University of Groningen

Molecular genetic studies in epithelial cells of lung cancer and COPD patientsBoelens, Mirjam Catharina

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GENERAL INTRoDUC TIoN 1

Lung cancer

Lung cancer is the leading cause of cancer mortality worldwide and is for over 90% caused by smoking. In the Netherlands, lung cancer was diagnosed in 9,014 patients (6,126 males, 2,888 females) in 2003 and almost the same number of patients (6,480 males and 2,855 females) died of lung cancer in 2004 (www.rivm.nl)1. The most recent data from Statistics Netherlands show a further increase with 9,426 patients dying from lung cancer in 2006 (www.cbs.nl). The staging system for lung cancer is based on the TNM (Tumor, Node, Metastasis)-classification, as summarized in Table 12. This classification provides information on the tumor load and spread, and is used as a measure for prognosis and treatment. Based on the TNM status, lung cancer is subdivided into discernable stages, ranging from stage IA (early stage) to stage IV (advanced stage) (Table 2)3. In general, higher stages correlate with worse survival outcomes. At the time of diagnosis most lung cancer patients have already developed distant metastases (stage IV). It is important to look for metastases during staging of the patient, since metastases, rather than the primary tumors are responsible for most lung cancer deaths. Lung cancer often metastasizes via the lymphatic system to regional lymph nodes and/or hematologically to distant organs (mainly to bone, brains, liver and adrenal glands). The 5-year overall survival for lung cancer patients is strongly dependent on the tumor stage, decreasing from 65-70% for stage IA to 1-2% for stage IV with an average 5-year survival of about 10-15%3,4.

Lung cancer can be subdivided into small cell lung cancer (SCLC) (15%) and non-small lung cancer (NSCLC) (85%) based on the global histological characteristics of the tumor cells. SCLC grows and metastasizes rapidly and tends to arise in the larger airways. While SCLC is initially more sensitive to chemotherapy, patients have a worse prognosis due to chemo resistance at tumor relapse and due to the high frequency of metastases at presentation. This type of lung cancer is in almost all cases associated with smoking5. NSCLC encompasses several distinct subtypes including the most common squamous cell (SCC) and adenocarcinoma (AC) subtypes. The SCC subtype is also strongly associated

Figure 1. Transformation from normal bronchial epithelium to SCC6. Hyperplasia and squamous metaplasia may transform back to normal epithelium when the patient quits smoking. The next transformation steps are first mild dysplasia and then moderate to severe dysplasia and carcinoma in situ. The latter stages are considered to be irreversible steps towards malignancy and subsequent invasion of the basement membrane results in (invasive) squamous cell carcinoma (SCC).

NormalEpithelium

Hyperplasia Invasive Carcinoma

Carcinoma in Situ

DysplasiaSquamous Metaplasia

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with smoking, whereas the AC subtype is also observed in non-smokers. The bronchial epithelial cell is considered to be the precursor cell of SCC6. As a result of smoking, normal bronchial epithelium can transform into metaplastic squamous epithelial cells that may become dysplastic. Advanced dysplasia is considered to be a premalignant stage for SCC (Figure 1). AC is suggested to originate from bronchial glandular epithelial cells or the surrounding stem cells. SCC used to be the most common histological subtype (40%), but recently a shift has been observed towards the AC subtype (Figure 2)1,7,8.

The choice of treatment in patients with NSCLC does not only depend on the stage of the disease but also on comorbidity and the condition of the patient. For early stage NSCLC, surgery with adjuvant chemotherapy is the most effective approach. Generally, one or more lobes are removed from the lung (lobectomy or pneumectomy), together with the draining lymph nodes. When the primary tumor can not be removed completely, or when dissemination of the primary tumor cells has been identified, combination therapy will be given (Table 1).

