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Ann Thorac Surg 2005;79:1180-1188
© 2005 The Society of Thoracic Surgeons

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Original articles: General thoracic

Can Aberrant Promoter Hypermethylation of CpG Islands Predict the Clinical Outcome of Non-Small Cell Lung Cancer After Curative Resection?

Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital, Cancer Research Institute, Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, Korea

Accepted for publication September 21, 2004.

^_* Address reprint requests to Dr Y. T. Kim, Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital, 28 Yongon-Dong, Chongno-Gu, Seoul, 110–744, Republic of Korea (E-mail: ytkim{at}snu.ac.kr).

Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2003.

	Abstract

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

 
BACKGROUND: Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one cause for the loss of gene function. We examined whether aberrant DNA hypermethylation could be used to predict the clinical outcomes of patients with primary nonsmall cell lung cancer (NSCLC) after curative resection.

METHODS: We tested 61 patients with NSCLC using methylation-specific polymerase chain reaction (MSP) and searched for promoter hypermethylation of the genes p16^INK4a, retinoic acid receptor ß-promoter (RARßP2), death-associated protein kinase (DAPK), and O⁶-methylguanine-DNA-methyltransferase (MGMT). The clinical data, the presence of DNA hypermethylation, and clinical outcomes were analyzed.

RESULTS: Hypermethylation in the tumor samples was detected in 67% (41 of 61) for p16^INK4a, 49% (30 of 61) for RARßP2, 30% (18 of 61) for DAPK, and 62% (38 of 61) for MGMT. Thirty patients (49%) developed recurrence within 33 months; 16 in the remaining lung, 10 in other organs, and 4 in both. We found no correlation between the specific DNA hypermethylation and any of the clinicopathological characteristics of the patients. DNA hypermethylation was not associated with a different survival or recurrence rate. However, the aberrant hypermethylation of RARßP2 seemed to be related to the location of cancer recurrence. Although advanced T stage and preoperative chemotherapy were statistically significant in univariate analysis, unmethylation of DAPK (p = 0.030) and hypermethylation of RARßP2 (p = 0.014), as well as advanced T stage (p = 0.075) and preoperative chemotherapy (p = 0.025), were significant risk factors in multivariate analysis for early recurrence in the remaining lung.

CONCLUSIONS: The P2 hypermethylation of the RARß gene and unmethylation of DAPK seem to be important factors in predicting early cancer recurrence in the remaining lung and could be used as a prognostic marker in NSCLC. However, the clinical implications of this finding need further investigation.

	Introduction

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

 
Nonsmall cell lung cancer (NSCLC) is one of the most common malignancies in the world and is the leading cause of cancer-related death in many countries [1]. Surgical resection has been the most effective treatment for this serious disease. However, even after curative resection, many patients develop recurrences in either the remaining lung or in distant organs. For example, more than 30% of patients recur at various sites within 5 years even though the patients are in the early stages. Thus, significant research time has been spent developing a new molecular marker, which could accurately predict the treatment outcome. To date, none of the methods have proven to be effective for predicting long-term outcome.

Aberrant methylation of CpG islands acquired in the promoter regions of tumor cells within a specific gene is one cause for the loss of gene function. The silencing of tumor suppressor genes by promoter hypermethylation is a common feature in human cancer and hypermethylation of normally unmethylated CpG island in the promoter region of many cancer suppressor genes correlates with their loss of transcription in various human tumors [2]. In primary lung cancer, the inactivation of the tumor suppressor gene p16^INK4a [2], retinoic acid receptor beta [3], the DNA repair gene MGMT [4], detoxifying gene GSTP1 [5], and DAPK gene [6] by promoter hypermethylation has been well described.

