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Aberrant Promoter Methylation of Multiple Genes in Non-Small Cell Lung Cancers¹

Hamon Center for Therapeutic Oncology Research [S. Z-M., A. K. V., A. F. G., J. D. M.], Departments of Pathology [A. F. G.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], The University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75390-8593; Department of Thoracic Medicine, The Prince Charles Hospital, Chermside, Brisbane 4032, Australia [K. M. F.]; and Nuffield Department of Pathology & Bacteriology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom [J. G.]

	ABSTRACT

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Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one method for loss of gene function. We determined the frequency of aberrant promoter methylation (referred to as methylation) of the genes retinoic acid receptor ß-2 (RARß), tissue inhibitor of metalloproteinase 3 (TIMP-3), p16^INK4^a, O⁶-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14^ARF, and glutathione S-transferase P1 (GSTP1) in 107 resected primary non-small cell lung cancers (NSCLCs) and in 104 corresponding nonmalignant lung tissues by methylation-specific PCR. Methylation in the tumor samples was detected in 40% for RARß, 26% for TIMP-3, 25% for p16^INK4^a, 21% for MGMT, 19% for DAPK, 18% for ECAD, 8% for p14^ARF, and 7% for GSTP1, whereas it was not seen in the vast majority of the corresponding nonmalignant tissues. Moreover, p16^INK4^a methylation was correlated with loss of p16^INK4a expression by immunohistochemistry. A total of 82% of the NSCLCs had methylation of at least one of these genes; 37% of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes methylated, 13% of the NSCLCs had three genes methylated, 8% of the NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes methylated. Methylation of these genes was correlated with some clinicopathological characteristics of the patients. In comparing the methylation patterns of tumors and nonmalignant lung tissues from the same patients, there were many discordancies where the genes methylated in nonmalignant tissues were not methylated in the corresponding tumors. This suggests that the methylation was occurring as a preneoplastic change. We conclude that these findings confirm in a large sample that methylation is a frequent event in NSCLC, can also occur in smoking-damaged nonmalignant lung tissues, and may be the most common mechanism to inactivate cancer-related genes in NSCLC.

	INTRODUCTION

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Alterations of the pattern of DNA methylation have been recognized as common changes in human cancers (1) . These are thought to have important implications for abnormalities of gene expression, chromosome structure, timing of DNA replication, and chromatin organization (1 , 2) . Aberrant methylation of normally unmethylated CpG-rich areas, also known as CpG islands, which are located in or near the promoter region of many genes, has been associated with transcriptional inactivation of defined TSGs³ in human cancer (3) . Thus, aberrant methylation serves as an alternative to the genetic loss of a TSG function by deletion or mutation (4) .

Aberrant promoter methylation (referred to as methylation) has been described for several genes in various malignant diseases including lung cancer (5, 6, 7, 8, 9, 10, 11, 12, 13) . In lung cancer, methylation of the TSG p16^INK4, the DNA repair gene MGMT, and the detoxification gene GSTP1 has been found in primary tumors (3 , 7 , 12 , 14) . Moreover, methylation of these genes and the apoptosis-associated gene DAPK has been described in serum DNA of NSCLC patients (15) . Interestingly, p16^INK4^a methylation has also been observed in precursor lesions of lung carcinomas, which makes it a reasonable candidate biomarker for the early diagnosis of lung cancer (16) . Methylation of TIMP-3 has been described in 4 of 21 (19%) NSCLCs (11) , and TIMP-3 is thought to suppress primary tumor growth (17 , 18) . The RARß-2 gene, RARß, which may function as a TSG (19 , 20) , has been observed to be silenced by methylation in colon cancer and breast cancer (6 , 9) . ECAD, which plays a role in invasion suppression, has been found methylated in breast and prostate carcinomas (5) . Esteller et al. (13) recently reported methylation of p14^ARF in 28% of primary colorectal carcinomas and suggested that methylation-associated inactivation of p14^ARF is independent of p16^INK4^a methylation and p53 mutational status.

Although several reports about methylation of various genes in lung cancer have been published, in most cases, the methylation status has been investigated for just a single gene or in a small number of samples (3 , 7 , 11 , 12 , 15 , 21) . Therefore, we decided to investigate methylation of multiple genes in a large sample collection of primary resected NSCLCs and their associated nonmalignant lung tissues, for which we also had clinical data and results about certain other molecular abnormalities. We determined the frequency of methylation of the eight genes RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, ECAD, p14^ARF, and GSTP1 in 107 primary NSCLCs and 104 corresponding nonmalignant lung tissues by MSP. Methylation of these genes was shown to occur in confirmed promoter regions or in 5' CpG islands in or near putative promoter regions. This analysis also provided us with the opportunity to determine whether the methylation status of the individual genes occurred independently of one another or with other molecular abnormalities. Finally, we wanted to know whether methylation of these genes was correlated with clinical features such as sex, age, smoking history, tumor stage, histology, and overall survival of the patients.