Tumor stage (T)

T1 Tumor 3 cm or less in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus (i.e., not in the main bronchus)

T2 Tumor with any of the following features of size or extent: More than 3 cm in greatest dimension; Involves main bronchus, 2 cm or more distal to the carina; Invades the visceral pleura; Associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung

T3 Tumor of any size that directly invades any of the following: chest wall (including superior sulcus tumors), diaphragm, mediastinal pleura, parietal pericardium; or tumor in the main bronchus less than 2 cm distal to the carina, but without involvement of the carina; or associated atelectasis or obstructive pneumonitis of the entire lung

T4 Tumor of any size that invades any of the following: mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina; or tumor with a malignant pleural or pericardial effusion, or with satellite tumor nodule(s) within the ipsilateral primary tumor lobe of the lung

Lymph Node stage (N)

N0 No regional lymph node metastasis

N1 Metastasis to ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, and intrapulmonary nodes involved by direct extention of the primary tumor

N2 Metastasis to ipsilateral mediastinal and/or subcarinal lymph node(s)

N3 Metastasis to contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s)

Distant Metastasis stage (M)

M0 No distant metastasis

M1 Distant metastasis present

Table 1. TNM classification for lung cancer

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Chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality worldwide. The major risk factor for the development of COPD is tobacco smoke, which accounts for 80-90% of the COPD cases. Genetic factors, such as α1-antitrypsin deficiency, present in about 1-5% of the COPD population, influences the susceptibility to COPD9. However, many other genetic factors may contribute to the development of COPD. In the Netherlands, COPD is diagnosed in about 33,000 patients (17,200 males, 16,400

Table 2. Tumor staging and treatment

Stage TNM stage Treatment

IA T1 N0 M0 surgery

IB T2 N0 M0 surgery + adjuvant chemotherapy

IIA T1 N1 M0 surgery + adjuvant chemotherapy

IIB T2 N1 M0, T3 N0 M0 surgery + adjuvant chemotherapy

IIIA T3 N1 M0 surgery + adjuvant chemotherapy

T1-3 N2 M0 combination chemo-radiotherapy

IIIB T1-4 N3 M0, T4 N0-2 M0 combination chemo-radiotherapy

IV anyT anyN M1 chemotherapy

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Figure 2. Age-standardized incidence of lung cancer in the Netherlands according to sex and histological subtype (1989-2003), for male (A) and female (B). The presence of SCC decreases through the years in males, while in females AC is an increasing lung cancer subtype. Squares, SCC; triangles, AC; circles, SCLC. Adapted from1.

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females) in 2003 and 5,662 patients (3,381 males, 2,281 females) died of COPD in 2004 (www.rivm.nl). COPD is an irreversible chronic airway disease characterized by persistent and irreversible airway obstruction. The diagnosis COPD is made – as defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) – by spirometry when the postbronchodilator Forced Expiratory Volume in one second/Forced Vital Capacity (FEV1/FVC) ratio is below 70%. COPD can be classified in different GOLD stages of severity based on FEV1% predicted (Table 3)10.

Depending on the disease severity, COPD is treated with bronchodilator/anti-inflammatory medications (anticholinergics, sympaticomimetics and inhaled or systemic corticosteroids), supplemental oxygen or surgery (lung volume reduction or

lung transplantation). In very advanced disease, i.e. patients with GOLD stage IV, lung transplantation is the only alternative to increase life expectancy.

COPD includes two main disease manifestations, chronic bronchitis/bronchiolitis, with obstruction in large and small airways, and emphysema with destruction of alveolar walls and enlargement of airspaces. Most COPD patients have a combination of both chronic bronchitis/bronchiolitis and emphysema. The airflow limitation is usually progressive and associated with an abnormal chronic reaction of the lungs, such as inflammation, oxidative stress and tissue remodeling, due to the toxic smoking particles11. Pathologic changes underlying the clinical symptoms of COPD can be found in the large and small airways, and in lung parenchyma. In the airways, inflammatory cells are recruited to the subepithelial layer and these cells can infiltrate also in the surface epithelium12,13. In addition, chronic inflammation can lead to ongoing cycles of injury and repair (remodeling) of the airway wall and hyperplasia of goblet cells and mucus glands in the airways results in mucus hypersecretion14. Destruction of the lung parenchyma in patients with smoking-

