Originally published as JCO Early Release 10.1200/JCO.2004.11.135 on July 26 2004

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Promoter Methylation of Retinoic Acid Receptor Beta 2 and the Development of Second Primary Lung Cancers in Non–Small-Cell Lung Cancer

From the Center for Genome Research, Samsung Biomedical Research Institute; Division of Pulmonary and Critical Care Medicine, Department of Thoracic Surgery, and Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; Department of Molecular Cell Biology, Sungkyunkwan University, School of Medicine, Suwon, Korea

Address reprint requests to Duk-Hwan Kim, MD, ScD, Center for Genome Research, Samsung Biomedical Research Institute, Rm B155, #50 Ilwon-dong, Kangnam-Ku, Seoul, Korea, 135-710; e-mail: dukhwan{at}samsung.co.kr

	ABSTRACT

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PURPOSE: To investigate whether the promoter hypermethylation of retinoic acid receptor beta 2 (RARß2) is associated with the development of second primary lung cancers (SPLCs) differentially according to smoking status in primary non–small-cell lung cancer (NSCLC).

PATIENTS AND METHODS: We retrospectively analyzed the relationship between RARß2 methylation and the SPLC development in a total of 342 NSCLCs. The methylation status of RARß2 was determined by using methylation-specific polymerase chain reaction. The difference in the time to SPLC development was analyzed by using the log-rank test and the Cox proportional hazards model. The median follow-up was 4.1 years.

RESULTS: SPLCs developed in 19 (5.6%) of the 342 NSCLCs, and overall incidence rate of SPLC development was 1.54 per 100 patient-years. SPLCs did not occur in 39 patients who had not smoked. After controlling for possible confounding factors, the hazard of failure for former smokers with RARß2 hypermethylation was about 2.87 (95% CI, 0.92 to 13.64; P = .08) times higher compared to those without RARß2 methylation. However, for current smokers, hypermethylation of the RARß2 was found to have a protective effect against the SPLC development (hazard ratio = 0.23; 95% CI, 0.11 to 0.87; P = .03).

CONCLUSION: Hypermethylation of RARß2 promoter had a differential effect on the development of SPLCs in NSCLC, and this was dependent on smoking status. Our study suggests that a combination of retinoids and/or a demethylating agent may be effective in the prevention of SPLCs in never-smokers and former smokers with NSCLC.

	INTRODUCTION

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Lung cancer is one of the most common malignancies in the world.¹ Despite advances in the detection and treatment of lung cancer, the overall 5-year survival rate remains under 15%.² Moreover, surgically treated patients with early stage non–small-cell lung cancer (NSCLC) have a high risk of developing second primary tumors (SPTs), which is one of the major causes of poor survival rate.

Retinoid, a class of natural and synthetic vitamin A analogs, is known to play a role in the regulation of cell proliferation, cell differentiation, and homeostasis.^3-5 Retinoids are recognized as promising agents for the prevention of SPTs in head and neck cancer.⁶ However, conflicting results have been issued on the effects of retinoic acid on the prevention of SPTs in NSCLC. A small study suggested that the adjuvant administration of retinyl palmitate in curatively resected NSCLC patients is effective at preventing SPTs.⁷ In contrast, two large-scale phase III retinoid trials reported that the exogenous administration of isotretinoin or retinyl palmitate and/or N-acetylcysteine in smokers with early stage NSCLC results in no preventive benefit on SPTs in NSCLC.^8,9

Retinoic acid is known to play its role by interacting with nuclear retinoic acid receptors (RARs) and retinoic X receptors (RXRs).^3-5 Both receptors have three subtypes—alpha, beta, and gamma—which have distinct functions. Of these receptors, the retinoic acid receptor beta (RARß) gene is expressed in three isoforms (ß1, ß2, and ß4), the expression of which is determined by two different promoters (P1 and P2) and alternative splicing.^5,10 RARß is implicated in mediating the growth inhibitory effect of retinoids in many different types of cancer cells, including lung cancer.^11-18 RARß expression is lost or reduced in a large percentage of patients with lung cancer and in a population at high risk of lung cancer.^12,19-25 Among RARß isoforms, RARß2 is primarily responsible for limiting the growth of lung cancer cells.^26-28 Transgenic mice expressing the antisense RARß2 sequences developed lung cancer,²⁷ and restoration of RARß2 into RARß-negative lung cancer cell lines inhibited the development of lung cancer.²⁸