We examined whether aberrant DNA hypermethylation could be used to predict the clinical outcomes of patients with primary NSCLC after surgical resection.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

 
Sample Collection
Tumor specimens from 61 patients (mean age, 59 years old; range, 38 to 79 years old), with no signs of distant metastasis, and who underwent surgical resection for primary NSCLC from 2000 to 2001 at Seoul National University Hospital were utilized for this study. Lobectomy was performed in 49 patients, pneumonectomy in 9, sleeve lobectomy in 4, and wedge resection in 1 patient. Radical mediastinal lymph node dissection was performed in all patients. Seven patients received preoperative chemotherapy. Patients whose mediastinal lymph node was affected by the tumor received additional radiation or chemotherapy. Lung cancer tissues were obtained from each patient immediately after lung resection had been performed, then quickly frozen in liquid nitrogen and stored at –80°C until analysis.

DNA Preparation and Bisulfite Modification
Tumors samples were digested overnight by proteinase K, and DNA was prepared by phenol-chloroform extraction and ethanol precipitation. One microgram of DNA was digested with restriction enzyme (Hind III, Intron biotechnology, Korea) and bisulfite modification was performed as reported by Herman and colleagues [7]. The DNA was denatured with 0.2 moles/L NaOH and 10 mmol/L hydroquinone (Sigma Chemical Company, St. Louis, MO) modified by 3 moles/L sodium bisulfite (Sigma) and incubated at 50°C for 16 hours [8, 9]. Modified DNA was then purified using a commercially available purification kit (Promega Wizard DNA Clean-Up System; Promega Corp, Madison, WI). The samples were treated again with NaOH, precipitated with ethanol, resuspended in water, and then stored at –80°C until used.

Methylation-Specific Polymerase Chain Reaction
Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines to uracil, whereas it does not convert methylated cytosines. The converted uracils are then converted to thymidine during the subsequent polymerase chain reaction (PCR) step, giving sequence differences between methylated and unmethylated DNA. PCR primers specific for either methylated or modified unmethylated DNA sequences were used. Primer sequences of p16^INK4a were 5'-TTA TTA GAG GGT GGG GTG GAT TGT-3' (sense) and 5'-CAA CCC CAA ACC ACA ACC ATA A-3' (antisense) for the unmethylated reaction, and 5'-TTA TTA GAG GGT GGG GCG GAT CGC-3' (sense) and 5'-GAC CCC GAA CCG CGA CCG TAA-3' (antisense) for methylated reaction. The primer sequences of RARßP2 for the unmethylated reaction were 5'-TTG AGA ATG TGA GTG ATT TGA-3' (sense) and 5'-AAC CAA TCC AAC CAA AAC AA-3' (antisense), and for methylated reaction they were 5'-TCG AGA ACG CGA GCG ATT CG-3' (sense) and 5'-GAC CAA TCC AAC CGA AAC GA-3' (antisense). The primer sequences of DAPK were 5'-GGA GGA TAG TTG GAT TGA GTT AAT GTT-3' (sense) and 5'-CAA ATC CCT CCC AAA CAC CAA-3' (antisense) for the unmethylated reaction, and 5'-GGA TAG TCG GAT CGA GTT AAC GTC-3' (sense) and 5'-CCC TCC CAA ACG CCG A-3' (antisense) for methylated reaction. The primer sequences of MGMT for the unmethylated reaction were 5'-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3' (sense) and 5'-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3' (antisense), and for methylated reaction the sequences were 5'-TTT CGA CGT TCG TAG GTT TTC GC-3' (sense) and 5'-GCA CTC TTC CGA AAA CGA AAC G-3' (antisense). All PCR amplifications were performed using the Ependorf thermocycler (Mastercycler; Eppendorf Corp, Germany) with tube control for accurate annealing temperatures. All reactions were performed with the hot start method using the Qiagen HotStart Master Mix Kit (Qiagen Corp, Leusden, The Netherlands). The PCR conditions of the four genes were as follows: 95°C for 15 minutes; then 35 cycles of 95°C for 30 seconds, the specific annealing temperature for 1 minute and 72°C for 1 minute; and a final extension of 7 minutes at 72°C. The specific annealing temperatures of the four genes were 69°C for the methylated p16^INK4a gene, 64°C for the unmethylated p16^INK4a, 59°C for the methylated RARßP2 gene, 54°C for the unmethylated RARßP2 gene, 64°C for both methylated and unmethylated DAPK, and 59°C for both MGMT reactions.