Table 3. GOLD classification of severity of COPD

GOLD stage FEV1

I: Mild COPD FEV1 ≥ 80% predicted

II: Moderate COPD 50% ≤ FEV1 < 80% predicted

III: Severe COPD 30% ≤ FEV1 < 50% predicted

IV: Very Severe COPDFEV1 < 30% predicted, or FEV1 < 50%

predicted plus chronic respiratory failure

FEV1, forced expiratory volume in one second

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induced COPD typically occurs as centrilobular emphysema. This involves dilatation and destruction of the peribronchial alveolar attachments15.

Smoking, lung cancer and COPD

In 2006, about 28% of the Dutch population smoked (31% male and 25% female) with an average of 15 cigarettes per day (www.stivoro.nl). Almost 20,000 people died in 2005 as a consequence of smoking. Smoking increases the risk of lung cancer (relative risk of 7.9-29.3) and COPD (relative risk of 2.3-13.7). About 90% of the patients with lung cancer and/or COPD have a long smoking history16,17. Despite the clear link with smoking, only 15-20% of the cigarette smokers develop lung cancer and/or COPD which suggests that other risk factors contribute to the susceptibility of these diseases and to their differential development18,19. Independent of the smoking history, COPD itself is also a risk factor for the development of lung cancer. Several epidemiological studies showed that the relative risk for developing lung cancer increases from at least 2-fold with moderate COPD to 10-fold with severe COPD20-27. Furthermore, COPD seems to be especially a risk factor for the SCC subtype21.

Cigarette smoke is a complex heterogeneous mixture containing more than 5,000 chemical compounds among which carbon oxide, nitric oxide, free radicals, and tar (aromatic hydrocarbons containing nicotine, hydroquinones, catechols and several

Figure 3. Smoking can activate bronchial epithelial cells to excrete chemokines, free radicals and proteases. This may lead to CoPD-related processes such as inflammation (neutrophils, macrophages, CD8+ T-cells), mucus excretion, fibrosis and emphysema.

epithelial cells

chemokines

neutrophils

macrophages

CD8+ T-cells

proteases, free radicals

COPD

fibrosis

emphysema

mucusexcretion

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carcinogens)28. There is sufficient evidence based on animal studies and humans cell lines that over 60 components present in cigarette smoke have a carcinogenic potential29.

The airways are covered with a layer of epithelial cells. These epithelial cells are the first line of defense against inhaled toxic substances, such as cigarette smoke and air pollution. This subsequently leads to a variety of signals to adjacent cell types in the bronchial wall. Bronchial epithelial cells are considered to give rise to the SCC subtype6.

In COPD, the airway epithelial cells can produce various chemokines that contribute to the recruitment of inflammatory cells, leading to the initiation of a local inflammatory process and fibrosis, which are the main pathogenetic processes in the airways of patients with COPD30-32 (Figure 3). The bronchial epithelium of COPD patients often shows squamous metaplasia, which is the result of chronic irritation, in case of COPD by the combination of cigarette smoke and inflammation. Proliferation of basal epithelial cells is increased in some healthy smokers, but is markedly increased in patients with COPD33.

Molecular genetic studies in epithelial cells of lung cancer and COPD patients

Microarray technologyMicroarray is a frequently used technique to screen genome-wide changes/alterations at

the RNA or DNA level in a wide variety of tissue samples or cells originating from all kind of species (Figure 4). Since its first application in the mid 1990s34, microarray technology has been successfully applied to almost every aspect of biomedical research35-40. Microarrays can be used to study chromosomal alterations41 using array based comparative genomic hybridization (array-CGH) and to study gene expression changes in cell types of interest in relation to specific characteristics.

