Abstract

ABSTRACT Lung cancer is the leading cause of cancer-related death worldwide. Given its impact on human health, extensive research is being conducted in an effort to reduce the global lung cancer death toll. Specifically, much interest has been placed on the development of biomarkers and the discovery of novel prognostic factors. Over the past 2 decades it has become evident that the cancer genome is not only affected by genetic abnormalities, such as mutations, deletions and chromosomal rearrangements, but also by epigenetic changes such as DNA methylation in gene promoter sequences. Following this line of investigation, we hypothesize the characterizations of whole genomic changes in lung cancer using new microarray base technology while for the first time molecular profiling in (NSCLC) was performed with three different platforms (CGH Microarray , SNPs Microarray and DNA Methylation ). Our study were involve in several stages first to isolate tumor genomic DNA from biopsy specimens from patients with NSCLC to be marking place of genomic tumor DNA and to optimize method of new microarray base technology , comparative genomic hybridization Microarray, SNPs Microarray and Methylation Microarray. Than to scanning of hybridization microarrays, the fluorescent image processing and analysis software the digitized results for each tumor sample, and summarize the results of all samples and determine the frequency of genetic gain , losses, loss of heterozygotes and Methylations DNA ,than identifing the the most common regional aberrations on cytogenetic bands.And otheres aberrations such LOH SNPs and Methylations DNA according the technology. In chapter tow we have performed whole genome oligonucleotide microarrays-based comparative genomic hybridization in ten samples of non-small cell lung cancer. Trisomies were discovered for chromosomes 1, 13, 18 and 20. Affected by genetic gain were chromosome arms 5p, 7p, 11q, 20q and Хq, and by genetic losses - 1p, 5q, 10q and 15q. Microstructural (<5 Mbp) genomic aberrations were revealed in regions 7p (containing EGFR) and 12p (containing KRAS) for gains and in 3p26 and 4q34 for losses. Based on high amplitude of alterations and small overlapping regions, new potential oncogenes have been suggested - NBPF4 (1p13.3); ETV1, AGR3 and TSPAN13 (7p21.3 -7p21.1); SOX5 and FGFR1OP2 (12p12.1-12p11.22); GPC6 (13q32.1). Significant genetic losses were assumed as containing potential tumor-suppressor genes: DPYD (1p21.3); CLDN22, CLDN24, ING2,CASP3, SORBS2 (4q34.2-q35.1); DEFB (8p23.1). Our results complement the picture of genomic characterization of non-small cell lung cancer. In chapter 3we used SNPs Microarrays. based on single nucleotide polymorphisms is one such technology. It has made possible genome-wide allelic association studies of predisposition to common clinical problems. This approach is also being used to identify somatic changes in cancer. We analyzed non-small cell lung cancer (NSCLC) tumors by Genome-wide SNP (Single Nucleotide Polymorphism) genotyping experiment. We found different duplications in all tumors (trisomy, duplications of whole arms and microduplications) except in tumor samples of patient No 2. Seven from 8 patients had a trisomy. Trisomy of 10 different chromosomes were found in two patients, trisomy of 4 chromosomes were found in three patients, trisomy of three different chromosomes were found in one patient and trisomy of tow chromosomes were found in one patient. We obtained loss of heterozygosity(LOH) in five patients and deletion of chromosome 17 in tumor No2.Molecular profiling of tumors revealed high chromosomal instability of lungcancer.In chapter four performed Methylation Microarrays approach This approach is also being used to identify the DNA methylation in gene promoter sequences so after methylation microarray analysis we identified five genes with different methylation statues between patients and controls (Znf569, MAOA, LAS1L, SIM1, DHP). Specific methylation statues was found in Grad 3 tumors were 27 hiper methylated gene (10 in promoters , 3 downstream and 14 in gene body) and on hypo methylated in gene body. Finally in our study we characterized an new candidate oncogenes, tumor suppressor genes and genes with high methylation in promoter and LOH were identified as molecular mechanism in lang cancer patients . SNPs Microarray study reviewed predominantly genetic duplications. The investigation in these area will contained in the near future