The aberrant methylation of CpG islands is an epigenetic change that induces the transcriptional silencing of tumor suppressor genes.^29,30 The RARß2 promoter is characterized by a CpG island, which is located in the 5'-untranslated region. The promoter methylation of the RARß2 gene is found in approximately 40% of NSCLCs^31-33 and is considered a major cause of the loss of RARß expression.^27,32

In contrast to many findings about tumor suppressive effect of RARß in lung cancer, recently Khuri et al³⁴ reported that strong RARß expression is associated with worse outcome of stage I NSCLC. Based on those reports, we hypothesized that an adverse effect of RARß expression in NSCLC depends on the exposure to tobacco smoke. In an effort to investigate whether hypermethylation of RARß2 contributes to the development of SPLCs differentially according to smoking status in primary NSCLC, we analyzed the methylation status of RARß2 and the occurrence of SPLCs in primary NSCLC.

	PATIENTS AND METHODS

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Study Population
A total of 342 NSCLC patients who had been treated by surgical resection between May 1994 and August 1999 in the Department of Thoracic Surgery at the Samsung Medical Center (Seoul, Korea) participated in this study. The tumor was located in the right lung in 191 patients (56%) and in the left lung in 151 patients (44%). The NSCLC was most frequently located in the upper lobe of the lung (63%). Lobectomy was performed in 298 patients (87%), pneumonectomy in 10 patients (3%), and wedge resection in 34 patients (10%). Written informed consent for the usage of paraffin-embedded tissues, as approved by the Institutional Board at Samsung Medical Center, was obtained from each patient before the operation. Postoperative follow-up was scheduled at 1 month, 2 months, and every 3 months during the first 2 years after surgery, and thereafter every 6 months, or more frequently if needed. Chest x-ray, chest computed tomography scan, carcinoembryonic antigen, and other serum chemistry were scheduled at every follow-up visit. Whenever patients did not keep the postoperative follow-up schedule, specialized nurses called patients and checked their health status.

All patients were followed retrospectively for this study, based on the methylation status of RARß2 promoter. Primary end point was the development of SPLCs. Information on the development of SPLCs and smoking status was collected from medical records. Never-smokers were defined as patients who had smoked fewer than 100 cigarettes in their lifetime, and current smokers were defined as active smokers and those who had stopped smoking less than 1 year before surgical resection of primary NSCLC. Former smokers were defined as patients who had stopped at least 1 year before surgery of primary NSCLC. Patients who did not experience SPLC development before the study ended, or who were lost to follow-up during the study period or who died without the development of SPLCs, were used as censored data in calculating the hazard of the SPLC development. The criteria used for SPLCs were described by Martini and Melamed³⁵: a new cancer with a different histologic type or with the same histology as initial NSCLC if (1) the second cancer occurred at least 2 years after the primary tumor; (2) the second cancer originated from a carcinoma-in-situ; or (3) the second cancer was in different lobe or lung, but neither carcinoma in lymphatics common to both nor extrapulmonary metastases at time of diagnosis were found. The occurrence of SPLCs was evaluated as of September 31, 2003.

The 342 patients consisted of 210 men (61%) and 132 women (39%), ranging in age from 23 to 89 years. The mean age at the time of surgical resection for the first primary NSCLC was 59.6 years. Two hundred fifty-eight patients of 342 primary NSCLC had stage I disease, 69 patients stage II, 10 patients stage III, and five patients had stage IV disease. The histologic distribution of the first primary cancers at the time of surgical resection was 39% adenocarcinomas, 48% squamous cell carcinomas, and 13% other cell types.