Laser dissection microscopy (LDM) is a recently developed technique that allows analysis of specific cell types from complex tissue samples. It has become an important tool to isolate pure cell populations from heterogeneous tissue42. In combination with the more recently developed DNA and RNA amplification procedures, analysis of LDM samples using microarray technology has been implemented in many studies43-50.

Comparative genomic hybridization (CGH) studies in NSCLCSpecific chromosomal aberrations have been found in many subtypes of cancers. In solid

tumor these aberrations usually are copy number changes (gains or losses) of chromosomes. A gain or loss of genomic DNA may result in a change in expression levels of genes that are located on the altered chromosomal regions. Such a region may harbor an oncogene that

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represents a driver gene on which these NSCLC tumors remain dependent with respect to their proliferation and survival, a phenomenon called “oncogene addiction”. Moreover, a loss of tumor suppressor genes may play an important role in the ultimate biological behavior of the tumor. During the development of cancer, copy number changes at specific chromosomal regions occur. Over the past years many techniques have been developed and applied to characterize specific chromosomal aberrations (LOH, FISH) or to identify these aberrations in a genome wide approach (multi color FISH, MLPA, CGH, aCGH, SNP array). Based on the accurate mapping data of the oligo’s / BACs on the arrays, the aCGH technique presents a powerful tool to identify the candidate target genes in genomic regions with copy number aberrations. In NSCLC, several common chromosomal copy number changes have been described, i.e. gains of 3q, 5p, 7q, 8q, 11q and 16p, and losses of 3p, 4q, 5q, 6q, 8p, 9p, 13q and 17q as reviewed by Balsara et al.51.

Gene expression studies in bronchial epithelium and NSCLCA pitfall of gene expression profiling studies is the large number of probes on the array

(many thousands) in relation to the relative limited number of samples (usually less than 100). This unbalance between the number of genes and the number of samples lowers the statistical power to detect significant differences for individual genes. This problem asks

Hybridize

to microarray SCAN

DNA/RNAlabeled with Cy3

DNA/RNAlabeled with Cy5

Figure 4. The principle of the microarray technology is based on simultaneous hybridization of a fluorescently labeled test (Cy3) and reference sample (Cy5) (either RNA or DNA) on a microarray slide containing thousands of oligonucleotide, cDNA or BAC probes. The intensity of the signals for both Cy3 and Cy5 are translated to gene expression or copy number changes of single genes/loci in the whole genome.

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for robust statistical analyses including multiple testing correction and validation of the results at RNA and/or protein level to extract the relevant differences. More and more tools are becoming available to extract significant differences related to specific pathways, gene ontologies and chromosomal regions. Moreover, microarray studies are frequently used to generate new hypotheses which can be the subject of new studies.

Gene expression profiling in normal bronchial epithelium requires the use of airway brushes or laser microdissection of lung tissue in order to enrich pure bronchial epithelial cells. Most published studies so far have used brush-derived epithelial samples for microarray studies and investigated smoke-related52-54 gene expression profiles. Many genes with significant changes in expression levels were observed in normal bronchial epithelial cells from smokers and non-smokers; these genes were mainly related to oxidative stress responses. After smoking cessation of at least two years, the altered expression levels for most of these genes returned back to the levels of a non-smoker52. In relation to COPD, one study analyzed oxidative stress related genes and observed that part of these genes were upregulated in COPD patients as compared to healthy smokers54. Other genes or proteins that have been described as differentially expressed between epithelium of patients with and without COPD include growth factors and their receptors and adhesion molecules55-

57. Gene expression profiling in brush-derived normal epithelial cells from patients with and without lung cancer resulted in a subset of 80 genes that may serve as a lung cancer biomarker prediction set58.