	MATERIALS AND METHODS

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Tumor Samples.
Primary tumor samples (n = 107) and corresponding nonmalignant lung tissues (n = 104) were obtained from NSCLC patients who had been treated with curative resectional surgery in The Prince Charles Hospital (Brisbane, Australia) between June 1990 and March 1993. This cohort of patients had been investigated previously for various genetic abnormalities (22, 23, 24, 25, 26, 27, 28) . There were 76 males and 31 females (age, 28–81 years; mean age at diagnosis, 61 years). Sixty-one patients had stage I disease, 21 patients had stage II disease, 24 patients had stage IIIA disease, and 1 patient had stage IIIB disease. Histological subtypes included 45 adenocarcinomas, 43 squamous cell carcinomas, 11 adenosquamous carcinomas, 4 large cell carcinomas, 3 atypical carcinoids, and 1 typical carcinoid. Ninety-eight patients were smokers (mean pack-years, 31), and the rest of patients were never smokers or nonsmokers. Survival data of 5 or more years were available on most patients.

MSP.
DNA was extracted as described previously (22) , and bisulfite modification of genomic DNA was performed as reported by Herman et al. (29) . Briefly, 1 µg of genomic DNA was denatured with NaOH (final concentration, 0.2 M), and 10 mM hydroquinone (Sigma) and 3 M sodium-bisulfite (Sigma) were added and incubated at 50°C for 16 h. Afterward, modified DNA was purified using Wizard DNA purification resin (Promega) followed by ethanol precipitation. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines (but not methylated cytosines) to uracil, which are then converted to thymidine during the subsequent PCR step, giving sequence differences between methylated and unmethylated DNA. PCR primers that distinguish between these methylated and unmethylated DNA sequences were then used. Primer sequences of all genes for both the methylated and the unmethylated form, annealing temperatures, and the expected PCR product sizes are summarized in Table 1 . The PCR mixture contained 10x PCR buffer (Qiagen), deoxynucleotide triphosphates (1.25 mM), primers (final concentration, 0.6 µM each per reaction), 1 unit of HotStarTaq (Qiagen), and bisulfite-modified DNA (150 ng). Amplification was carried out in a 9700 Perkin-Elmer Thermal Cycler. DNA from peripheral blood lymphocytes of healthy individuals and water blanks were used as a negative control for methylated genes. DNA from peripheral blood lymphocytes treated with SssI methyltransferase (New England Biolabs) was used as a positive control for methylated alleles. Fifteen µl of each PCR reaction were loaded onto a 2% agarose gel and visualized under UV illumination. The PCR for all samples demonstrating methylation for the individual genes was repeated to confirm these results.

Statistics.
Statistical analysis was performed using the ² and Fisher’s exact test for differences between groups and t tests between means. Overall survival was calculated using Kaplan-Meier log-rank testing. To determine the overall rate of methylation in individual samples, we used the MI. The MI is defined as a fraction representing the number of genes methylated/the number of genes tested. A previously designed Microsoft Visual Basic Program was used for color formatting and visualization of our data (30) .

	RESULTS

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Frequency of Methylation in Primary NSCLCs and Their Corresponding Nonmalignant Lung Tissues.
We determined the frequency of methylation of RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, ECAD, p14^ARF, and GSTP1 in 107 resected NSCLCs and in 104 corresponding nonmalignant lung tissues by MSP (Fig. 1 ; Table 2 ). In the corresponding nonmalignant lung tissues, methylation of p16^INK4^a, MGMT, ECAD, and GSTP1 was not detected, but it was seen at low frequencies for RARß (14%), TIMP-3 (8%), DAPK (6%), and p14^ARF (5%; Table 2 ). The bands that were seen for the methylated form in nonmalignant tissues, especially for RARß, were faint. The detailed results of methylation for each gene in all tumors compared with the corresponding nonmalignant lung tissues are shown in Fig. 2 . The unmethylated form of all genes was detected in 100% of samples in both tumors and nonmalignant tissues. Because the tumor specimens represented macroscopically isolated samples that contained both tumor and nonmalignant tissue, this was expected.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Methylation analysis of eight genes in primary NSCLCs and their corresponding nonmalignant lung tissues by MSP. The gene studied is given at the left of each panel. TU, tumor; NL, nonmalignant lung tissue; Lane U, amplified product with primers recognizing unmethylated sequence; Lane M, amplified product with primers recognizing methylated sequence. Peripheral blood lymphocytes (L) were used as a negative control. In vitro methylated DNA (IVD) was used as a positive control for methylation. H2O, water blanks. The PCR product sizes of all of the genes are summarized in Table 1 .