DNA Extraction From Paraffin Block
Formalin-fixed, paraffin wax-embedded tissues were cut into 10 µm thick sections. Before DNA extraction, the sections were placed on slides and stained with hematoxylin-eosin to locate the tumor areas. Areas corresponding to tumor or surrounding normal lung tissue were microdissected separately. Microdissected tissues were collected in 15 mL centrifuge tubes, and xylene was added to the tissues. After centrifugation at full speed for 5 minutes, the supernatant was removed. Ethanol was added to the pellet to remove residual xylene, and then removed by centrifugation. After ethanol evaporation, the tissue pellet was resuspended in lysis buffer ATL (DNeasy Tissue kit; Qiagen, Valencia, CA) and the DNA was isolated according to manufacturer's instruction.

Methylation-Specific Polymerase Chain Reaction
Genomic DNA was modified with sodium bisulfite, as described by Herman et al.³⁶ Briefly, 1 µg of genomic DNA was denatured by incubation with 0.2M NaOH for 10 minutes at 37°C. Aliquots of 3M sodium bisulfite (pH 5.0; Sigma Chemical Co, St Louis, MO) and 10 mmol/L hydroquinone (Sigma Chemical Co) were then added, and the solution was incubated at 50°C for 16 hours. The modified DNA was purified by use of a Wizard DNA Purification System (Promega Corporation, Madison, WI), followed by ethanol precipitation. Modified DNA was stored in aliquots at –20°C until required. Treating genomic DNA with sodium bisulfite deaminates all unmethylated cytosines and converts them to uracil, whereas 5-methylcytosines remain unaltered. Thus, after bisulfite treatment, alleles that were originally methylated have DNA sequences that differ from those of their corresponding unmethylated alleles, and these differences can be used to design polymerase chain reaction (PCR) primers that are specific for methylated or unmethylated alleles.

The amplification of the bisulfite-modified DNA of the RARß gene promoter P2 was performed by PCR, as described by Virmani et al,³⁶ with primers specific for the methylated or unmethylated RARß2 sequences. The primer sequences used to amplify the methylated RARß2 gene were 5'-TCGAGAACGCGAGCGATTCG-3' (sense) and 5'-GACCAATCCAACCGAAACGA-3' (antisense), and the primer sequences used for the unmethylated RARß2 gene were 5'-TTGAGAATGTGAGTGATTTGA-3' (sense) and 5'-AACCAATCCAACCAAAACAA-3' (antisense). The PCR mixture contained 1x PCR buffer (50 mmol/L KCl, 67 mmol/L Tris, pH 8.7, 1.5 mmol/L MgCl₂), deoxynucleotide triphosphates (each 1.25 mmol/L), primers (300 ng each per reaction), 2.5 unit of Taq polymerase, and bisulfite-modified DNA (50 ng). Reactions were hot started at 94°C before adding 2.5 units of Taq polymerase. Amplification was carried out over 35 cycles (1 minute at 94°C, 1 minute at the annealing temperature, 1 minute at 72°C), followed by 4 minutes at 72°C. Fifteen µL of the PCR reaction were loaded on 2% agarose gel, stained with ethidium bromide (Life Technologies Inc, Rockville, MD), and visualized under ultraviolet illumination. DNA from peripheral blood lymphocytes of a healthy individual was treated with SssI methyltransferase (New England Biolabs Inc, Beverly, MA), subjected to bisulfite modification, and used as a positive-control for methylated alleles. Negative control samples without DNA were included for each set of PCR. DNA from normal lymphocytes served as a negative control for methylated alleles.