In NSCLC, several studies identified a specific gene expression profiles that could discriminate between or was related to specific histological subtypes of NSCLC59-61. Other studies have focused on the relationship of gene expression profiles with clinical characteristics this resulted in specific gene expression signatures in relation to time of survival59,62-64, chemosensitivity65,66, or lymph node metastasis65,67,68. Furthermore, many studies contributed to an enhanced pathogenetic understanding in cellular processes involved in oncogenesis of NSCLC among which apoptosis and cell-cycle progression69-74. With respect to smoking, it is observed that NSCLC developed in smokers have a different gene expression profile than NSCLC developed in never-smokers, indicating that smoking leads to a different pathogenesis of NSCLC75-77.

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Scope of the thesis

The aim of this thesis is to explore potential smoking- and/or COPD-related gene expression signatures in histologically normal bronchus epithelium and in SCC. We started with a pilot experiment to test the feasibility of using routinely collected tissue from normal bronchial wall, SCC and AC (chapter 2). As a next step, we tested the laser dissection microscopy (LDM) approach to allow isolation of pure normal bronchial epithelium and pure SCC tumor cells in combination with gene expression arrays. To examine the extent of the experimental bias due to amplification procedures required to gain sufficient RNA from LDM samples we compared the expression profiles from single and double amplification rounds (chapter 3). After these two studies, we collected a number of normal bronchial epithelial and SCC tissue samples from patients in such a way that these patients were matched for their smoking history (smoking status and packyears), COPD status and presentation of lymph node or distant metastasis. This enabled us to investigate whole genome gene expression in normal bronchial epithelium and in SCC and copy number changes in SCC in relation to these features (Figure 5). In chapter 4, we investigated the smoking related effects in normal bronchial epithelium and SCC by comparing expression profiles of current and ex-smokers. The genes that showed a marked smoking-induced effect in normal bronchial epithelium were also analyzed in SCC. In chapter 5, we investigated whether a difference exists between gene expression signatures in patients with and without COPD in both normal bronchial epithelium and SCC. In chapter 6, we compared genome-wide DNA copy number alterations in primary SCC in relation to metastatic behavior.

Figure 5. Smoking is a risk factor for the development of CoPD and lung cancer. CoPD is independent of smoking behavior also a risk factor for the development of lung cancer, especially the SCC subtype.

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ReferencesSiesling S, Visser O, van Dijck JA, Coebergh JW. [Trends in the incidence and death from cancer from 1989-2003 in The Netherlands]. Ned Tijdschr Geneeskd 2006; 150(45):2490-2496.

Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997; 111(6):1710-1717.

Goldstraw P, Crowley J, Chansky K et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol 2007; 2(8):706-714.

Gloeckler Ries LA, Reichman ME, Lewis DR, Hankey BF, Edwards BK. Cancer survival and incidence from the Surveillance, Epidemiology, and End Results (SEER) program. Oncologist 2003; 8(6):541-552.

Barbone F, Bovenzi M, Cavallieri F, Stanta G. Cigarette smoking and histologic type of lung cancer in men. Chest 1997; 112(6):1474-1479.

Minna JD, Roth JA, Gazdar AF. Focus on lung cancer. Cancer Cell 2002; 1(1):49-52.

Travis WD, Lubin J, Ries L, Devesa S. United States lung carcinoma incidence trends: declining for most histologic types among males, increasing among females. Cancer 1996; 77(12):2464-2470.

Janssen-Heijnen ML, Nab HW, van Reek J, van der Heijden LH, Schipper R, Coebergh JW. Striking changes in smoking behaviour and lung cancer incidence by histological type in south-east Netherlands, 1960-1991. Eur J Cancer 1995; 31A(6):949-952.

Carrell RW, Lomas DA, Sidhar S, Foreman R. Alpha 1-antitrypsin deficiency. A conformational disease. Chest 1996; 110(6 Suppl):243S-247S.

Rabe KF, Hurd S, Anzueto A et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007; 176(6):532-555.

Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Oxidative Stress Study Group. Am J Respir Crit Care Med 1997; 156(2 Pt 1):341-357.