Normal lung tissue DNA was treated in vitro with an excess of SssI methyltransferase (New England Biolabs, Boston, MA) to generate completely methylated DNA at all CpGs, processed with bisulfite modification, and then used as a positive control for methylated alleles of each gene. Water control was added as a negative control. The PCR products were analyzed on a 2.5% agarose gel, stained with ethidium bromide, and visualized by ultraviolet illumination (Fig 1).

View larger version (47K): [in this window] [in a new window]   Fig 1. Representative MSP of the promoter of p16INK4a, RARßP2, DAPK, and MGMT in NSCLC. The presence of product in Lane U indicates the presence of unmethylated genes; the presence of product in Lane M indicates the presence of methylated genes. (bp = base pair; DAPK = death-associated protein kinase; M = methylation; MGMT = O6-methylguanine-DNA-methyltransferase; MSP = methylation-specific polymerase chain reaction; N = negative control; Nor = normal; NSCLC = nonsmall cell lung cancer; P = positive control; RARßP2 = retinoic acid receptor ß-promoter; Tum = tumor; U = unmethylation.)

Clinical Data
Informed consents were obtained from all patients. All 61 patients had follow-ups at the clinic every 3 months. The mean follow-up period was 27.9 months (range, 4 to 50 months). On every visit, chest X-rays and sputum cytology examinations were performed and chest CT scans were obtained on an annual basis. If there were any symptoms or observations suggesting recurrence, additional evaluations were performed.

The clinical outcomes and DNA hypermethylation patterns were analyzed along with clinical variables such as gender, cell type, pathologic tumor node metastasis (TNM) stage, status of resection margin, preoperative and postoperative chemotherapy, and postoperative radiation. Statistical differences between groups were examined by use of the ² test and Fisher's exact test with continuity corrections. Risk factors of overall survival and recurrence patterns were estimated by means of the Kaplan-Meier method and the differences between them were determined by the log-rank test. For multivariate analysis, independent prognostic factors were assessed by using the Cox proportional hazard model with enter method. A p value less than 0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

 
Frequency of Methylation in Lung Cancer Tissue
We determined the frequency of methylation of p16^INK4a, RARßP2, DAPK, and MGMT in 61 resected NSCLC tissues (Table 1, Fig 1). Fifty-nine patients (96.7%) exhibited abnormal promoter hypermethylation in at least one gene. The unmethylated form of all genes was detected in 100% of the NSCLC samples. There was no statistically significant correlation between the status of each gene methylation. We compared immunohistochemical staining results for RARß and RARßP2 methylation status. However, we could not find any significant correlation between the two results. (p = 0.718, Fig 2).

View larger version (120K): [in this window] [in a new window]   Fig 2. Immunohistochemical staining for RARß (original magnification x200). There was no correlation between the methylation status and immunohistochemical staining result: (A) strong positive; (B) negative. (RARßP2 = retinoic acid receptor ß-promoter.)

View larger version (7K): [in this window] [in a new window]   Fig 3. Cox hazard model analysis for freedom from recurrence in the remaining lung according to the (A) RARßP2 and (B) DAPK methylation status. - - - = unmethylated; — = methylated. (DAPK = death-associated protein kinase; RARßP2 = retinoic acid receptor ß-promoter.)

	Comment

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

None of the previous studies had analyzed the pattern of recurrence related to gene methylation. It is rational to consider that when there is a certain epigenetic change in the tracheobronchial tree and if the development of the cancer had been related to that event, there would be a high risk of having recurrence in the remaining lung after surgical resection of the primary tumor. Based on this hypothesis, we analyzed the risk of recurrence in the remaining lung with the gene methylation results. Univariate and multivariate analyses showed that advanced T staging and preoperative performance of adjuvant chemotherapy were statistically significant risk factors for developing a recurrence in the remaining lung. It is reasonable that an advanced T stage will have a higher risk of locoregional recurrence. However, it does not explain the recurrence in the contralateral remaining lung other than the fact that a higher T stage represents a more advanced stage. Preoperative chemotherapy most likely appeared as a significant risk factor because those who received neoadjuvant chemotherapy would have been at the advanced stage.