Statistical Analysis
The clinicopathologic difference between patients with and without SPLCs was analyzed using the Wilcoxon rank sum test for continuous variables or Fisher's exact test (or Pearson's ² test) for categoric variables, such as smoking status, histology, pathologic stage, and RARß2 methylation. Incidence rate of SPLC development was calculated as the ratio of SPLC cases over 100 patient-years of follow-up. Data were stratified by smoking status for further analysis, since the aim of the study was to investigate whether the association between RARß2 methylation and SPLC development depends on smoking status. The time to SPLC development was measured from the time of surgery for the first primary tumor until the time of SPLC diagnosis. The effect of RARß2 methylation on time to SPLC development was estimated by the Kaplan-Meier method, and differences in time to development of SPLC between patients with and without RARß2 methylation were compared using the log-rank test. Stratified Cox proportional hazard regression analysis was performed to estimate the hazard ratio of independent factors for the time to SPLC development, after controlling for potential confounding factors such as age, sex, pathologic stage, and histology. All statistical analyses were two-sided, with a 5% type I error rate.

	RESULTS

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Clinicopathologic Characteristics
Methylation status of CpG island at the promoter of RARß2 gene in paraffin-embedded tissues was analyzed by methylation-specific polymerase chain reaction (Fig 1). Table 1 shows the clinicopathologic characteristics of 342 patients included in this study as a function of SPLC.

View larger version (19K): [in this window] [in a new window]   Fig 1. Methylation-specific polymerase chain reaction of DNA from primary tumors. The numbers shown are sample identification numbers. Pos represents positive control for methylated (M) and unmethylated (U) allele. Neg, negative.

Incidence Rate of SPLC Development by Smoking and RARß2 Methylation
Incidence rate of SPLC development in terms of smoking status and RARß2 methylation is shown in Table 2. The overall incidence rate of SPLC development was 1.54 per 100 patient-years. The incidence rate of SPLCs was 2.2 per 100 patient-years for the first 5 years of postoperative follow-up and 0.7 per 100 patient-years thereafter (data not shown). The incidence rate for former smokers and current smokers was 1.12 and 2.58 per 100 patient-years, respectively (P = .03). The incidence rate of SPLCs for patients without and with RARß2 methylation was 1.68 per 100 patient-years and 1.56 per 100 patient-years, respectively (P = .81). Patients with stage II showed higher incidence rate than those with stage I, but the difference did not reach a statistical significance (1.96 v 1.37, respectively; rate ratio [RR] = 1.41; 95% CI, 0.89 to 5.67; P = .37; data not shown). Squamous cell carcinoma had a higher incidence rate of SPLC than adenocarcinoma (1.62 v 1.14 per 100 patient-years, respectively), but this difference was not statistically significant (RR = 1.41; 95% CI, 0.74 to 3.80; P = .27; data not shown). The incidence rate of SPLC development by RARß2 methylation, stratified by smoking status, is shown in Figure 2. SPLC did not develop in 39 never-smokers. For former smokers, incidence rate of SPLC development was 0.76 and 2.14 per 100 patient-years in patients without and with RARß2 methylation, respectively (P = .08). In contrast, incidence rate of SPLC development in current smokers was lower in those with RARß2 methylation than those without RARß2 methylation (1.31 v 2.97, respectively; P = .04).

View larger version (15K): [in this window] [in a new window]   Fig 2. Incidence rate of second primary lung cancers by retinoic acid receptor beta 2 methylation, stratified by smoking status.

View larger version (16K): [in this window] [in a new window]   Fig 3. Kaplan-Meier plots of the time to second primary lung cancer (SPLC) development by retinoic acid receptor beta 2 (RARß2) methylation, stratified by smoking status. (A) former smokers; (B) current smokers. The gray and black lines denote the groups with and without RARß2 methylation, respectively.

	DISCUSSION

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Many groups have conducted intervention studies to assess the tertiary chemopreventive effect of retinoids in patients with lung cancer or head and neck cancer, but the effect of retinoids on the prevention of SPTs in NSCLC remains controversial. Two small studies suggested beneficial effects for isotretinoin and retinyl palmitate in terms of preventing SPTs in NSCLC and in head and neck cancer patients.^6,7 However, the results of subsequent large trials specifically undertaken to confirm the positive outcomes of these prior smaller studies did not support the preventive effect for isotretinoin or retinyl palmitate and/or N-acetylcysteine in patients with NSCLC or head and neck cancer.^8,9 The EUROSCAN study reported that a 2-year supplement of retinyl palmitate (300,000 U/d for 1 year, then at 150,000 U/d for 1 year) and/or N-acetylcysteine (at 600 mg/d) does not result in a benefit in terms of preventing SPTs in lung cancer. Lippsman et al⁸ also found no beneficial effect of isotretinoin (30 mg/d for 3 years) on SPT prevention in stage I NSCLC in a large phase III randomized controlled trial.

