Identification of novel genetic alterations in non-small cell lung cancer progression

• Main Author: Siwoski, Arek
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/12241

To improve the survival rate in lung cancer, novel molecular targets that facilitate early detection and intervention are required. This thesis aims at screening multiple loci from distinct stages of lung cancer using genome scanning methods to identify frequently occurring genetic alterations that could potentially reveal novel regions of chromosomal instability. Due to the tissue heterogeneity in biopsies, tissue microdissection is necessary to isolate specific populations of cells from formalin-fixed, paraffin-embedded tissues representing progression stages of lung cancer. Since some of the commonly used histological stains such as Hematoxylin & Eosin have been shown to degrade DNA due to their acidic nature, a panel of histological stains have been tested for minimal degradative effect on DNA. To work with the minute quantities of DNA recovered from premalignant lesions, we have adapted a Randomly Amplified Polymorphic DNAPolymerase Chain Reaction (RAPD-PCR) assay to assess the quality of DNA extracted from the microdissected specimens, as well as a Southern hydridization assay to assess DNA quantity. DNA samples have been extracted from normal epithelial, hyperplastic, dysplastic, CIS and invasive carcinoma cells originated from 41 patients at various bronchial sites. DNA yield varied from 20ng to 2000ng depending on the amount of cells available in each lesion. Typical molecular techniques such as loss of heterozygosity (LOH) assay, DNA microarrays and fluorescent in situ hybridization (FISH) require large amounts of DNA to detect genomic changes. Using RAPD-PCR as a multi-loci fingerprinting technique, it is possible to screen for genetic alterations using minute DNA samples from microdissected specimens. RAPD-PCR involves simultaneous amplification of genomic DNA at multiple loci using short oligonucleotide primers. Gains and losses of PCR signals that occur in multiple patients identify regions potentially harboring candidate tumour suppressor genes or oncogenes. Our results show that twelve patients showed a gain of PCR signal in carcinoma in situ (CIS) tissue compared to normal tissue. By cloning, sequencing, and mapping the fragments, the recurring gains in PCR signal in multiple patients corresponded to regions at 8q24.3 (6/17), lq23.3 (2/18), 5q33.1 (2/16), and 7q36.2 (2/15). Microsatellite markers were chosen surrounding all four regions, however, due to minimal amounts of patient DNA, only regions 8q24.3 and 7q36.2 were verified for allelic imbalance in multiple patients using LOH. The groundwork has been laid for others to define the boundaries of these unstable regions as both regions contain many potential oncogenes and tumor suppressor genes.