Genome-wide screening of chromosomal imbalance in NSCLC by array-based comparative genomic hybridization(array-CGH)

Abstract

Lung cancer is a leading cause of cancer deaths throughout the world. Non-small cell lung cancer (NSCLC), the major histological subgroup, shows widespread chromosomal aberrations. Genome-wide screening for DNA gains/losses is a useful approach to identify genetic loci harboring potential oncogene/tumor suppressor gene (TSG). Currently, comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) analysis are the most commonly used screening methods, but an array-based CGH approach enables a higher resolution power for detecting DNA copy number changes spanning a single BAC clone. The aim of this study is to identify chromosomal aberrations in NSCLC by array-CGH as a preliminary step towards the identification of oncogene/TSG. 48 cases of NSCLC from 31 non-smokers (NS) and 17 smokers (SM), including 20 males and 28 females were studied. 3200 BAC clones with minimal repetitive sequences chromosomally spaced at about 1Mb interval were arrayed in triplicate on glass slides and the hybridization results read by a microarray scanner. For each case, duplicate sets of hybridizations were performed between tumour and sex-matched reference DNA pooled from peripheral blood mononuclear cells of 15 normal young NS. In the second hybridization, reverse-labeling of samples was performed to obtain reciprocal readings for the calculation of results. Hybridizations using reference DNA only were performed to control for non-random background aberrations. The frequency of genomic gain/loss for each locus was calculated, and 60 clones showing high frequency imbalances were selected for sequence-verification of their chromosomal locations. DNA copy number alterations were observed in all tumors studied. Overall, DNA losses were more frequent than gains. The most frequent locus of DNA gain and loss, respectively, was 1q32 (29/48, 60%) and 4q35 (26/48, 54%). Tumors of SM harbored more changes than NS, with significant differences (p<0.05) at 4q35, 6p21.2-21.3, 10p15, 10q23, 11p14, 13q32 and 16q22; squamous cell carcinoma (SCC) showed more frequent alterations than adenocarcinoma (AD) at 4p15.3, 6p21.2-21.3, 7q11-21, 10p12, 10p15, 10q26, 11q13 and 15q25-26 (p<0.05). Patients with advanced stage showed more changes than those at early stage at 11q13, 18q22.1 and 19q13.3 (p<0.05). Fluorescence in-situ hybridization (FISH) study was performed on 2 primary lung cancer cell lines using BAC clone probes at 1q32, 1q21 and 6p12, verifying DNA gains at 1q32 and 1q21 and loss at 6p12. In conclusion, the results suggest that array CGH is a valuable first step to target the genome for DNA imbalances and to further identify candidate genes involved in the development of NSCLC.