View larger version (103K): [in this window] [in a new window]   Fig. 2. Summary of methylation of RARß, TIMP-3, p16INK4a, MGMT, DAPK, ECAD, p14ARF, and GSTP1 in resected primary NSCLCs (left) and corresponding nonmalignant lung tissues (right). Green boxes represent samples that are not methylated; blue boxes represent samples that are methylated.

Clinicopathological Correlations.
We analyzed the methylation changes in the tumors and the clinical data obtained from these patients (Table 3) . Overall, we found no correlation between the MI (overall fraction of genes methylated) and any of the clinicopathological characteristics of the patients. A significantly longer overall survival was found for patients whose tumors showed methylation of ECAD (P = 0.005, Kaplan-Meier log-rank test). This result was seen particularly in stage I disease. We found no association between methylation of RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, p14^ARF, or GSTP1 and survival, regardless of whether we analyzed all stages combined or performed a separate analysis for stage I, II, and III disease. Lymph node involvement is a well-established prognostic indicator for resected NSCLC, and we found lymph nodes were involved with the tumor in 41% of samples with any gene methylated, but in only 11% of samples in which no genes showed methylation (P = 0.012). To summarize our findings regarding other clinical parameters, methylation of TIMP-3 was detected more frequently in women than in men, DAPK methylation and p16^INK4^a methylation were more frequent in men than in women, and p16^INK4^a methylation was more frequent in squamous carcinomas than in adenocarcinomas and was seen only in smokers. When making multiple comparisons of clinical data with multiple biomarkers like the methylated genes, such as was done in this study, caution must be used with conservative statistical corrections (such as the Bonferroni, Tukey, and Newman-Kauls post tests) before deciding that significant correlations exist. These also need to be confirmed in other data sets and larger series. Thus, we feel the most conservative approach is to present the data in tabular form for future reference without drawing any statistical conclusions of significance (Table 3) .

	DISCUSSION

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This report describes the frequency of methylation of the genes RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, ECAD, p14^ARF, and GSTP1 as well as the correlation of these methylation changes with clinicopathological characteristics and molecular abnormalities in a large number of patients. Methylation of RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, and GSTP1 had been described previously in lung cancer cell lines or small numbers of primary lung tumors, and our results are similar to the previously reported data (3 , 7 , 11 , 12 , 14 , 15 , 21) . However, methylation of ECAD and p14^ARF had not been reported in lung cancer. We also investigated methylation in the corresponding nonmalignant lung tissues. Several studies reported the lack of methylation in nonmalignant tissues and described methylation as a tumor-restricted event. However, we found methylation of RARß, TIMP-3, DAPK, and p14^ARF in some of the matched nonmalignant lung tissues. We cannot exclude that this may be due in some cases to contamination with adjacent malignant cells. However, from many previous studies, we have learned that preneoplastic/preinvasive and even histologically normal smoking-damaged epithelium has suffered genetic changes (31, 32, 33) . Thus, a possible explanation for detecting methylated alleles in the nonmalignant lung samples is that they represent premalignant changes. To begin to address this issue, we compared the specific genes methylated in the tumors and nonmalignant tissues from the same patient (Fig. 2 ; Table 4 ). In the majority of cases, the genes methylated in the nonmalignant tissues were not those methylated in the corresponding tumors from the same patients. This would indicate that the methylation changes in these cases do not represent tumor contamination but are more likely premalignant changes. This was especially true in the case of RARß methylation, which we found relatively frequently but with weak signals in the corresponding nonmalignant lung tissues. RARß mRNA and protein expression have been lost in some smoking-damaged lungs (34, 35, 36) . These findings are also in agreement with the results reported by Cote et al. (6) and Bovenzi et al. (9) , who described RARß methylation in a few samples of corresponding normal colon and breast tissues associated with cancers. The studies by Belinsky et al. (16) , who reported the appearance of p16^INK4^a methylation in hyperplasias associated with squamous lung cancers, and Esteller et al. (13) , who reported a similar rate of p14^ARF methylation in colorectal adenomas and colorectal carcinomas, are other examples where methylation may be an early event in cancer development. However, we found no examples of p16^INK4^a methylation and only a few examples of p14^ARF methylation in nonmalignant lung samples from lung cancer patients. It has been described that aging is associated with methylation of certain genes (4) . Therefore, the aging mechanism could also be a possible explanation for detecting methylation in nonmalignant lung tissues.