RARß is a member of the retinoic acid receptor family that is primarily responsible for mediating the effects of retinoic acid [13]. Reduced expression of RARß has been widely reported in lung tumor patients [14–16], which supports the hypothesis that the RARß gene may exert tumor-suppressive effects in lung cancer. Aberrant methylation of the RARß promoter gene has been well studied in lung cancer as well as in the other types of cancers [3, 17, 18]. Our observations demonstrated a high frequency of aberrant DNA methylation of the RARßP2 in lung cancer. What is not clear is the mechanism for the high rate of recurrence in the remaining lung in patients with P2 promoter methylation of RARß. We tested the pathology slides of 39 patients with immunohistochemical staining for RARß and could not demonstrate a significant correlation between the protein expression and its promoter methylation status. It may be difficult to show a definite correlation with the hypermethylation. The methylation of the specific location we tested may not be fully responsible for the expression of the corresponding protein [19]. In addition, when only a small number of tumor cells in the mass contain hypermethylation of the promoter area, the MSP will detect that methylation and the tumor will still express the protein. In order to evaluate the expression of each gene and thus comprehend the functional significance of the gene, a further comprehensive study is needed.

Our findings indicate that P2 methylation of the RARß gene seems to be an important factor in predicting early cancer recurrence in the remaining lung and could be used as a prognostic marker in NSCLC. A recent paper found that among former smokers, the RARßP2 methylation was about 2.87 times higher of a risk for developing a second primary lung cancer compared to those without RARßP2 methylation. The study also reports that for current smokers, hypermethylation of the RARßP2 was found to have a protective effect against second primary lung cancer [20]. This result is of interest as there is no clear way to differentiate between cancer recurrences in the remaining lungs from second primary lung cancer. Of even greater interest is that they defined the second primary lung cancer when the disease-free interval was greater than 2 years if the histology was the same as the first cancer. It means their cases are a relatively longer event compared to ours. When we analyzed the effect of RARßP2 with time effect using Cox regression with the time-dependent covariate model, we found that the effect of RARßP2 hypermethylation to the development of recurrence at the remaining lung decreased.

DAPK is a pro-apoptotic serine/threonine kinase involved in apoptosis. Aberrant methylation of DAPK was reported in lung cancers by methylation-specific PCR by many researchers. However, none of them demonstrated a relationship between DAPK methylation and clinical outcome [12, 21]. Our result is interesting because unmethylation, instead of hypermethylation of DAPK, was a risk factor for recurrence in the remaining lung.

Although we found that unmethylation of DAPK and hypermethylation of RARßP2 were risk factors for the development of a specific recurrence pattern, we were unable to correlate this finding with down-regulation of a specific protein. A recent paper suggests that considerable heterogeneity of methylation is present and there may be several mechanisms for down-regulation of DAPK as well as RARßP2 [19, 22].

Many researchers are investigating methylation status of normal tissue adjacent to the tumor [21, 23]. It is relevant to think that hypermethylation of normal tissue may suggest the presence of premalignant area and may have an effect on the prognosis. We tested our methylation analysis for normal tissue and analyzed their impact on the clinical outcome. However, we were unable to find any significance at all.

Although we could not demonstrate the functional mechanism, we believe our results suggest that the investigation of promoter hypermethylation may be a promising tool to predict the clinical outcome of lung cancer. As a result, future research should be directed towards investigating the detailed mechanism of promoter hypermethylation on lung cancer prognosis.

	DISCUSSION

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

 
DR W. ROY SMYTHE (Houston, TX): I have one question for you, Dr Kim. When you look at most solid tumors by genomic analysis, you find that there are usually many more genes that are upregulated than downregulated from an expression standpoint, and that has been pretty consistent with most solid tumors. We certainly understand the concept of tumor suppressor hypermethylation, but it has been suggested that hypomethylation of some genes may be important in malignant progression. I wanted to know if you could comment on that or if you had plans to look from a genomic standpoint at genes that might be upregulated in hypomethylation events.