O’Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 1997; 155(3):852-857.

Saetta M, Di Stefano A, Maestrelli P et al. Activated T-lymphocytes and macrophages in bronchial mucosa of subjects with chronic bronchitis. Am Rev Respir Dis 1993; 147(2):301-306.

Saetta M, Di Stefano A, Turato G et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157(3 Pt 1):822-826.

McLEAN KH. The pathogenesis of pulmonary emphysema. Am J Med 1958; 25(1):62-74.

Pauwels RA, Rabe KF. Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 2004; 364(9434):613-620.

Proctor RN. Tobacco and the global lung cancer epidemic. Nat Rev Cancer 2001; 1(1):82-86.

Sethi JM, Rochester CL. Smoking and chronic obstructive pulmonary disease. Clin Chest Med 2000; 21(1):67-86, viii.

Mayer AS, Newman LS. Genetic and environmental modulation of chronic obstructive pulmonary disease. Respir Physiol 2001; 128(1):3-11.

Spina D, Ferri L, Cesario A, Granone P. Chronic obstructive pulmonary disease as a prognostic factor in non-small cell lung cancer. Eur J Cardiothorac Surg 2005; 28(3):505-506.

Papi A, Casoni G, Caramori G et al. COPD increases the risk of squamous histological subtype in smokers who develop non-small cell lung carcinoma. Thorax 2004; 59(8):679-681.

Skillrud DM, Offord KP, Miller RD. Higher risk of lung cancer in chronic obstructive pulmonary disease. A prospective, matched, controlled study. Ann Intern Med 1986; 105(4):503-507.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

chapter1

20

Tockman MS, Anthonisen NR, Wright EC, Donithan MG. Airways obstruction and the risk for lung cancer. Ann Intern Med 1987; 106(4):512-518.

Wasswa-Kintu S, Gan WQ, Man SF, Pare PD, Sin DD. Relationship between reduced forced expiratory volume in one second and the risk of lung cancer: a systematic review and meta-analysis. Thorax 2005; 60(7):570-575.

Malhotra S, Lam S, Man SF, Gan WQ, Sin DD. The relationship between stage 1 and 2 non-small cell lung cancer and lung function in men and women. BMC Pulm Med 2006; 6:2.

Mannino DM, Aguayo SM, Petty TL, Redd SC. Low lung function and incident lung cancer in the United States: data From the First National Health and Nutrition Examination Survey follow-up. Arch Intern Med 2003; 163(12):1475-1480.

Kishi K, Gurney JW, Schroeder DR, Scanlon PD, Swensen SJ, Jett JR. The correlation of emphysema or airway obstruction with the risk of lung cancer: a matched case-controlled study. Eur Respir J 2002; 19(6):1093-1098.

Brunnemann KD, Hoffmann D. Analytical studies on tobacco-specific N-nitrosamines in tobacco and tobacco smoke. Crit Rev Toxicol 1991; 21(4):235-240.

Hoffmann D, Hoffmann I, El-Bayoumy K. The less harmful cigarette: a controversial issue. a tribute to Ernst L. Wynder. Chem Res Toxicol 2001; 14(7):767-790.

Mio T, Romberger DJ, Thompson AB, Robbins RA, Heires A, Rennard SI. Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells. Am J Respir Crit Care Med 1997; 155(5):1770-1776.

Hellermann GR, Nagy SB, Kong X, Lockey RF, Mohapatra SS. Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells. Respir Res 2002; 3:22.

Takizawa H, Tanaka M, Takami K et al. Increased expression of transforming growth factor-beta1 in small airway epithelium from tobacco smokers and patients with chronic obstructive pulmonary disease (COPD). Am J Respir Crit Care Med 2001; 163(6):1476-1483.

Demoly P, Simony-Lafontaine J, Chanez P et al. Cell proliferation in the bronchial mucosa of asthmatics and chronic bronchitics. Am J Respir Crit Care Med 1994; 150(1):214-217.

Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995; 270(5235):467-470.

Alizadeh AA, Eisen MB, Davis RE et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403(6769):503-511.

Miki R, Kadota K, Bono H et al. Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays. Proc Natl Acad Sci U S A 2001; 98(5):2199-2204.

Chu S, DeRisi J, Eisen M et al. The transcriptional program of sporulation in budding yeast. Science 1998; 282(5389):699-705.

Whitfield ML, Sherlock G, Saldanha AJ et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol Biol Cell 2002; 13(6):1977-2000.

van de Vijver MJ, He YD, van’t Veer LJ et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 2002; 347(25):1999-2009.

Boldrick JC, Alizadeh AA, Diehn M et al. Stereotyped and specific gene expression programs in human innate immune responses to bacteria. Proc Natl Acad Sci U S A 2002; 99(2):972-977.

Pollack JR, Perou CM, Alizadeh AA et al. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 1999; 23(1):41-46.

Emmert-Buck MR, Bonner RF, Smith PD et al. Laser capture microdissection. Science 1996; 274(5289):998-1001.

Luo L, Salunga RC, Guo H et al. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat Med 1999; 5(1):117-122.

Matsui H, Suzuki K, Hasumi M et al. Gene expression profiles of human BPH (II): Optimization of laser-capture microdissection and utilization of cDNA microarray. Anticancer Res 2003; 23(1A):195-200.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

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1

Yim SH, Ward JM, Dragan Y et al. Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles. Toxicol Pathol 2003; 31(3):295-303.

Nakamura T, Furukawa Y, Nakagawa H et al. Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection. Oncogene 2004; 23(13):2385-2400.

Luzzi V, Mahadevappa M, Raja R, Warrington JA, Watson MA. Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. J Mol Diagn 2003; 5(1):9-14.

Febbo PG, Thorner A, Rubin MA et al. Application of oligonucleotide microarrays to assess the biological effects of neoadjuvant imatinib mesylate treatment for localized prostate cancer. Clin Cancer Res 2006; 12(1):152-158.

Yang F, Foekens JA, Yu J et al. Laser microdissection and microarray analysis of breast tumors reveal ER-alpha related genes and pathways. Oncogene 2006; 25(9):1413-1419.

Alevizos I, Mahadevappa M, Zhang X et al. Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis. Oncogene 2001; 20(43):6196-6204.

Balsara BR, Testa JR. Chromosomal imbalances in human lung cancer. Oncogene 2002; 21(45):6877-6883.

Spira A, Beane J, Shah V et al. Effects of cigarette smoke on the human airway epithelial cell transcriptome. Proc Natl Acad Sci U S A 2004; 101(27):10143-10148.

Hackett NR, Heguy A, Harvey BG et al. Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers. Am J Respir Cell Mol Biol 2003; 29(3 Pt 1):331-343.

Pierrou S, Broberg P, O’Donnell RA et al. Expression of genes involved in oxidative stress responses in airway epithelial cells of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007; 175(6):577-586.

de Boer WI, Hau CM, van Schadewijk A, Stolk J, van Krieken JH, Hiemstra PS. Expression of epidermal growth factors and their receptors in the bronchial epithelium of subjects with chronic obstructive pulmonary disease. Am J Clin Pathol 2006; 125(2):184-192.

Schulz C, Petrig V, Wolf K et al. Upregulation of MCAM in primary bronchial epithelial cells from patients with COPD. Eur Respir J 2003; 22(3):450-456.

Kranenburg AR, Willems-Widyastuti A, Mooi WJ et al. Chronic obstructive pulmonary disease is associated with enhanced bronchial expression of FGF-1, FGF-2, and FGFR-1. J Pathol 2005; 206(1):28-38.

Spira A, Beane JE, Shah V et al. Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer. Nat Med 2007; 13(3):361-366.