Although the reason for the failure of retinoids to demonstrate tertiary chemopreventive effect in cigarette smokers in these clinical trials is unclear, our data supports the finding of the large intervention studies^8,9 that show that the exogenous administration of retinoids in smokers results in no preventive benefit on SPTs in NSCLC. Based on our data that aberrant methylation of the RARß2 promoter has a differential effect on the development of SPLCs in NSCLC according to the exposure to tobacco smoke, it is likely that the retinoic acid receptors in current smokers may interact with smoking at the level of post-translation.

Several clinical trials that examined the chemopreventive effectiveness of conventional retinoids in lung cancer have suggested the existence of a smoking-retinoids interaction. A trial of isotretinoin in chemoprevention of bronchial squamous metaplasia showed differential treatment trends according to smoking status.⁴¹ Active smokers on isotretinoin had slightly worse metaplasia outcomes than active smokers on placebo, whereas patients who had recently quit smoking had slightly better metaplasia outcomes on isotretinoin than recent quitters on the placebo. A trial of fenretinide (N-[4-hydroxyphenyl]retinamide), a retinoid analog with antitumor and chemopreventive activities, did not find a beneficial effect in terms of reversing squamous metaplasia and dysplasia in the bronchial epithelium of current smokers.⁴² Two large intervention studies, the EUROSCAN and the National Cancer Institute intergroup phase III trial, also reported a differential effect of retinoids according to smoking status.

What is the mechanism responsible for the interaction between retinoids and smoking? Cigarette smoke is a source of free radicals that can induce oxidative DNA damage in humans.⁴³ The major free radical species in cigarette smoke is a quinone-hydroquinone complex, and this free radical complex causes redox cycling that generates superoxide anions from molecular oxygen and leads to the formation of hydrogen peroxide and hydroxyl radicals.⁴⁴ Hydrogen peroxide induces cell death with nuclear condensation and DNA fragmentation, typical of apoptosis in various cell types.^45,46 It is known that the constitutive activation of the JNK-AP-1 pathway results in apoptotic cell death, and that dominant-negative inactivation of JNK or c-Jun prevents the apoptotic processes.^47,48 Accumulative data have suggested that c-Jun/AP-1 is involved in the intracellular signaling pathway to apoptosis triggered by hydrogen peroxide. H₂O₂ induces the expression of c-Jun and the activation of AP-1, and the downregulation of c-Jun/AP-1 inhibits H₂O₂-initiated apoptosis.⁴⁵ All-trans-retinoic acid (ATRA) is known to induce apoptosis and also to function as an inhibitor of apoptosis in some cell types.⁴⁹ The ATRA inhibits the apoptosis of T cells, leukemic cells, and hematopoietic cells,^50-53 and also attenuates H₂O₂-induced apoptosis in various cell types, including mesangial cells and fibroblasts.⁵⁴

