P-290 Aberrant promoter hypermethylation of RUNX3 in non-small cell lung cancer confers adverse prognosis

Poster Session 2/Molecular

Biology

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phocytes obtained from healthy volunteers. Methylation was detected 1 of 40 nonmalignant lung tissues, of which corresponding tumor sample was methylated. The expression level of the hDAB2lP quantified with real time RT-PCR was down-regulated in methylated cell lines compared with unmethylated cell line. Furthermore the hDAB2lP expression was up-regulated in four methylated cell lines treated with 5-Aza-2deoxycytidine. We conclude that the hDAB2IP gene expression is frequently down regulated by aberrant promoter methylation in lung cancers and less expression of DAB2lP may result in lung carcinogeneSk

nent. A total of 98 biopsies from 30 base-line bronchoscopies have been analyzed thus far. We studied four chromosomal loci: 3p14,3p21, 9p21 and 17~13, using two microsatellite markers for each locus. In general, LOH and Al were increased with histological severity of the lesions, but no obvious differences were found between biopsies obtained from cancer patients and those obtained from smoking volunteers. The most affected loci were 3~14 and 9p21, followed by 3~21 and 17~13. Prospective follow-up of these high-risk patients should establish the value of these and other markers to assess the risk for second primary lung cancers.

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Chromosomal abnormalities in bronchial epithelium from smokers, non-smokers and lung cancer patients

Robbert J.C. Slebos’, Elizabeth Livanos’, Hyeon Woo Yim’, Scott H. Randella, Alden M. Parsons*, Frank C. Detterbeckz, M Patricia Riveraa, Jack A. Taylor’ ’ National institute of Environmental Health Sciences, N/H, Research niangle Park, USA; p University of North Carolina at Chapel Hi//, Chapel Hi//, USA Lung cancer risk goes up dramatically in individuals who smoke, although only about 1 out of 12 smokers eventually develop lung cancer. The identification of individuals who are at greatest risk of developing lung cancer would greatly improve diagnosis, early treatment and eventually life expectancy. One such marker for cancer risk might be the accumulated genetic damage to human bronchial epithelial (HBE) cells. Loss of chromosomal material as determined by microsatellite analysis is common in bronchial epithelium from smokers, but it is not normally found in cells from non-smokers. However, these studies do not detect larger chromosomal abnormalities such as duplications or translocations. Such abnormalities have been reported in small series of bronchial cells from lung cancer patients, but no direct comparisons have been made between chromosomal status in non-smokers, smokers and lung cancer patients. In this study, we used standard Trypsin-Giemsa staining to assess abnormal karyotypes in short-term HBE cell cultures obtained from lung cancer patients and from discarded native lungs at the time of lung transplantation. More than 30 HBE cell cultures were obtained from airway brushes performed during fluorescent bronchoscopy, from specimens of normal bronchus obtained during lung cancer surgery or from unused transplant lung tissue or excised native lungs at transplant. Twenty-five metaphases were scored for each HBE cell culture. Preliminary data show that of 2 non-smokers (6- and 35 years of age), one had a single abnormal metaphase lacking chromosome 17, while the other one had 4 abnormal metaphases lacking chromosome IO, X, 3p, and 9q. One 61-year old smoker had 3 abnormal metaphases, one lacking X, and 2 with reciprocal translocations between chromosome arms 7q and 13q, and 2q and IOq. One lung cancer patient had 3 abnormal metaphases with loss of chromosomes 13, 17 and Y, while a second did not show any abnormalities in 18 metaphases analyzed. Analysis of the full set will test the utility of karyotype analysis in the assessment of precancerous lesions in individuals at risk for lung cancer.

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P 289

Loss of heterozygosity in bronchial epithelium obtained with fluorescent bronchoscopy from patients at high risk for lung cancer

Robbert J.C. Slebos’, M Patricia Rivera2, David M. Urnbach’, Gordon P. Flake’, William K. Funkhouse?, Frank C. Detterbeck’, Jack A. Taylor’. ’ National Institute of Environmental Health Sciences, N/H, Research Triangle Park, USA; 2 University of North Carolina at Chapel Hi//, Chapel Hi//, USA; 3 University of North Carolina at Chapel Hi//, Chapel H//I, USA Patients who are surgically cured from Stage I or II NSCLC remain at increased risk for the development of a new primary carcinoma. Screening by fluorescent bronchoscopy is indicated for these patients. It is currently unknown how the histologic and molecular abnormalities found in premalignant lesions are associated with the risk for progression to overt malignancies. To study these questions, we initiated a screening study based on following high-risk patients by fluorescent bronchoscopy through a base-line bronchoscopy and a followup bronchoscopy after 2 years. Four biopsies were taken from pre-determined sites, while any abnormal areas either under white or fluorescent light were also biopsied. To date, 36 patients have been enrolled, of which 21 were ex-cancer patients, and 15 heavy smokers with no cancer history. Some of these patients had an extensive family history of lung cancer. Biopsies were snap frozen and used for Laser Capture Microdissection to obtain pure preparations of epithelial cells. We amplified the DNA equivalent of at least 100 cells by time-release PCR, in which DNA is amplified using 60 PCR cycles performed in a single reaction. PCR products were sized and quantified by capillary electrophoresis using fluorescent oligonucleotides. A calibration curve was constructed from DNA mixing experiments to decide cut-off values for loss of heterozygosity (LOH) and allelic imbalance (Al). In these experiments, intra- and inter-individual variation was approximately equal and generally lower than the error variance compo-

