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Identifier 39628
Title Μελέτη της αστάθειας μικροδορυφορικού DNA σε κυτταρολογικά δείγματα ασθενών με νεοπλασίες του πνεύμονα
Creator Papadakis, Emmanouel D
Abstract Lung cancer is a leading cause of cancer death among both men and women. Alterations at the DNA level occur and contribute significantly to the development of lung cancer. These alterations include an increase in the mutation rate, the presence of activated oncogenes and loss of onco-suppressor genes. Simple DNA repeat alterations such as microsatellite DNA alterations, are useful markers for the detection of both potent onco-suppressor genes, by reduction to hemizygosity of particular chromosomal regions, which presumably include the onco-suppressors and an increase in the mutation rate that characterizes cancer, in the form of microsatellite instability. Aim of the present study was to perform a genetic analysis of cytological specimens from lung cancer patients using polymerase chain reaction (PCR) amplification of microsatellite sequences. Cytological specimens such as sputum, or specimens obtained by bronchoscopy, such as bronchial washing or lavage, provide a simple and effective means of diagnosing lung cancer. However, conventional cytology has not been helpful in the early detection of lung cancer. Identification of cancer specific molecular markers would have a major impact in the detection of lung cancer in an early and possibly curable stage. The aim of the first part of the study was to investigate whether microsatellite alterations are a detectable phenomenon in fresh cytological material from lung cancer patients. We initially performed microsatellite instability (MI) and loss of heterozygosity (LOH) analysis of 20 microsatellite markers located on 7 different chromosomal arms, in cytological specimens of lung cancer patients mainly obtained by bronchoscopy or fine needle aspiration biopsy. The microsatellite loci assayed were located on chromosomes 2q, 8p, 9p, 9q, 13q (flanking or within BRCA2 gene), 14q and 17q (flanking or within BRCA1 gene). Based on our results MI was a common event in cytological specimens derived from lung cancer patients. MI was detected in 68% of cases, and 21% of cases showed instability in more than one microsatellite loci. Loss of heterozygosity most frequently affected chromosomes 8p(54% of cases), 13q(48% of cases) and 17q(44% of cases), indicating the presence of candidate tumour suppressor genes on these loci. We then proceeded to evaluate the incidence of microsatellite alterations in lung cancer patients in microsatellite loci adjacent to mismatch repair genes, since MI was a commonly occurring phenomenon in lung cancer. We used a set of 48 microsatellite DNA markers, located on 1p, 1q, 2p, 2q, 3p, 5q, 7p, 7q, 9p, 9q, 13q, 14q and 17p. In our study we included 54 squamous cell, 47 adenocarcinomas and 23 small cell carcinomas. The material assayed included matched bronchial washing and sputum specimens obtained from the same patient. 36 healthy donors were included in the study and sputum samples were obtained from them in order to evaluate the incidence of microsatellite alterations in a presumably healthy population. Microsatellite alterations were detected in at least one locus in all cancer patients but only in 22.2% of sputum specimens from healthy donors. Loss of heterozygosity (LOH) in bronchial washings from non small cell lung cancer patients (NSCLC) predominantly affected chromosomal loci 17p13.1-p13.3 (69.7%), 9p13.3-p24.1 (63.3%), 1p34.2-p36.22 (48.5%), 13q12.1-q13.1 (47.7%) and 3p22.3-p23 (42.7%). Bronchial washings from small cell lung cancer (SCLC) exhibited a different pattern of LOH, and the chromosomal arms mainly affected were 3p22.3-p23 (88.6%), 17p13.1-p13.2 (82.3%), 5q32-q33.1 (66.6%), 13q12.2-q13.1 (65.6%) and 9q22.33-q31.3 (52.9%). These findings suggest the participation of diverse molecular mechanisms in the pathogenesis of NSCLC and SCLC. Microsatellite instability (MIN) was detected in 65.2% of SCLC and 56.4% of NSCLC bronchial washings in at least one locus, and in only 8.7% and 4.0% respectively in at least three loci. Thus, even though MI is a common phenomenon in lung cancer, it does not seem to contribute in lung carcinogenesis through the expression of the replication error phenotype. Coincidences of LOH or MIN detection in sputum and bronchial washing from the same patient were 80.3% and 56.2% respectively. The fractional allele loss (FAL) mean value of all cancer specimens was 0.24±0.02 compared to 0.01±0.01 of healthy donors. Only 3 out of 124 lung cancer specimens (1.2%) exhibited FAL value less than 0.05, the highest observed in the healthy donors group. This suggests that, even though a “gray area” does exist, a threshold for FAL values can be calculated to disclose cancer prone individuals with the use of the appropriate micosatellite markers. LOH at the TP53 locus was found in approximately two thirds of lung cancer patients. Certain p53 polymorphisms have been associated with increased susceptibility to cancer. We examined the genotypic frequency of p53 codon 72 polymorphism in lung cancer patients, in order to determine the lost p53 allele whether being Arginine (Arg) or Proline (Pro). LOH at the TP53 locus was found in 51.85% of Arg/Pro patients. The Pro allele was lost in 78.6% of cases while the Arg allele was lost in 21.4%. The preferential retention of the Arg allele in heterozygous patients motivated us to examine the genotypic frequency of this polymorphism in normal controls too. p53 Arg/Arg genotype was significantly increased in lung cancer patients compared 154 to normal controls (50% versus 24.2%, P<0.002). Sputum and bronchial washing samples from each patient were assayed for the presence of human papillomavirus (HPV) since the Arg allele has been associated with HPV induced carcinogenesis. No patient had HPV infection of bronchial washing specimens. Our results suggest that p53 codon 72 Arg homozygosity is associated with advanced lung cancer, and that the Arg allele is preferentially retained in patients heterozygous for this polymorphism. On the other hand, HPV infection does not seem to play an important role in lung carcinogenesis.
Language Greek
Issue date 2003-04-01
Date available 2003-07-10
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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