The molecular mechanism involved in the antiapoptotic action of ATRA in H₂O₂- triggered cells is not fully understood, but many observations suggest that ATRA might inhibit H₂O₂-induced apoptosis in several ways: (1) by suppressing AP-1; (2) by sequestering AP-1; and (3) by activating NF-B. Although the manner in which retinoic acid affects the JNK-AP-1 pathway is different, and is dependent on cell type and the nature of triggering signal, many groups have reported that ATRA inhibits the AP-1 pathway by suppressing AP-1 expression. ATRA inhibits the expressions of c-fos and c-jun in synovial fibroblasts and the growth factor-induced activation of JNK in human bronchial epithelial cells.^55,56 In NSCLC cells expressing functional retinoic acid receptors, ATRA inhibits JNK activity by increasing dual-specificity phosphatase activity.⁵⁷ ATRA also inhibits H₂O₂-stimulated AP-1 activation by suppressing c-fos/c-jun expression and by phosphorylating JNK; moreover, the dominant-negative inhibition of JNK attenuates H₂O₂-induced apoptosis.⁵⁸ The biologic actions of retinoic acid are mediated by RARs and RXRs. RXR is implicated in the inhibition of T-cell apoptosis by retinoic acid,⁵¹ and both RAR and RXR are required for the antiapoptotic effect of ATRA in H₂O₂-exposed cells.⁵⁸ In addition to its suppressive effects on JNK and c-fos/c-jun, ATRA may be involved in the antiapoptotic action by sequestering AP-1 proteins.⁵⁹ It is also likely that ATRA inhibits apoptosis via the upregulation of NF-B, a potent antiapoptotic molecule.⁶⁰ The antiapoptotic effect of ATRA was observed in H₂O₂-stimulated SM/IkBM cells,⁶⁰ and ATRA was found to attenuate mesangial cell apoptosis induced by the NF-B inhibitor pyrrolidine dithiocarbamate.⁵⁸ Based on those observations that cigarette smoke forms H₂O₂ and that retinoic acid inhibits H₂O₂-induced apoptosis, the expression of RARß may have an adverse effect on the development of SPLCs in current smokers with NSCLC.

Is it reasonable to recommend the intake of retinoids for chemoprevention of SPLCs to former and never-smokers who have undergone surgical resection for NSCLC? Retinoids are known to play a role in inhibiting the proliferation of bronchial epithelial cells. Retinoids regulate the proliferation of bronchial epithelial cells in vivo and in vitro by inhibiting JNK/AP-1 activity,⁵⁶ the activity of which is associated with cell proliferation and tumor progression, or by triggering G₁ cell cycle arrest in bronchial epithelial cells.^61,62 In addition, ATRA causes bronchial epithelial cells to exit the cell cycle by activating degradation of CDK4 or cyclins through the ubiquitin-protesome pathway.^61,63,64 Our finding that the hypermethylation of RARß2 gene in former smokers has a detrimental effect on the development of SPLCs in NSCLC suggests that the administration of retinoids in former smokers remains an effective way to prevent SPLCs in NSCLC. In the present study, the log-rank test for former smokers did not show a statistically significant difference in the time to SPLC development between those with and without the hypermethylation of RARß2 gene (P = .08). This may result from the small sample size and the short duration of follow-up. It needs further evaluation through longer follow-up of a large sample. Recently Kurie et al⁶⁵ reported that treatment of former smokers with 9-cis-retinoic acid reversed the loss of RARß expression in the bronchial epithelium, suggesting that 9-cis-retinoic acid is effective at preventing SPLCs in former smokers. Accordingly, the combination of retinoids and demethylating agent and smoking cessation is recommended for the prevention of SPLCs in former smokers with NSCLC. The SPLCs did not develop in the never-smokers with NSCLC, irrespective of the methylation status of RARß2. However, considering the finding that transfection of RARß into RARß-negative lung cancer cell lines inhibits tumorigenicity in nude mice,¹² the combination of retinoids and/or demethylating agent may be beneficial to nonsmokers. In the present study, the analyzed person-years are a little higher in patients without hypermethylation of RARß2 than in those with (data not shown), which may provide a bias. Loss to follow-up is a particular concern for cohort studies. A low or a high follow-up rate that is noncomparable for exposed and nonexposed individuals may not provide interpretable results. Accordingly, minimizing losses to follow-up is very important in this kind of study. In conclusion, our study suggests that hypermethylation of RARß2 has a differential effect on the development of SPLCs in NSCLC, and that this is dependent on smoking status.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We gratefully acknowledge Jin-Hyuk Kim for critical reading of the manuscript. We also thank Eunkyung Kim for assistance with data collection and management.

	NOTES

 
Supported by grants from the Samsung Biomedical Research Institute and the Samsung Advanced Institute of Technology.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted November 21, 2003; accepted April 20, 2004.

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