Affiliations of authors: A. Lamy, T. Frébourg, R. Sesboüé, Institut National de la Santé et de la Recherche Médicale (INSERM) Equipe Mixte INSERM (EMI) 9906, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides (IFRMP), Faculté de MédecinePharmacie, Rouen, France; J. Métayer, Service d'Anatomie et Cytologie Pathologiques, Centre Hospitalier Universitaire Régional (CHUR) de Rouen; L. Thiberville, INSERM EMI 9906, IFRMP, Faculté de MédecinePharmacie, and Service de Pneumologie, CHUR de Rouen.
Correspondence to: Richard Sesboüé, M.D., INSERM EMI 9906, Faculté de MédecinePharmacie, 22 Boulevard Gambetta, 76183 Rouen Cedex, France (e-mail: richard.sesboue{at}univ-rouen.fr).
Virmani et al. (1) recently reported that methylation of the retinoic acid receptor- (RAR
) gene promoter P2 was a frequent event in lung carcinomas and suggested that demethylation could be of therapeutic interest in these tumors. The timing of this epigenetic alteration in the transformation of normal bronchial cells to invasive cancer cells is, therefore, important to determine. Although many investigators have suggested that alteration of RAR
expression is an early phenomenon in lung carcinogenesis, it is difficult to study such alterations in precancerous stages because most intraepithelial neoplastic lesions are not detectable under conventional white-light bronchoscopy. The recent introduction of the light-induced fluorescence endoscopy (LIFE) system (2) greatly enhances the ability to detect preneoplastic lesions and allowed us to collect a large series of bronchial biopsy specimens from patients at high risk (because of asbestos and/or tobacco exposure or a history of head and neck cancer) for the development of lung cancer.
After giving written informed consent, patients were examined under fluorescence bronchoscopy (LIFE-Lung Imaging System; Xillix Co., Richmond, BC, Canada). This study has been approved by the regional Ethics Committee (CCPPRB-HN: Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Haute-Normandie). Biopsy specimens were taken from abnormal and, whenever possible, normal areas. Two samples were taken from each site: one was used for pathologic examination, and the other was cultured as described previously (3) to obtain pure bronchial cell populations. Biopsy specimens were collected from 38 patients, and bronchial cells were derived from 71 different biopsy specimens. Histopathologic examination subsequently revealed that these 71 biopsy specimens included normal epithelium (nine cases), inflammation (13 cases), hyperplasia (19 cases), mild dysplasia (16 cases), moderate to severe dysplasia (11 cases), and carcinoma in situ (three cases), classified according to the international nomenclature (4). After sodium bisulfite treatment of genomic DNA extracted from cultured cells at the second passage, the methylation status of the RAR gene promoter P2 was analyzed by the methylation-specific polymerase chain reaction according to the procedure of Côté et al. (5). In contrast to the DLD-1 colon carcinoma cell line used as a positive control (5), none of the 71 samples displayed a methylated RAR
promoter P2. To exclude false-negative results caused by possible interference of in vitro culture with the methylation process, we analyzed the same samples for the methylation status of CpG islands within the CDKN2A/p16INK4a gene (6) and found that this gene was indeed methylated in 13 of 71 samples.
The lack of methylation of the RAR gene promoter P2 in this series, which included 30 dysplastic lesions, indicates that methylation is probably not an early event in lung carcinogenesis, at least in non-small-cell lung carcinoma (NSCLC). Although further investigations exploring the kinetics of genetic or epigenetic alterations in the development of lung cancer are clearly needed, these results suggest that retinoids may be more appropriate than demethylating agents for chemoprevention of NSCLC.
NOTES
Supported by l'INSERM (grant PARMERCA 9611), l'Association pour la Recherche sur le Cancer, and La Ligue Nationale Contre le Cancer.
REFERENCES
1
Virmani AK, Rathi A, Zochbauer-Muller S, Sacchi N, Fukuyama Y, Bryant D, et al. Promoter methylation and silencing of the retinoic acid receptor- gene in lung carcinomas. J Natl Cancer Inst 2000;92:13037.
2 Lam S, Hung JY, Kennedy SM, Leriche JC, Vedal S, Nelems B, et al. Detection of dysplasia and carcinoma in situ by ratio fluorometry. Am Rev Respir Dis 1992;146:145861.[Medline]
3 Franklin WA, Folkvord JM, Varella-Garcia M, Kennedy T, Proudfoot S, Cook R, et al. Expansion of bronchial epithelial cell populations by in vitro culture of explants from dysplastic and histologically normal sites. Am J Respir Cell Mol Biol 1996;15:297304.[Abstract]
4 Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E, editors. Histological typing of lung and pleural tumours with contributions by pathologists from 14 countries. World Health Organization International Histological Classification of Tumors, XIII, 3rd ed. Berlin/Heidelberg (Germany): Springer-Verlag; 1999.
5 Cote S, Sinnett D, Momparler RL. Demethylation by 5-aza-2`-deoxycytidine of specific 5-methylcytosine sites in the promoter region of the retinoic acid receptor beta gene in human colon carcinoma cells. Anticancer Drugs 1998;9:74350.[Medline]
6
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:98216.
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