A COPD-related gene expression signature in squamous cell lung cancer

Chronic obstructive pulmonary disease (COPD) and squamous cell lung carcinoma (SCC) are both smoking-related diseases. Interestingly, COPD itself is a risk factor for SCC, and this is independent of smoking history. Inferring from this knowledge, it is plausible to assume that patients with COPD may have a different gene expression profile in normal bronchial epithelium and in SCC. To test this hypothesis, we compared gene expression profiles in bronchial epithelium and in SCC from patients with and without COPD. Gene expression profiles were generated using whole human genome oligo microarrays for 28 laser microdissected histologically normal bronchial epithelium samples (10 without COPD, 18 with COPD) and 35 laser microdissected SCC samples (17 without COPD, 18 with COPD). We found no significant differences in gene expression levels between normal bronchial epithelium from patients with and without COPD. Remarkably, 374 genes were differentially expressed in SCC obtained from patients with or without COPD; 295 showed a higher expression level in SCC from patients with COPD and 79 genes showed higher expression levels in SCC from patients without COPD. Genes related to mitochondrial localization (n=44) and genes located on chromosomal arm 5q (n=34) were significantly overrepresented. For both categories, all genes showed a higher expression level in SCC from patients with COPD than from those without COPD. In conclusion, SCC from patients with COPD show a different gene expression profile compared to SCC from patients without COPD. This suggests that COPD related factors affect the gene expression signature in SCC. PATIENTS AND TISSUE SPECIMENS We collected resected bronchial tissues of the larger airways (bronchus diameter > 2 mm, surrounded by cartilage) from 10 individuals without COPD (“no COPD”) and 18 patients with COPD (“COPD”). We collected centrally located primary squamous cell lung carcinoma (SCC) from 35 patients; 17 patients without COPD (“no COPD”) and 18 patients with COPD (“COPD”). COPD stages were assessed according the Global initiative for chronic Obstructive Lung Disease (GOLD) classification. Briefly, individuals with a normal spirometry (FEV1/FVC greater than or equal to 70%) are classified as “no COPD”. Patients with FEV1/FVC < 70% are classified as “COPD”. For every patient, the most recent (post-bronchodilator) spirometric results before surgery were used to assess the GOLD stage. Bronchial tissues from 10 individuals without COPD were obtained during thoracotomy for non-small cell lung carcinoma (NSCLC); bronchial tissues from 18 patients with COPD were obtained during thoracotomy for NSCLC (n=11) or lung transplantation (n=7). Bronchus samples of patients with or without COPD were matched for current smoking status and packyears. Patients with SCC who were treated with chemotherapy, used intravenous and/or oral steroids before surgery, and patients with COPD due to alpha-1-antitrypsin deficiency were excluded. All tissue samples were snap frozen in liquid isopentane, and stored at -80 ºC until further processing. The study protocol was consistent with national ethical and professional guidelines (“Code of Conduct; Dutch Federation of Biomedical Scientific Societies”). LASER MICRODISSECTION MICROSCOPY For bronchus samples, only histologically normal bronchial epithelium that consists of basal epithelial, ciliated epithelial and goblet cells was laser microdissected. For SCC samples, only vital tumour cells without apparent admixture of inflammatory cells through the tumour fields were isolated using laser microdissection. Areas containing hyperplastic, metaplastic or dysplastic epithelial cells were excluded. An area of approximately 20 – 25 x106 μm2 was microdissected from frozen sections of 8 μm by the P.A.L.M. Microlaser Technology system, according to the manufacturer’s instructions (P.A.L.M., Bernried, Germany). Microdissected cells were immediately collected in lysis buffer (Macherey-Nagel, Düren, Germany). RNA ISOLATION Total RNA was isolated and purified from the laser-dissected cells with a Nucleospin RNA II kit (Macherey-Nagel), according to the manufacturer’s instructions, including DNase treatment. The quantity of DNA-free total RNA was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed with RNA 6000 PicoChip (Agilent, Palo Alto, CA) on the 2100 bioanalyzer (Agilent) by the ratio of the 18S and 28S rRNA bands combined with a low baseline. MICROARRAY APPROACH Agilent Whole Human Genome (WHG) Oligo Microarrays (GPL1708) (Agilent, Palo Alto, CA), containing 44K 60-mer oligonucleotides representing over 41,000 human genes and transcripts, were used to determine gene expression levels in laser microdissected bronchial epithelial cells. 150 ng of total RNA was amplified for one round using MessageAmp II (Ambion, Austin, USA). mRNA amplification, ULS labelling, Cy-dye coupling, hybridization and data extraction were performed at ServiceXS (Leiden, the Netherlands) according to manufacturer’s instructions. A total of 63 samples (28 bronchus, 35 SCC) were hybridized randomly, including a dye-swap, on 63 Agilent WHG arrays resulting in data from 126 individual hybridizations, based on the non-competitive conditions of a two-colour array (table of randomization will be added to this series as a supplementary table).