Response Effect of Budesonide Independent of Response and Smoking Statusand Smoking Status

Example of genes with increased (left) and decreased expression (right) in complete responders. Among the up-regulated genes are: caveolin 2 (CAV2), hydroxyprostaglandin dehyrogenase 15-(NAD) (HPGD), TU3A protein (TU3A) and the down-regulated genes are: toll-like receptor 3 (TLR3), TIR domain containing adaptor inducing interferon-beta (TRIF), mitogen-activated protein kinase kinase 6 (MAP2K6).HPGD has been recently shown to mediate anti-inflammatory effects in lung adenocarcinoma cell lines [Arch Biochem Biophys. 2005;435(1):50-5]TRIF and TLR3 potentially implicate down-regulation of the Toll-like receptor pathway

Subset of Genes Differentially Expressed Between Current and Subset of Genes Differentially Expressed Between Current and Former SmokersFormer Smokers

Analysis of SAGE data affords more discovery of significant genes as compared to the Affymetrix U133A platformBudesonide exhibits anti-inflammatory effects as evidenced by increased HPGD, furthermore this gene is increased even higher in complete responders. Expression changes of some of the genes appeared as soon as one month after budesonide exposure, while others do not appear until six monthsInhaled budesonide has a differential effect in current versus smokers. Such differences such as effects on Phase I & II enzymes gene expression may account for differences in response between current and former smokers

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CYP1B1 CYP1A1 GST2 GPX2 CYP/(GST+GPX)

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Phase I enzymes and cell cycle control genes was observed in current smokers compared to former smokers

Examples of Differentially Expressed Genes - Phase Examples of Differentially Expressed Genes - Phase 1& 2 Toxin Exposure Genes1& 2 Toxin Exposure Genes

Increased in Current Smokers, Decreased in Former Smokers

Increased in Former Smokers, Decreased in Current Smokers

MYO6 - Myosin VI TM7SF4 - transmembrane 7 superfamily member 4

MAP4K5 - mitogen-activated protein kinase kinase kinase kinase 5

CORO1A - coronin, actin binding protein, 1A

TPD52 - tumor protein D52 NMT2 - N-myristoyltransferase 2

CYP1B1 - cytochrome P450, family 1, subfamily B, polypeptide 1

PLA2 - phospholipase A2, group IVB (cytosolic)