Aberrant Promoter Hypermethylation of RUNX3 in Non-small Cell Lung Cancer Confers Adverse Prognosis

Hisavuki Shiaematsu’, Makoto Suzuki”, Kuniharu Miyajima3, Takao Takahashi4, Ubaradka G. Sathyanarayanas, Elizabeth M.P. Brambillaa, John D. Minna7, Adi F. Gazdar7. ’ Okayama University, Okayama, JAPAN; ’ Chiba University. Chiba, JAPAN; 3 Tokyo Medical University: Tokyo, JAPAN; 4 Gifu Univrsity, Gifu, JAPAN; s Hamon Center Therapeutic Oncology Research Univ of Texas Southwestern Medical Center, Dallas, USA; s Laboratoire de Pathologic Ce//u/aire, Cenfre Hospilalier Regional Universitaire, Grenoble, FRANCE; 7 Hamon Center for Therapeutic Oncology Research Univ of Texas Southwestern Medical Center, Da//as, USA RUNX3 located at 1~36 is a member of the Runt domain transcription factor family which are integral components of signaling cascades mediated by both TGF-6 and bone morphogenetic proteins (Ito Curr Opin Gen Dev 13:43, 2003). RUNX3 is a tumor suppressor gene (TSG) of gastric cancer and is an important component of the TGF-6 induced tumor suppressor pathway (lto ibid, and Li et al. Cell 109:113, 2002). 1~36 is a frequent site of allele loss in lung cancers with no TSG yet identified. Thus, we examined the promoter methylation of RUNX3 in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines and primary tumors by methylation-specific PCR (MSP) and expression in tumor cell lines by RT-PCR. Loss of RUNX3 mRNA expression was observed in 43% of 23 NSCLC lines, while RUNX3 expression were detected in all 14 SCLC lines (PC 0.001). Treatment of expression negative lung cancer cell lines with 5-aza-2’-deoxycytidine restored RUNX3 expression. Promoter methylation frequencies were 35% of NSCLC lines, 0% of SCLC lines, 23% of primary 348 NSCLCs, 0% of primary 9 SCLCs, and 0% of 8 bronchial carcinoids. Aberrant methylation status was significant associated with loss of RUNX3mRNA expression (concordance ratio: 92%; P< 0.001). DNA promoter methylation of RUNX3 was found in 28% (N = 201) adenocarcinomas, 12% (N = 134) squamous cell carcinomas, and 58% (N = 12) large cell carcinomas. RUNX3 methylation was not detected in normal corresponding lung tissues. The proportion of RUNX3 methylation tended to increase with clinical stage progression (P=O.O61). However, patients with RUNX3 methylation had a significantly poorer overall survival than those without methylation (P= 0.006; log-rank test), and in multivariate analysis, RUNX3methylation was a significant independent prognostic factor for overall survival (P= 0.002; risk ratio 1.80; 95% Cl, 1.24-2.61). In conclusion, RUNX3 is inactivated by promoter methylation in NSCLC but not SCLC and the frequencies of methylation are different among histologipal NSCLC subtypes indicating that RUNX3 methylation contributes to the pathogenesis of NSCLC where it is a prognostic molecular marker.

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Distinct DNA Methylation Profiles in Malignant Mesothelioma, Lung Aclenocarcinoma, and Non-Tumor Lung

Jeffrey A. Tsou’, Linda Y.C. Shen’, Kimberly D. Siegmund’, Tiffany I. Long’, Peter W. Laird’, Chandrika Seneviratne’, Michael N. Koss’, Harvey I. Pass?, Jeffrey A. Hagen’, Ite A. Laird-Offrinoa’ ’ University of Southern California, Los Angeles, USA; ’ Karmanos Comprehensive Cancer Center, Detroit, USA Malignant mesothelioma (MM), which can be difficult to distinguish from lung adenocarcinoma, is an aggressive cancer strongly associated with asbestos exposure. In an attempt to identify molecular markers for MM and adenocarcinoma, we examined the DNA methylation status of 14 loci. Analysis of methylation profiles in 10 MM and 8 adenocarcinoma cell lines showed that methylation levels in APC were significantly higher in adenocarcinoma than MM cell lines (P=O.OOOS), while methylation of CDHI was higher in MM (P