Bhattacharjee A, Richards WG, Staunton J et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci U S A 2001; 98(24):13790-13795.

Garber ME, Troyanskaya OG, Schluens K et al. Diversity of gene expression in adenocarcinoma of the lung. Proc Natl Acad Sci U S A 2001; 98(24):13784-13789.

Meyerson M, Franklin WA, Kelley MJ. Molecular classification and molecular genetics of human lung cancers. Semin Oncol 2004; 31(1 Suppl 1):4-19.

Wigle DA, Jurisica I, Radulovich N et al. Molecular profiling of non-small cell lung cancer and correlation with disease-free survival. Cancer Res 2002; 62(11):3005-3008.

Beer DG, Kardia SL, Huang CC et al. Gene-expression profiles predict survival of patients with lung adenocarcinoma. Nat Med 2002; 8(8):816-824.

Ikehara M, Oshita F, Sekiyama A et al. Genome-wide cDNA microarray screening to correlate gene expression profile with survival in patients with advanced lung cancer. Oncol Rep 2004; 11(5):1041-1044.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

chapter1

22

Kikuchi T, Daigo Y, Katagiri T et al. Expression profiles of non-small cell lung cancers on cDNA microarrays: identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs. Oncogene 2003; 22(14):2192-2205.

Oshita F, Ikehara M, Sekiyama A et al. Genomic-wide cDNA microarray screening to correlate gene expression profile with chemoresistance in patients with advanced lung cancer. J Exp Ther Oncol 2004; 4(2):155-160.

Hoang CD, D’Cunha J, Tawfic SH, Gruessner AC, Kratzke RA, Maddaus MA. Expression profiling of non-small cell lung carcinoma identifies metastatic genotypes based on lymph node tumor burden. J Thorac Cardiovasc Surg 2004; 127(5):1332-1341.

Takada M, Tada M, Tamoto E et al. Prediction of lymph node metastasis by analysis of gene expression profiles in non-small cell lung cancer. J Surg Res 2004; 122(1):61-69.

Singhal S, Amin KM, Kruklitis R et al. Differentially expressed apoptotic genes in early stage lung adenocarcinoma predicted by expression profiling. Cancer Biol Ther 2003; 2(5):566-571.

Singhal S, Amin KM, Kruklitis R et al. Alterations in cell cycle genes in early stage lung adenocarcinoma identified by expression profiling. Cancer Biol Ther 2003; 2(3):291-298.

Kettunen E, Anttila S, Seppanen JK et al. Differentially expressed genes in nonsmall cell lung cancer: expression profiling of cancer-related genes in squamous cell lung cancer. Cancer Genet Cytogenet 2004; 149(2):98-106.

Wikman H, Kettunen E, Seppanen JK et al. Identification of differentially expressed genes in pulmonary adenocarcinoma by using cDNA array. Oncogene 2002; 21(37):5804-5813.

Nakamura H, Saji H, Ogata A et al. cDNA microarray analysis of gene expression in pathologic Stage IA nonsmall cell lung carcinomas. Cancer 2003; 97(11):2798-2805.

Kobayashi K, Nishioka M, Kohno T et al. Identification of genes whose expression is upregulated in lung adenocarcinoma cells in comparison with type II alveolar cells and bronchiolar epithelial cells in vivo. Oncogene 2004; 23(17):3089-3096.

Powell CA, Spira A, Derti A et al. Gene expression in lung adenocarcinomas of smokers and nonsmokers. Am J Respir Cell Mol Biol 2003; 29(2):157-162.

Lam DC, Girard L, Ramirez R et al. Expression of nicotinic acetylcholine receptor subunit genes in non-small-cell lung cancer reveals differences between smokers and nonsmokers. Cancer Res 2007; 67(10):4638-4647.

Fukumoto S, Yamauchi N, Moriguchi H et al. Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers’ non-small cell lung carcinomas. Clin Cancer Res 2005; 11(5):1776-1785.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

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