Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas

Abstract

We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment.

Introduction

Lung cancer is responsible for the highest cancer-related morbidity and mortality worldwide [1]. Non-small cell lung cancer (NSCLC) comprises approximately 80% of all lung cancers, among which adenocarcinoma (AdC) and squamous cell carcinoma (SqC) are now the two most common histological subtypes. Cigarette smoking continues to be an important aetiological factor, with a clear implication of involvement in over 95% of male cases [2]. A correlation between the incidence of lung cancer in females and smoking habit has also been observed in recent years [3].

Due to the high incidence and mortality rates, much effort has been drawn towards investigating the cause and course of NSCLC. Although classical cytogenetic studies by banding analysis can provide an overall view of structural and numerical abnormalities, the frequent presence of karyotypic complexity has precluded more accurate interpretation. Conversely, molecular characterization by comparative genomic hybridization (CGH) and allelotyping has shown common over-representations of 1q, 3q, 5p, 8q and 20, and loss of heterozygosity (LOH) on 3p, 8p, 13q and 17p in NSCLC [4], [5], [6]. Despite the reportedly frequent genomic imbalances, the pattern of karyotypic alterations associated with AdC and SqC remains uncertain.

Comparative genomic hybridization (CGH) allows screening of the entire genome in order to identify regions of gain or loss of DNA and map them according to chromosomal location [7]. A major advantage of CGH is that tissue culture and preparation of metaphase spreads are not required as DNA extracted from a tumor specimen is used. Previous CGH studies of NSCLC cell lines and primary tumors have identified chromosomal regions that are commonly over- or under-represented, including gains of 1q, 3q, 5p, 7p and 8q and losses of 3p, 6q, 8p, 9p and 17p [8], [9], [10]. Multicolor fluorescence in situ hybridization (M-FISH) is a karyotyping technique that uses whole chromosome paints (WCP) to label each chromosome with a unique color [11]. Use of this technique facilitates the karyotyping of tumor cells and allows the identification of marker chromosomes and complex chromosomal rearrangements that would previously have been uninterpretable. Additionally, the use of color has opened up the field to non-cytogeneticists. Despite these technological advances, acquisition of suitable metaphase spreads is still a problem and consequently much of the work on solid tumors with M-FISH has been carried out using established cell lines.

We have now conducted a comprehensive molecular cytogenetic characterization of AdC and SqC tumors. By utilizing M-FISH we analysed one established lung adenocarcinoma cell line (A549) and another squamous cell lung carcinoma cell line (H520). Eighty primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples are detected by CGH. Our combined M-FISH and CGH analysis shows distinct patterns of genetic aberration in the two specific NSCLC subtypes, which might in turn influence the different pathogenetic pathways adopted by these tumors. It is hoped that the identification of such changes may eventually lead to improvements in diagnosis and identification of prognostic factors for lung adenocarcinomas and squamous cell lung carcinoma.