Another aim of this study was to investigate whether methylation of RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, ECAD, p14^ARF, and GSTP1 in NSCLC is associated with clinicopathological parameters, particularly survival, of these patients. With the exception of ECAD, the presence of methylation of these genes or a group of genes was not associated with different survival. The longer survival associated with ECAD methylation will need to be confirmed by other series. However, a possible explanation for this surprising finding might be the fact that methylation of ECAD seems to be dynamic and heterogenous as described by Graff et al. (37) . In the Graff et al. (37) study, breast cancers under conditions favoring invasion (with loss of adhesion) exhibited densely methylated ECAD promoter with reduced ECAD expression, whereas their survival and growth under conditions similar to metastatic deposits (spheroids) requiring cell adhesion actually showed loss of ECAD methylation with reestablished ECAD expression. It will be of interest to see whether similar findings occur in lung cancer primary lesions and metastatic deposits. Although it was not correlated with survival, the presence of finding any gene methylated was correlated with lymph node positivity. Methylation of TIMP-3 was seen more frequently in women, whereas methylation of DAPK and p16^INK4^a was more common in men. The reason for this gender difference is unknown. Methylation of p16^INK4^a was more frequent in squamous cell carcinomas than in adenocarcinomas. These results demonstrate that methylation of certain genes may be associated with some clinicopathological characteristics of these patients. These clinical correlations need to be confirmed in other independent studies.

For p16^INK4^a and RB, which are involved in the p16^INK4a/cyclin D1/cyclin-dependent protein kinase 4/RB pathway, data about protein expression from immunohistochemistry studies were available. We found a statistically significant correlation between loss of p16^INK4^a expression and methylation of p16^INK4^a and, as expected, an inverse correlation between loss of RB expression and p16^INK4^a methylation. These findings confirm previous data showing that methylation is a mechanism for gene silencing of p16^INK4^a. This finding is also in agreement with finding either p16^INK4^a or RB mutations or loss of protein expression, but not both, in the same tumor (28 , 38) . Esteller et al. (13) reported that methylation-associated inactivation of p14^ARF is independent of p16^INK4^a methylation and p53 mutational status in colon carcinomas. In agreement with their study, we also found methylation-associated inactivation of p14^ARF to be independent of p16^INK4^a methylation and p53 mutational status.

In conclusion, our study about methylation of RARß, TIMP-3, p16^INK4^a, MGMT, DAPK, ECAD, p14^ARF, and GSTP1 in primary resected NSCLCs stresses the high frequency of methylation in a large collection of samples and demonstrates that methylation may be the most common mechanism to inactivate cancer-related genes in NSCLC. Why certain genes are targeted for methylation and the enzymes involved in this methylation merit special attention, and the answers should be of translational value. In the meantime, the detection of methylated genes is an attractive biomarker for testing the early detection of lung cancer and for monitoring chemoprevention efforts as proposed by Belinsky et al. (16) . In these studies, it will be necessary to show in prospective clinical trials that persons at high risk for developing lung cancer (such as smokers with a heavy smoking history who go on to develop lung cancer) have certain key genes methylated in the nonmalignant tissues (e.g., sputum or bronchial brushes and washings) before the cancer becomes clinically evident.

	ACKNOWLEDGMENTS

 
We thank Luc Girard for analysis with the Visual Basic Program.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Grants J1658-MED and J1860-MED from the Austrian Science Foundation, Lung Cancer SPORE (Special Program of Research Excellence) P50 CA70907, and The G. Harold and Leila Y. Mathers Charitable Foundation.

² To whom requests for reprints should be addressed, at Hamon Center for Therapeutic Oncology Research, The University of Texas, Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4900; Fax: (214) 648-4940; E-mail: John.Minna{at}UTSouthwestern.edu

³ The abbreviations used are: TSG, tumor suppressor gene; NSCLC, non-small lung cancer; RAR, retinoic acid receptor; TIMP, tissue inhibitor of metalloproteinase; MGMT, O⁶-methylguanine-DNA-methyltransferase; DAPK, death-associated protein kinase; ECAD, E-cadherin; GSTP1, glutathione S-transferase P1; RB, retinoblastoma; LOH, loss of heterozygosity; MI, methylation index; MSP, methylation-specific PCR.

Received 6/ 5/00. Accepted 10/30/00.

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