DR MALCOLM V. BROCK (Baltimore, MD): I congratulate the authors, I really enjoyed their presentation.

I have three questions. The first question concerns the correlation between transcriptional inactivation and lack of gene expression with promoter hypermethylation. If your primers are not designed correctly within the promoter or if your polymerase chain reaction (PCR) conditions are pushed to the limit, you may throw off the correlation. My question is, did you perform any experiments, for example with immunohistochemistry, to show that in your hands promoter hypermethylation of the primary tumor samples actually correlated with lack of gene expression? I think that would be a very important experiment here as proof of principle to validate your PCR and the methylation frequencies observed.

The second question concerns paired adjoining nonmalignant tissue or "normal lung." Did you analyze paired "normal" lung for each of the primary tumors, and if so what was the methylation rate in these tissues? I appreciate that you used lung tissue as your normal control, but I was specifically wondering about the background methylation rates for nonmalignant adjoining lung tissue?

My third question regards the RARß story. Did you happen to look at any of the tumors that recurred to see if the RARß gene was methylated? Thank you.

DR DAVID S. SCHRUMP (Bethesda, MD): I enjoyed your presentation. I have two questions. The first is a follow-up to the question pertaining to normal tissues. What was the frequency of hypermethylation of these genes in the normal tissues? We know there is a field effect that is seen commonly with lung cancers, and a number of these genes may have been hypermethylated in histologically normal tissues.

My second question pertains to additional studies that you may have in progress. You looked at four of the six genes that are very commonly hypermethylated in lung cancer. Given the fact that K-ras mutations are known to correlate in a statistically significant adverse manner with survival, have you examined, or do you plan to investigate RASSF1A—a very pertinent tumor suppressor which is frequently hypermethylated in lung cancer? Thank you.

DR KIM: Thank you very much for your questions.

In answer to Dr Smythe's question regarding upregulated genes, I agree it would be a very promising subject. The reason I'm doing methylation studies for the analysis of gene research is as follows. If one tries to look at upregulated genes, one should perform reverse transcriptase–polymerase chain reaction (RT-PCR) or add quantitative measurement methods. By investigating the methylation, one can expect genomic expressions by a relatively simple method without such procedures. I agree that the genes, which are hypomethylated, may have some effect in cancer development. For example, I'm currently working on the CAGE gene, which is normally methylated, but hypomethylated in the cancer tissue.

Addressing Dr Brock's comments regarding immunohistochemistry, I am in complete agreement with you. However, other authors have already proved the genes that I studied in this report. They demonstrated that when these genes were hypermethylated, their gene expression was downregulated. I did not perform studies such as immunohistochemistry, quantitative RT-PCR, or protein studies.

I think that Dr Brock's and Dr Schrump's questions regarding the normal tissue are important questions. I have examined normal tissue for methylation of the RARß gene promoter and I found that about 25 cases of normal tissue were affected by hypermethylation. However, this result didn't correlate with any of the clinical findings. So I think we have to investigate this aspect a little bit more. To answer the third question of Dr Brock, regarding the recurred cases, we do not have a sufficient number of cancer tissue attained from reresection specimens. I'm more interested in finding out specific genes so that I can follow the patient by examining the DNA extracted from the patient's serum. Actually, this focus is my current research project. However, it's difficult to present the findings at this time, because the data are not yet sufficient.

In response to Dr Schrump's comments regarding the other genes, I'm currently working on the CAGE gene. There is evidence that other genes, such as E-cadherin, can affect long-term survival of lung cancer. Unfortunately, our series is just a 1-year follow-up so it is hard to comment on the long-term effect. Yes, I am planning to look at the other set of genes in a future study and I appreciate your suggestion of ras F1a. Thank you.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

	References

Top Abstract Introduction Material and Methods Results Comment DISCUSSION Acknowledgments References

 

1. Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999 CA Cancer J Clin 1999;49:8-31.[Abstract/Free Full Text]
2. Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia Adv Cancer Res 1998;72:141-196.[Medline]
3. Virmani AK, Rathi A, Zochbauer-Muller S, et al. Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas J Natl Cancer Inst 2000;92:1303-1307.[Abstract/Free Full Text]
4. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia Cancer Res 1999;59:793-797.[Abstract/Free Full Text]
5. Esteller M, Corn PG, Urena JM, Gabrielson E, Baylin SB, Herman JG. Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia Cancer Res 1998;58:4515-4518.[Abstract/Free Full Text]
6. Kissil JL, Feinstein E, Cohen O, et al. DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: possible implications for role as tumor suppressor gene Oncogene 1997;15:403-407.[Medline]
7. 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 USA 1996;93:9821-9826.[Abstract/Free Full Text]
8. Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands Proc Natl Acad Sci USA 1992;89:1827-1831.[Abstract/Free Full Text]
9. Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines Nucleic Acids Res 1994;22:2990-2997.[Abstract/Free Full Text]
10. Suzuki T, Sasano H, Kimura N, et al. Immunohistochemical distribution of progesterone, androgen and oestrogen receptors in the human ovary during the menstrual cycle: relationship to expression of steroidogenic enzymes Hum Reprod 1994;9:1589-1595.[Abstract/Free Full Text]
11. Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients Cancer Res 1999;59:67-70.[Abstract/Free Full Text]
12. Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in non-small cell lung cancers Cancer Res 2001;61:249-255.[Abstract/Free Full Text]
13. Clifford JL, Petkovich M, Chambon P, Lotan R. Modulation by retinoids of mRNA levels for nuclear retinoic acid receptors in murine melanoma cells Mol Endocrinol 1990;4:1546-1555.[Medline]
14. Lotan R. Aberrant expression of retinoid receptors and lung carcinogenesis J Natl Cancer Inst 1999;91:989-991.[Free Full Text]
15. Geradts J, Chen JY, Russell EK, Yankaskas JR, Nieves L, Minna JD. Human lung cancer cell lines exhibit resistance to retinoic acid treatment Cell Growth Differ 1993;4:799-809.[Abstract]
16. Xu XC, Sozzi G, Lee JS, et al. Suppression of retinoic acid receptor beta in non-small-cell lung cancer in vivo: implications for lung cancer development J Natl Cancer Inst 1997;89:624-629.[Abstract/Free Full Text]
17. Sirchia SM, Ferguson AT, Sironi E, et al. Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor beta2 promoter in breast cancer cells Oncogene 2000;19:1556-1563.[Medline]
18. Cote S, Momparler RL. Activation of the retinoic acid receptor beta gene by 5-aza-2'-deoxycytidine in human DLD-1 colon carcinoma cells Anticancer Drugs 1997;8:56-61.
19. Yang Q, Sakurai T, Yoshimura G, et al. Hypermethylation does not account for the frequent loss of the retinoic acid receptor beta2 in breast carcinoma Anticancer Res 2001;21:1829-1833.[Medline]
20. Kim JS, Lee H, Kim H, et al. Promoter methylation of retinoic Acid receptor Beta 2 and the development of second primary lung cancers in non-small-cell lung cancer J Clin Oncol 2004;22:3443-3450.[Abstract/Free Full Text]
21. Guo M, House MG, Hooker C, et al. Promoter hypermethylation of resected bronchial margins: a field defect of changes? Clin Cancer Res 2004;10:5131-5136.[Abstract/Free Full Text]
22. Toyooka S, Toyooka KO, Miyajima K, et al. Epigenetic down-regulation of death-associated protein kinase in lung cancers Clin Cancer Res 2003;9:3034-3041.[Abstract/Free Full Text]
23. Kim JS, Kim H, Shim YM, Han J, Park J, Kim DH. Aberrant methylation of the FHIT gene in chronic smokers with early stage squamous cell carcinoma of the lung. Carcinogenesis 2004;25:2165@001771..

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Young Tae Kim Seung Hee Lee Joo Hyun Kim Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, Y. T. Articles by Kim, J. H. Search for Related Content PubMed PubMed Citation Articles by Kim, Y. T. Articles by Kim, J. H. Related Collections Lung - cancer